US20040224323A1 - PAK5 screening methods - Google Patents

PAK5 screening methods Download PDF

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US20040224323A1
US20040224323A1 US10/725,329 US72532903A US2004224323A1 US 20040224323 A1 US20040224323 A1 US 20040224323A1 US 72532903 A US72532903 A US 72532903A US 2004224323 A1 US2004224323 A1 US 2004224323A1
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amino acid
human
kinase
sequence
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Gregory Plowman
Ricardo Martinez
David Whyte
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Sugen LLC
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel kinase polypeptides, nucleotide sequences encoding the novel kinase polypeptides, as well as various products and methods useful for the diagnosis and treatment of various kinase-related diseases and conditions.
  • Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells.
  • One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure and function.
  • Protein kinases can also be characterized by their location within the cell. Some kinases are transmembrane receptor-type proteins capable of directly altering their catalytic activity in response to the external environment such as the binding of a ligand. Others are non-receptor-type proteins lacking any transmembrane domain. They can be found in a variety of cellular compartments from the inner surface of the cell membrane to the nucleus.
  • kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state. Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway.
  • Protein kinases are one of the largest families of eukaryotic proteins with several hundred known members. These proteins share a 250-300 amino acid domain that can be subdivided into 12 distinct subdomains that comprise the common catalytic core structure. These conserved protein motifs have recently been exploited using PCR-based cloning strategies leading to a significant expansion of the known kinases.
  • mammalian members of the STE20-kinase family have been identified as part of the present invention.
  • Multiple alignment and parsimony analysis of the catalytic domain of all of these STE20-family members reveals that these proteins cluster into 9 distinct subgroups. Classification in this manner has proven highly accurate not only in predicting motifs present in the remaining non-catalytic portion of each protein, but also in their regulation, substrates, and signaling pathways.
  • the present invention includes the partial or complete sequence of new members of the STE20-family, their classification, predicted or deduced protein structure, and a strategy for elucidating their biologic and therapeutic relevance.
  • a first aspect of the invention features an isolated, enriched, or purified nucleic acid molecule encoding a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • isolated in reference to nucleic acid is meant a polymer of nucleotides conjugated to each other, including DNA and RNA, that is isolated from a natural source or that is synthesized.
  • the isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature.
  • Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
  • enriched in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term “significant” is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more.
  • the term also does not imply that there is no DNA or RNA from other sources.
  • the other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC 19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • cDNA synthetic substance
  • the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 6 -fold purification of the native message.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • kinase polypeptide 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; 250 (preferably 255, more preferably 260, most preferably 270) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; 27 (preferably 30, more preferably 40, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO: 18; 16 (preferably 20, more preferably 25, most preferably 35) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:
  • sequences for which the full-length sequence is not given the remaining sequences can be determined using methods well-known to those in the art and are intended to be included in the invention.
  • polypeptides of 100, 200, 300 or more amino acids are preferred.
  • the kinase polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained, not to include fragments containing only amino acids 1-22 of SEQ ID NO: 13 or only amino acids 1-33 of SEQ ID NO:107.
  • amino acid sequence will be substantially similar to the sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or fragments thereof, not to include fragments consisting only of the amino acid sequences 1-22 of SEQ ID NO:13 or 1-33 of SEQ ID NO:107.
  • a sequence that is substantially similar to the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID
  • identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. “Gaps” are spaces in an alignment that are the result of additons or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Blast (Altschul, et al. (1997) Nucleic Acids Res.
  • nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence.
  • domain refers to a region of a polypeptide which contains a particular function.
  • N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal.
  • Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
  • N-terminal domain refers to the extracatalytic region located between the initiator methionine and the catalytic domain of the protein kinase.
  • the N-terminal domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the N-terminal boundary of the catalytic domain.
  • the N-terminal domain may or may not play a regulatory role in kinase function.
  • An example of a protein kinase whose N-terminal domain has been shown to play a regulatory role is PAK65, which contains a CRIB motif used for Cdc42 and rac binding (Burbelo, P. D. et al. (1995) J. Biol. Chem. 270, 29071-290740).
  • the N-terminal domain spans amino acid residues 1-21 of the sequence set forth in SEQ ID NO:5, amino acid residues 1-31 of the sequence set forth in SEQ ID NO:6, amino acid residues 1-22 of the sequence set forth in SEQ ID NO: 13, amino acid residues 1-13 of the sequence set forth in SEQ ID NO: 18, amino acid residues 1-21 of the sequence set forth in SEQ ID NO:22, amino acid residues 1-25 of the sequence set forth in SEQ ID NO:24, amino acid residues 1-51 of the sequence set forth in SEQ ID NO:29, amino acid residues 1-25 of the sequence set forth in SEQ ID NO:31, amino acid residues 1-57 of the sequence set forth in SEQ ID NO:99, amino acid residues 1-52 of the sequence set forth in SEQ ID NO: 103, amino acid residues 1-24 of the sequence set forth in SEQ ID NO:105, or amino acid residues 1-33 of the sequence set forth in SEQ ID NO:107.
  • catalytic domain refers to a region of the protein kinase that is typically 25-300 amino acids long and is responsible for carrying out the phosphate transfer reaction from a high-energy phosphate donor molecule such as ATP or GTP to itself (autophosphorylation) or to other proteins (exogenous phosphorylation).
  • the catalytic domain of protein kinases is made up of 12 subdomains that contain highly conserved amino acid residues, and are responsible for proper polypeptide folding and for catalysis.
  • the catalytic domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database.
  • the catalytic domain spans amino acid residues 22-274 of the sequence set forth in SEQ ID NO:5, residues 32-308 of the sequence set forth in SEQ ID NO:6, residues 1-178 of the sequence set forth in SEQ ID NO:7, residues 23-289 of the sequence set forth in SEQ ID NO:13, residues 1-255 of the sequence set forth in SEQ ID NO:14, residues 1-255 of the sequence set forth in SEQ ID NO: 15, residues 14-273 of the sequence set forth in SEQ ID NO:18, residues 22-277 of the sequence set forth in SEQ ID NO:22, residues 1-66 of the sequence set forth in SEQ ID NO:23, residues 26-273 of the sequence set forth in SEQ ID NO:24, residues 394-658 of the sequence set forth in SEQ ID NO:29, residues 26-281 of the sequence set forth in SEQ ID NO:31, residues 1-278 of the sequence set forth in SEQ ID NO:97, residues 58-369 of the sequence set forth in SEQ ID NO
  • catalytic activity defines the rate at which a kinase catalytic domain phosphorylates a substrate.
  • Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a phosphorylated product as a function of time.
  • Catalytic activity can be measured by methods of the invention by holding time constant and determining the concentration of a phosphorylated substrate after a fixed period of time.
  • Phosphorylation of a substrate occurs at the active-site of a protein kinase.
  • the active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
  • substrate refers to a molecule phosphorylated by a kinase of the invention.
  • Kinases phosphorylate substrates on serine/threonine or tyrosine amino acids.
  • the molecule may be another protein or a polypeptide.
  • C-terminal domain refers to the region located between the catalytic domain or the last (located closest to the C-terminus) functional domain and the carboxy-terminal amino acid residue of the protein kinase.
  • functional domain is meant any region of the polypeptide that may play a regulatory or catalytic role as predicted from amino acid sequence homology to other proteins or by the presence of amino acid sequences that may give rise to specific structural conformations (i.e. coiled-coils).
  • the C-terminal domain can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C-terminal boundary of the catalytic domain or of any functional C-terminal extracatalytic domain.
  • the C-terminal domain may or may not play a regulatory role in kinase function.
  • An example of a protein kinase whose C-terminal domain may play a regulatory role is PAK3 which contains a heterotrimeric G b subunit-binding site near its C-terminus (Leeuw, T. et al (1998) Nature, 391, 191-195).
  • the C-terminal domain spans amino acid residues 275-416 of the sequence set forth in SEQ ID NO:5, residues 309-489 of the sequence set forth in SEQ ID NO:6, residues 179-414 of the sequence set forth in SEQ ID NO:7, residues 897-1239 of the sequence set forth in SEQ ID NO:13, residues 955-1297 of the sequence set forth in SEQ ID NO:14, residues 984-1326 of the sequence set forth in SEQ ID NO:15, residues 535-894 of the sequence set forth in SEQ ID NO: 18, residues 752-898 of the sequence set forth in SEQ ID NO:22, residues 279-330 of the sequence set forth in SEQ ID NO:97, residues 370-418 of the sequence set forth in SEQ ID NO:99, or residues 873-1227 of the sequence set forth in SEQ ID NO:105.
  • signal transduction pathway refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response.
  • the polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine binding (PTB and PH) domain containing proteins), proline-rich binding proteins (SH3 domain containing proteins), nucleotide exchange factors, and transcription factors.
  • coil-coil structure region refers to a polypeptide sequence that has a high probability of adopting a coiled-coil structure as predicted by computer algorithms such as COILS (Lupas, A. (1996) Meth. Enzymology 266:513-525). Coiled-coils are formed by two or three amphipathic ⁇ -helices in parallel. Coiled-coils can bind to coiled-coil domains of other polypeptides resulting in homo- or heterodimers (Lupas, A. (1991) Science 252:1162-1164).
  • Coiled-coil-dependent oligomerization has been shown to be necessary for protein function including catalytic activity of serine/threonine kinases (Roe, J. et al. (1997) J. Biol. Chem. 272:5838-5845).
  • the coiled-coil structure region spans amino acid residues 290-526 of the sequence set forth in SEQ ID NO: 13, residues 256-442 of the sequence set forth in SEQ ID NO:14, residues 256-476 of the sequence set forth in SEQ ID NO:15, residues 428-637 of the sequence set forth in SEQ ID NO:22, residues 216-425 or 540-786 of the sequence set forth in SEQ ID NO:23, residues 423-632 of the sequence set forth in SEQ ID NO:24, residues 431-640 or 755-901 of the sequence set forth in SEQ ID NO:31, residues 291-398 or 629-668 of the sequence set forth in SEQ ID NO: 105, or residues 473-724 or 725-968 of the sequence set forth in SEQ ID NO:107.
  • proline-rich region refers to a region of a protein kinase whose proline content over a given amino acid length is higher than the average content of this amino acid found in proteins (i.e., >10%). Proline-rich regions are easily discernable by visual inspection of amino acid sequences and quantitated by standard computer sequence analysis programs such as the DNAStar program EditSeq. Proline-rich regions have been demonstrated to participate in regulatory protein -protein interactions.
  • PxxP proline rich motif found in certain protein kinases (i.e., human PAK1) and the SH3 domain of the adaptor molecule Nck (Galisteo, M. L. et al. (1996) J. Biol. Chem. 271:20997-21000).
  • Other regulatory interactions involving “PxxP” (SEQ ID NO: 148) proline-rich motifs include the WW domain (Sudol, M. (1996) Prog. Biochys. Mol. Bio. 65:113-132).
  • the proline-rich region spans amino acid residues 527-640 of the sequence set forth in SEQ ID NO:13, residues 443-626 of the sequence set forth in SEQ ID NO:14, residues 477-680 of the sequence set forth in SEQ ID NO:15, residues 347-534 of the sequence set forth in SEQ ID NO: 18, residues 398-628 of the sequence set forth in SEQ ID NO:105, or residues 338-472 of the sequence set forth in SEQ ID NO:107.
  • spacer region refers to a region of the protein kinase located between predicted functional domains.
  • the spacer region has no detectable homology to any amino acid sequence in the database, and can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C- and N-terminal boundaries of the flanking functional domains.
  • Spacer regions may or may not play a fundamental role in protein kinase function. Precedence for the regulatory role of spacer regions in kinase function is provided by the role of the src kinase spacer in inter-domain interactions (Xu, W. et al. (1997) Nature 385:595-602).
  • the spacer region spans amino acid residues 641-896 of the sequence set forth in SEQ ID NO:13, residues 627-954 of the sequence set forth in SEQ ID NO:14, residues 681-983 of the sequence set forth in SEQ ID NO: 15, residues 274-346 of the sequence set forth in SEQ ID NO:18, residues 278-427 or 638-751 of the sequence set forth in SEQ ID NO:22, residues 67-215 or 426-539 of the sequence set forth in SEQ ID NO:23, residues 274-422 or 633-748 of the sequence set forth in SEQ ID NO:24, residues 225-393 of the sequence set forth in SEQ ID NO:29, residues 282-430 or 641-754 of the sequence set forth in SEQ ID NO:31, residues 174-307 of the sequence set forth in SEQ ID NO:103, residues 669-872 of the sequence set forth in SEQ ID NO:105, or residues 295-337 of the sequence set forth in SEQ ID NO:
  • Insert refers to a portion of a protein kinase that is absent from a close homolog. Inserts may or may not by the product alternative splicing of exons. Inserts can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Inserts may play a functional role by presenting a new interface for protein-protein interactions, or by interfering with such interactions. Inserts span amino acid residues 52-224 of the sequence set forth in SEQ ID NO:29 or residues 53-173 of the sequence set forth in SEQ ID NO:103.
  • C-terminal tail refers to a C-terminal domain of a protein kinase, that by homology extends or protrudes past the C-terminal amino acid of its closest homolog.
  • C-terminal tails can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Depending on its length, a C-terminal tail may or may not play a regulatory role in kinase function.
  • the C-terminal tail spans amino acid residues 490-516 of the sequence set forth in SEQ ID NO:6, residues 787-887 of the sequence set forth in SEQ ID NO:23, residues 659-681 of the sequence set forth in SEQ ID NO:29, residues 994-1093 of the sequence set forth in SEQ ID NO:31, or residues 573-591 of the sequence set forth in SEQ ID NO:103.
  • Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. These conditions are well-known to those skilled in the art. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides, more preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 50 contiguous nucleotides, most preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 100 contiguous nucleotides. In some instances, the conditions may prevent hybridization of nucleic acids having more than 5 mismatches in the full-length sequence.
  • stringent hybridization assay conditions hybridization assay conditions at least as stringent as the following: hybridization in 50% formamide, 5 ⁇ SSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5 ⁇ Denhart solution at 42° C. overnight; washing with 2 ⁇ SSC, 0.1% SDS at 45 DC; and washing with 0.2 ⁇ SSC, 0.1% SDS at 45° C. Under some of the most stringent hybridization assay conditions, the second wash can be done with 0.1 ⁇ SSC at a temperature up to 70° C. (Berger et al.
  • the invention features isolated, enriched, or purified nucleic acid molecules encoding kinase polypeptides, further comprising a vector or promoter effective to initiate transcription in a host cell.
  • the invention also features recombinant nucleic acid, preferably in a cell or an organism.
  • the recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO:106, or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell.
  • the recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a kinase polypeptide and a transcriptional termination region functional in a cell. Specific vectors and host cell combinations are discussed herein.
  • vector relates to a single or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome.
  • a circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes.
  • restriction enzymes An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art.
  • a nucleic acid molecule encoding a kinase can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
  • the term “transfecting” defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies.
  • promoter refers to nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to persons skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5′-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the kinase polypeptides selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, comprise, consist essentially of, or consist of at least at least 40, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 35, 40, 45, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ
  • the nucleic acid may be isolated from a natural source by cDNA cloning or by subtractive hybridization.
  • the natural source may be mammalian, preferably human, blood, semen, or tissue, and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer.
  • mice refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes, and most preferably to humans.
  • the nucleic acid is a conserved or unique region, for example those useful for: the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, obtaining antibodies to polypeptide regions, and designing antisense oligonucleotides.
  • conserved nucleic acid regions are meant regions present on two or more nucleic acids encoding a kinase polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding kinase polypeptides are provided in Abe, et al. (J. Biol. Chem. 19:13361-13368, 1992), hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables. Preferably, conserved regions differ by no more than 5 out of 20 nucleotides, even more preferably 2 out of 20 nucleotides or most preferably 1 out of 20 nucleotides.
  • nucleic acid region is meant a sequence present in a nucleic acid coding for a kinase polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide.
  • Such regions preferably encode 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; 250 (preferably 255, more preferably 260, most preferably 270) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; 27 (preferably 30, more preferably 40, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO: 18; 16 (preferably 20, more preferably 25, most preferably 35) or more contiguous amino acids set forth
  • a second aspect of the invention features a nucleic acid probe for the detection of nucleic acid encoding a kinase polypeptide in a sample, wherein said polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • the nucleic acid probe encodes a kinase polypeptide that is a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO: 107, or the corresponding full-length amino acid sequences, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO: 13 or amino acids 1-33 of SEQ ID NO:107.
  • the nucleic acid probe contains a nucleotide base sequence that will hybridize to a sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO: 102, SEQ ID NO: 104, or SEQ ID NO: 106, or the corresponding full-length sequence, or a functional derivative thereof.
  • the nucleic acid probe hybridizes to nucleic acid encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or functional derivatives thereof.
  • Methods for using the probes include detecting the presence or amount of kinase RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to kinase RNA.
  • the nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a kinase polypeptide may be used in the identification of the sequence of the nucleic acid detected (Nelson et al., in Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Kricka, ed., p. 275, 1992, hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables).
  • Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
  • the invention describes a recombinant cell or tissue comprising a nucleic acid molecule encoding a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • the nucleic acid may be under the control of the genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter.
  • exogenous it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the kinase polypeptides.
  • the polypeptide is preferably a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107.
  • fragment an amino acid sequence present in a kinase polypeptide.
  • a sequence comprises at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or of the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, OR SEQ ID NO:105, or of the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO: 103, or of the corresponding full-length amino acid sequence; 6 (preferably
  • the invention features an isolated, enriched, or purified kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • isolated in reference to a polypeptide is meant a polymer of amino acids (2 or more amino acids) conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized.
  • the isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature.
  • Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
  • enriched in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2-fold, more preferably at least 5- to 10-fold or even more.
  • the term also does not imply that there is no amino acid sequence from other sources.
  • the other source of amino acid sequences may, for example, comprise amino acid sequence encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to increase the proportion of the desired amino acid sequence.
  • an amino acid sequence be in purified form.
  • purified in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment. Compared to the natural level this level should be at least 2-5 fold greater (e.g., in terms of mg/mL). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
  • the kinase polypeptide is a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequences, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107.
  • the kinase polypeptide contains at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino
  • the kinase polypeptide comprises an amino acid sequence having (a) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107; (b) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:
  • the polypeptide can be isolated from a natural source by methods well-known in the art.
  • the natural source may be mammalian, preferably human, blood, semen, or tissue, and the polypeptide may be synthesized using an automated polypeptide synthesizer.
  • the isolated, enriched, or purified kinase polypeptide is preferably: a STLK2, STLK3, STLK4; STLK5, STLK6, or STLK7 polypeptide; a ZC1, ZC2, ZC3, or ZC4 polypeptide; a KHS2 polypeptide; a SULU1 or SULU3 polypeptide; a GEK2 polypeptide; or a PAK4 or PAK5 polypeptide.
  • the invention includes a recombinant kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • recombinant kinase polypeptide is meant a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
  • the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain or fragment where the polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • specific binding affinity is meant that the antibody binds to the target kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
  • Antibodies or antibody fragments are polypeptides that contain regions that can bind other polypeptides.
  • the term “specific binding affinity” describes an antibody that binds to a kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
  • polyclonal refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
  • various host animals may be immunized by injection with the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • “Monoclonal antibodies” are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art (Kohler et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110, both of which are hereby incorporated by reference herein in their entirety including any figures, tables, or drawings).
  • antibody fragment refers to a portion of an antibody, often the hyper variable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule.
  • a hyper variable region is a portion of an antibody that physically binds to the polypeptide target.
  • Antibodies or antibody fragments having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by probing the sample with the antibody under conditions suitable for kinase-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide.
  • Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for the kinase as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
  • An antibody or antibody fragment with specific binding affinity to a kinase polypeptide of the invention can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above.
  • Antibodies having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide.
  • Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
  • the invention features a hybridoma which produces an antibody having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain, where the polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • hybridoma is meant an immortalized cell line that is capable of secreting an antibody, for example an antibody to a kinase of the invention.
  • the antibody to the kinase comprises a sequence of amino acids that is able to specifically bind a kinase polypeptide of the invention.
  • the invention features a kinase polypeptide binding agent able to bind to a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK6, STLK7, STLK5, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • the binding agent is preferably a purified antibody that recognizes an epitope present on a kinase polypeptide of the invention.
  • Other binding agents include molecules that bind to kinase polypeptides and analogous molecules that bind to a kinase polypeptide. Such binding agents may be identified by using assays that measure kinase binding partner activity, such as those that measure PDGFR activity.
  • the invention also features a method for screening for human cells containing a kinase polypeptide of the invention or an equivalent sequence.
  • the method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying the kinases of the invention (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
  • the invention features methods for identifying a substance that modulates kinase activity comprising the steps of: (a) contacting a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5 with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide.
  • modulates refers to the ability of a compound to alter the function of a kinase of the invention.
  • a modulator preferably activates or inhibits the activity of a kinase of the invention depending on the concentration of the compound exposed to the kinase.
  • the term “activates” refers to increasing the cellular activity of the kinase.
  • the term inhibit refers to decreasing the cellular activity of the kinase.
  • Kinase activity is preferably the interaction with a natural binding partner.
  • modulates also refers to altering the function of kinases of the invention by increasing or decreasing the probability that a complex forms between the kinase and a natural binding partner.
  • a modulator preferably increases the probability that such a complex forms between the kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the kinase and the natural binding partner depending on the concentration of the compound exposed to the kinase, and most preferably decreases the probability that a complex forms between the kinase and the natural binding partner.
  • complex refers to an assembly of at least two molecules bound to one another.
  • Signal transduction complexes often contain at least two protein molecules bound to one another.
  • a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
  • natural binding partner refers to polypeptides, lipids, small molecules, or nucleic acids that bind to kinases in cells.
  • a change in the interaction between a kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of kinase/natural binding partner complex.
  • the term “contacting” as used herein refers to mixing a solution comprising the test compound with a liquid medium bathing the cells of the methods.
  • the solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the test compound or compounds into the cells of the methods.
  • DMSO dimethyl sulfoxide
  • the solution comprising the test compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device.
  • the invention features methods for identifying a substance that modulates kinase activity in a cell comprising the steps of: (a) expressing a kinase polypeptide in a cell, wherein said polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5; (b) adding a test substance to said cell; and (c) monitoring a change in cell phenotype or the interaction between said polypeptide and a natural binding partner.
  • the term “expressing” as used herein refers to the production of kinases of the invention from a nucleic acid vector containing kinase genes within a cell.
  • the nucleic acid vector is transfected into cells using well known techniques in the art as described herein.
  • the invention provides methods for treating a disease by administering to a patient in need of such treatment a substance that modulates the activity of a kinase selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • a kinase selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5.
  • the disease is selected from the group consisting of immune-related diseases and disorders, organ transplantation, myocardial infarction, cardiovascular disease, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer.
  • the immune-related diseases and disorders include, but are not limited to, rheumatoid arthritis, artherosclerosis, and autoimmune
  • the invention provides methods for treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, ZC4, KHS2, PAK4, and PAK5.
  • a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, ZC4, KHS2, PAK4, and PAK5.
  • the disease or disorder is selected from the group consisting of rheumatoid arthritis, artherosclerosis, autoimmune disorders, and organ transplantation.
  • the invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of STLK1, STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7.
  • a kinase polypeptide selected from the group consisting of STLK1, STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7.
  • the disease or disorder is selected from the group consisting of immune-related diseases and disorders, myocardial infarction, cardiomyopathies, stroke, renal failure, and oxidative stress-related neurodegenerative disorders.
  • the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation.
  • the invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, and ZC4.
  • a substance that modulates the activity of a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, and ZC4.
  • the disease is selected from the group consisting of immune-related diseases and disorders, cardiovascular disease, and cancer.
  • the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation.
  • Substances useful for treatment of kinase-related disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (Examples of such assays are provided in the references in section VI, below; and in Example 7, herein). Examples of substances that can be screened for favorable activity are provided and referenced in section VI, below.
  • the substances that modulate the activity of the kinases preferably include, but are not limited to, antisense oligonucleotides and inhibitors of protein kinases, as determined by methods and screens referenced in section VI and Example 7, below.
  • preventing refers to decreasing the probability that an organism contracts or develops an abnormal condition.
  • treating refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
  • a therapeutic effect refers to the inhibition or activation factors causing or contributing to the abnormal condition.
  • a therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.
  • a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.
  • Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
  • abnormal condition refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.
  • An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival.
  • Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
  • Abnormal differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
  • Abnormal cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.
  • a number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
  • administering relates to a method of incorporating a compound into cells or tissues of an organism.
  • the abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.
  • Cells existing outside the organism can be maintained or grown in cell culture dishes.
  • many techniques exist in the art to administer compounds including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
  • multiple techniques exist in the art to administer the compounds including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
  • the abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.
  • the effect of administering a compound on organism function can then be monitored.
  • the organism is preferably a mouse, rat, rabbit, guinea pig, or goat, more preferably a monkey or ape, and most preferably a human.
  • the invention features methods for detection of a kinase polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease.
  • a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a kinase polypeptide selected from the group consisting of STLK2, STLK
  • the disease or disorder is selected from the group consisting of rheumatoid arthritis, artherosclerosis, autoimmune disorders, organ transplantation, myocardial infarction, cardiomyopathies, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer.
  • the kinase polypeptide is selected from the group consisting of PAK4 and PAK5, or the polypeptide is selected from the group consisting of ZC1, ZC2, ZC3, and ZC4, and the disease is cancer.
  • the kinase “target region” is the nucleotide base sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO: 106, or the corresponding full-length sequences, a functional derivative thereof, or a fragment thereof to which the nucleic acid probe will specifically hybridize.
  • Target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database.
  • the nucleic acid probe hybridizes to a kinase target region encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or a functional derivative thereof.
  • Hybridization conditions should be such that hybridization occurs only with the kinase genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.
  • the diseases for which detection of kinase genes in a sample could be diagnostic include diseases in which kinase nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.
  • amplification is meant increased numbers of kinase DNA or RNA in a cell compared with normal cells.
  • kinases are typically found as single copy genes.
  • the chromosomal location of the kinase genes may be amplified, resulting in multiple copies of the gene, or amplification.
  • Gene amplification can lead to amplification of kinase RNA, or kinase RNA can be amplified in the absence of kinase DNA amplification.
  • RNA can be the detectable presence of kinase RNA in cells, since in some normal cells there is no basal expression of kinase RNA. In other normal cells, a basal level of expression of kinase exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, kinase RNA, compared to the basal level.
  • the diseases that could be diagnosed by detection of kinase nucleic acid in a sample preferably include cancers.
  • the test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.
  • the invention features a method for detection of a kinase polypeptide in a sample as a diagnostic tool for a disease or disorder, wherein the method comprises: (a) comparing a nucleic acid target region encoding the kinase polypeptide in a sample, where the kinase polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, or one or more fragments thereof, with a control nucleic acid target region encoding the kinase polypeptide, or one or more fragments thereof; and (b) detecting differences in sequence or amount between the target region and the control target region, as an indication of the disease or disorder.
  • the disease or disorder is selected from the group consisting of immune-related diseases and disorders, organ transplantation, myocardial infarction, cardiovascular disease, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer.
  • Immune-related diseases and disorders include, but are not limited to, those discussed previously.
  • comparing refers to identifying discrepancies between the nucleic acid target region isolated from a sample, and the control nucleic acid target region.
  • the discrepancies can be in the nucleotide sequences, e.g. insertions, deletions, or point mutations, or in the amount of a given nucleotide sequence. Methods to determine these discrepancies in sequences are well-known to one of ordinary skill in the art.
  • the “control” nucleic acid target region refers to the sequence or amount of the sequence found in normal cells, e.g. cells that are not diseased as discussed previously.
  • FIGS. 1A, 1B and 1 C show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 84-85, 5-7, respectively, in order of appearance) of the STE20-STE20 family kinases.
  • FIGS. 2A and 2B show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 84, 86-87 & 8, respectively, in order of appearance) of the STE20-STLK5 family kinases.
  • FIGS. 3A, 3B, 3 C, 3 D, 3 E, 3 F and 3 G show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 88-89, 13-16, respectively, in order of appearance) of STE20-ZC family kinases.
  • FIGS. 4A, 4B and 4 C show a pairwise sequence (SEQ ID NOS 91 & 18, respectively, in order of appearance) alignment of STE20-KHS family kinases.
  • FIGS. 5A, 5B, 5 C and 5 D show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 90, 22, 24 & 151 respectively, in order of appearance) of STE20-SULU family kinases.
  • FIGS. 6A, 6B and 6 C show a pairwise sequence (SEQ ID NOS 92 & 26, respectively, in order of appearance) alignment of STE20-GEK family kinases.
  • FIGS. 7A, 7B and 7 C show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 93-95, 29-30 respectively, in order of appearance) of STE20-PAK family kinases.
  • FIGS. 8A, 8B, 8 C, 8 D, 8 E, 8 F and 8 G show the amino acid sequences of human STLK2(SEQ ID NO:5), human STLK3(SEQ ID NO:6), human STLK4(SEQ ID NO:7), human STLK5(SEQ ID NO:8), human ZC1(SEQ ID NO:13), human ZC2(SEQ ID NO:14), human ZC3(SEQ ID NO:15), human ZC4(SEQ ID NO:16), human KHS2(SEQ ID NO:18), human SULU1(SEQ ID NO:22), human SULU3(SEQ ID NO:23), murine SULU3(SEQ ID NO:24), human GEK2(SEQ ID NO:26), human PAK4(SEQ ID NO:29), and human PAK5(SEQ ID NO30).
  • FIGS. 9A, 9B, 9 C, 9 D, 9 E, 9 F, 9 G, 9 H, 91 , 9 J, 9 K, 9 L, 9 M, 9 N, 90 , 9 P, 9 Q, 9 R, 9 S, 9 T, 9 U and 9 V show the nucleic acid sequences of human STLK2(SEQ ID NO: 1), human STLK3(SEQ ID NO:2), human STLK4(SEQ ID NO:3), human STLK5(SEQ ID NO:4), human ZC1(SEQ ID NO:9), human ZC2(SEQ ID NO:10), human ZC3(SEQ ID NO:11), human ZC4(SEQ ID NO:12), human KHS2(SEQ ID NO:17), human SULU1(SEQ ID NO:19), human SULU3(SEQ ID NO:20), murine SULU3(SEQ ID NO:21), human GEK2(SEQ ID NO:25), human PAK4(SEQ ID NO:27),
  • FIGS. 10A, 10B and 10 C show the full-length amino acid sequences of human STLK5 (SEQ ID NO: 97), human PAK5 (SEQ ID NO:103), and human ZC4 (SEQ ID NO: 105), as well as the partial amino acid sequences of human full-length STLK6 (SEQ ID NO: 99) and human STLK7 (SEQ ID NO: 101) and human GEK2 (SEQ ID NO: 107).
  • FIGS. 11A, 11B, 11 C, 11 D, 11 E, 11 F, 11 G and 11 H show the full-length nucleic acid sequences of human STLK5 (SEQ ID NO:96), human PAK5 (SEQ ID NO:102), and human ZC4 (SEQ ID NO:104), as well as the partial nucleic acid sequences of human STLK6 (SEQ ID NO: 98) and human STLK7 (SEQ ID NO: 100) and human GEK2 (SEQ ID NO: 106).
  • FIGGS. 12A and 12B show a multiple sequence alignment among human SPAK (SEQ ID NO: 153), human STLK6 (SEQ ID NO: 99), human STLK7 (SEQ ID NO: 101) and full-length human STLK5 (SEQ ID NO: 152).
  • FIGS. 13A, 13B and 13 C show a multiple sequence alignment among human PAK1 (SEQ ID NO: 93), human PAK4 (SEQ ID NO: 29) and human PAK5 (SEQ ID NO: 103).
  • FIGS. 14A, 14B and 14 C show a pair-wise sequence alignment between human ZC1 (SEQ ID NO: 15) and human ZC4 (SEQ ID NO: 105).
  • FIGS. 15A, 15B and 15 C show a pair-wise sequence alignment between LOK1 (SEQ ID NO: 154) and full-length GEK2 (SEQ ID NO: 155).
  • the present invention relates in part to kinase polypeptides, nucleic acids encoding such polypeptides, cells containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing.
  • the present invention is based upon the isolation and characterization of new kinase polypeptides.
  • the polypeptides and nucleic acids may be produced using well-known and standard synthesis techniques when given the sequences presented herein.
  • STE20-related protein kinases have been implicated as regulating a variety of cellular responses, including response to growth factors or cytokines, oxidative-, UV-, or irradiation-related stress pathways, inflammatory signals (i.e., TNF ⁇ ), apoptotic stimuli (i.e., Fas), T and B cell costimulation, the control of cytoskeletal architecture, and cellular transformation.
  • TNF ⁇ inflammatory signals
  • Fas apoptotic stimuli
  • T and B cell costimulation the control of cytoskeletal architecture
  • the STE20-related kinases serve as upstream regulators of MAPK cascades.
  • HPK1 a protein-serine/threonine kinase (STK) that possesses a STE20-like kinase domain that activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK
  • PAK1 an STK with an upstream CDC42-binding domain that interacts with Rac and plays a role in cellular transformation through the Ras-MAPK pathway
  • murine NIK which interacts with upstream receptor tyrosine kinases and connects with downstream STE11-family kinases.
  • the STE20-kinases possess a variety of non-catalytic domains that are believed to interact with upstream regulators. Examples include proline-rich domains for interaction with SH3-containing proteins, or specific domains for interaction with Rac, Rho, and Rab small G-proteins. These interactions may provide a mechanism for cross-talk between distinct biochemical pathways in response to external stimuli such as the activation of a variety of cell surface receptors, including tyrosine kinases, cytokine receptors, TNF receptor, Fas, T cell receptors, CD28, or CD40.
  • nucleic acid molecules include the functional equivalents of the herein-described isolated nucleic acid molecules.
  • the degeneracy of the genetic code permits substitution of certain codons by other codons that specify the same amino acid and hence would give rise to the same protein.
  • the nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon.
  • portions or all of the kinase genes of the invention could be synthesized to give a nucleic acid sequence significantly different from that shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO: 104, and SEQ ID NO: 106.
  • the encoded amino acid sequence thereof would, however, be preserved.
  • the nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5′-end and/or the 3′-end of the nucleic acid formula shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO:106, or a derivative thereof.
  • nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, which is encoded by the nucleotide sequence.
  • the present invention is intended to include any nucleic acid sequence resulting from the addition of ATG as an initiation codon at the 5′-end of the inventive nucleic acid sequence or its derivative, or from the addition of TTA, TAG or TGA as a termination codon at the 3′-end of the inventive nucleotide sequence or its derivative.
  • the nucleic acid molecule of the present invention may, as necessary, have restriction endonuclease recognition sites added to its 5′-end and/or 3′-end.
  • nucleic acid sequence affords an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto.
  • All variations of the nucleotide sequence of the kinase genes of the invention and fragments thereof permitted by the genetic code are, therefore, included in this invention.
  • the full-length human STLK2 cDNA (SEQ ID NO: 1) is 3268 bp long and consists of a 1248 bp open reading frame (ORF) flanked by a 181 bp 5′ untranslated region (UTR; 1-181) and a 1784 bp 3′ UTR (1433-3216) that is followed by a 52 nucleotide polyadenylated region.
  • a polyadenylation signal (AATAAA) is found at positions (3193-3198).
  • the sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M., Nucleic Acids Res.
  • the partial human STLK3 cDNA (SEQ ID NO:2) is 3030 bp long and consists of a 1548 bp ORF flanked by a 1476 bp 3′ UTR (1550-3025) and a 5 nucleotide polyadenylated region.
  • a potential polyadenylation signal begins at position 3004. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • the partial human STLK4 cDNA (SEQ ID NO:3) is 3857 bp long and consists of a 1242 bp ORF flanked by a 2596 bp 3′ UTR (1244-3839) and an 18 nucleotide polyadenylated region.
  • a potential polyadenylation signal (AATAAA) is found at positions 2181-3822. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • a near full-length murine STLK4 cDNA is represented in the 1773 bp EST AA117438.
  • the full-length human STLK5 cDNA (SEQ ID NO:96) is 2110 bp long and consists of a 1119 bp ORF flanked by a 229 bp 5′ UTR and a 762 bp 3′ UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for STLK5.
  • Several EST fragments span the complete STLK5 sequence with AA297059 and F07734 at the 5′ end, and R46686 and F03423 and others at the 3′ end.
  • the full-length human STLK6 cDNA (SEQ ID NO:98) is 2,001 bp long and consists of a 1,254 bp ORF flanked by a 75 bp 5′ UTR and a 673 bp 3′ UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for STLK6.
  • the partial human STLK7 cDNA (SEQ ID NO: 100) is 311 bp long and consists of a 309 bp ORF. Since the coding region is open throughout both the 5′ and 3′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine and C-terminal stop codon.
  • the full-length human ZC1 cDNA (SEQ ID NO:9) is 3798 bp long and consists of a 3717 bp ORF (7-3723) flanked by a 6 bp 5′ UTR and a 75 bp (3724-3798) 3′ UTR. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human ZC1.
  • the partial human ZC2 cDNA (SEQ ID NO:10) is 4055 bp long and consists of a 3891 bp ORF (1-3891) and a 164 bp (3892-4055) 3′ UTR. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR.
  • the partial human ZC3 cDNA (SEQ ID NO:11) is 4133 bp long and consists of a 3978 bp ORF (1-3978) and a 152 bp (3979-4133) 3′ UTR region. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR.
  • the full-length human ZC4 cDNA (SEQ ID NO:104) is 3,684 bp long and was originally assembled from X chromosome genomic DNA sequence.
  • the full-length human KHS2 cDNA (SEQ ID NO:17) is 4023 bp long and consists of a 2682 bp ORF (6-2687) flanked by a 5 bp (1-5) 5′UTR and a 1336 bp (2688-4023) 3′ UTR.
  • a potential polyadenylation signal (AATAAA) is found at positions 4008-4013. No polyadenylated region is present in the 3′UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human KHS2.
  • the full-length human SULU1 cDNA (SEQ ID NO:19) is 4177 bp long and consists of a 2694 bp ORF (415-3108) flanked by a 414 bp (1-414) 5′UTR and a 1069 bp (3109-4177) 3′ UTR followed by a 19 nucleotide polydenylated region.
  • a potential polyadenylation signal (AATAAA) is found at positions 4164-4169.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human SULU1.
  • the partial murine SULU3 cDNA (SEQ ID NO:21) is 2249 bp long and consists of a 2244 bp ORF (6-2249) flanked by a 5 bp (1-5) 5′UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for murine SULU3.
  • the 3′ end of the murine SULU3 cDNA shares 90% DNA sequence identity over 1620 nucleotides with human SULU3, suggesting that these two genes are functional orthologues.
  • the partial human SULU3 cDNA (SEQ ID NO:20) is 3824 bp long and consists of a 2358 bp ORF (2-2359) flanked by a 1465 bp (2360-3824) 3′ UTR followed by a 19 nucleotide polydenylated region.
  • a potential polyadenylation signal (AATAAA) is found at positions 2602-2607. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • the 5′ end of the human SULU3 cDNA shares 90% DNA sequence identity over 1620 nucleotides with murine SULU3, suggesting that these two genes are functional orthologues.
  • the full-length human GEK2 cDNA (SEQ ID NO:106) is 2962 bp long and consists of a 2737 bp ORF (59-2795) flanked by a 58 bp (1-58) 5′UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human GEK2.
  • the full-length human PAK4 cDNA (SEQ ID NO:27) is 3604 bp long and consists of a 2043 bp ORF (143-2185) flanked by a 142 bp (1-142) 5′UTR and a 1419 3′UTR followed by a 22 nucleotide polydenylated region.
  • a potential polyadenylation signal (AATTAAA) is found at positions 3582-3588.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human PAK4.
  • the full-length human PAK5 cDNA (SEQ ID NO:102) is 2806 bp long and consists of a 1773 bp ORF flanked by a 201 bp 5′ UTR and a 833 bp 3′ UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for PAK5.
  • a nucleic acid probe of the present invention may be used to probe an appropriate chromosomal or cDNA library by usual hybridization methods to obtain other nucleic acid molecules of the present invention.
  • a chromosomal DNA or cDNA library may be prepared from appropriate cells according to recognized methods in the art (cf. “Molecular Cloning: A Laboratory Manual”, second edition, Cold Spring Harbor Laboratory, Sambrook, Fritsch, & Maniatis, eds., 1989).
  • nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions of the amino acid sequence of the polypeptide of interest.
  • the synthesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, “A Guide to Methods and Applications”, Academic Press, Michael, et al., eds., 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment of the present invention.
  • PCR polymerase chain reaction
  • hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes may be visualized using known methods.
  • the nucleic acid probes of the present invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art.
  • the nucleic acid probe may be immobilized on a solid support.
  • solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
  • test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
  • One method of detecting the presence of nucleic acids of the invention in a sample comprises (a) contacting said sample with the above-described nucleic acid probe under conditions such that hybridization occurs, and (b) detecting the presence of said probe bound to said nucleic acid molecule.
  • One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue.
  • a kit for detecting the presence of nucleic acids of the invention in a sample comprises at least one container means having disposed therein the above-described nucleic acid probe.
  • the kit may further comprise other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe.
  • detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin).
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like.
  • wash reagents such as phosphate buffered saline, Tris-buffers, and the like
  • the present invention also relates to a recombinant DNA molecule comprising, 5′ to 3′, a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules.
  • the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecule.
  • the present invention also relates to a nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in said cell.
  • the above-described molecules may be isolated and/or purified DNA molecules.
  • the present invention also relates to a cell or organism that contains an above-described nucleic acid molecule and thereby is capable of expressing a polypeptide.
  • the polypeptide may be purified from cells which have been altered to express the polypeptide.
  • a cell is said to be “altered to express a desired polypeptide” when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels.
  • One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
  • a nucleic acid molecule such as DNA
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation.
  • Such regions will normally include those 5′-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3′ to the sequence encoding a kinase of the invention may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3′-region naturally contiguous to the DNA sequence encoding a kinase of the invention, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3′ region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a gene sequence encoding a kinase of the invention, or (3) interfere with the ability of the gene sequence of a kinase of the invention to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of a gene encoding a kinase of the invention (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system for kinases of the invention.
  • Prokaryotes most frequently are represented by various strains of E. Coli . However, other microbial strains may also be used, including other bacterial strains.
  • plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
  • suitable plasmid vectors may include pBR322, pUC118, pUC 119 and the like; suitable phage or bacteriophage vectors may include ⁇ gt10, ⁇ gt11 and the like; and suitable virus vectors may include pMAM-neo, pKRC and the like.
  • the selected vector of the present invention has the capacity to replicate in the selected host cell.
  • Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia , and the like. However, under such conditions, the polypeptide will not be glycosylated. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • a kinase of the invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the kinase of the invention to a functional prokaryotic promoter.
  • promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ -lactamase gene sequence of pBR322, and the cat promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the trp, recA, ⁇ acZ, ⁇ acI, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and the c-28-specific promoters of B.
  • subtilis (Gilman et al., Gene Sequence 32:11-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478, 1986).
  • Prokaryotic promoters are reviewed by Glick (Ind. Microbiot. 1:277-282, 1987), Cenatiempo (Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
  • progeny Proper expression in a prokaryotic cell also requires the presence of a ribosome-binding site upstream of the gene sequence-encoding sequence.
  • ribosome-binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981).
  • the selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
  • “cell”, “cell line”, and “cell culture” may be used interchangeably and all such designations include progeny.
  • the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell.
  • Host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the kinase polypeptide of interest. Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332, which may provide better capacities for correct post-translational processing.
  • eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459, 1988).
  • baculovirus vectors can be engineered to express large amounts of kinases of the invention in insect cells (Jasny, Science 238:1653, 1987; Miller et al., In: Genetic Engineering, Vol. 8, Plenum, Setlow et al., eds., pp. 277-297, 1986).
  • Any of a series of yeast expression systems can be utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes that are produced in large quantities when yeast are grown in mediums rich in glucose.
  • Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
  • Yeast provides substantial advantages in that it can also carry out post-translational modifications.
  • Yeast recognizes leader sequences on cloned mammalian genes and secretes peptides bearing leader sequences (i.e., pre-peptides).
  • Several possible vector systems are available for the expression of kinases of the invention in a mammalian host.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).
  • a nucleic acid molecule encoding a kinase of the invention and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome.
  • a vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Okayama (Mol. Cell. Biol. 3:280-, 1983).
  • the introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, ⁇ VX; “Molecular Cloning: A Laboratory Manual”, 1989, supra).
  • Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, NY, pp. 307-329, 1982).
  • Suitable Streptomyces plasmids include plJ101 (Kendall et al., J. Bacteriol.
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982; Broach, In: “The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance”, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon et al., J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980).
  • the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene(s) results in the production of a kinase of the invention, or fragments thereof.
  • polypeptides of the present invention may be purified from tissues or cells that naturally produce the polypeptides.
  • isolated nucleic acid fragments could be used to express the kinases of the invention in any organism.
  • the samples of the present invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The samples will vary based on the assay format, the detection method, and the nature of the tissues, cells or extracts used as the sample.
  • source organism refers to the original organism from which the amino acid sequence of the subunit is derived, regardless of the organism the subunit is expressed in and ultimately isolated from.
  • STLK2 is an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • STLK2 contains a 21 amino acid N-terminal domain, a 253 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, followed by a 142 amino acid C-terminal domain.
  • STLK2 is most closely related to human STE20-subfamily kinases, MST3 (GB:AF024636) and SOK-1 (GB:X99325) and a C. elegans kinase yk34b11.5 (GB:U53153) sharing 72.7%, 68.7%, and 69.3% amino acid identity, respectively.
  • the 21 amino acid N-terminal domain of human STLK2 is 71.4% identical to the N-terminus of MST3 (GB:AF024636). Human STLK2 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • the 253 amino acid catalytic domain of human STLK2 is most related to human SOK-1 (X99325), MST3 (GB:AF024636), C. elegans yk32b11.5 (GB:U53153), and STLK3 (SEQ ID NO:6) sharing 88.9%, 87.4%, 78.3%, and 49% amino identity respectively, placing it in the STLK-subfamily of STE20-related kinases.
  • the STLK2 kinase domain displayed lesser homology to other STE20-related kinases including: 55.9% to human MST2 (GB:U26424), 49.2% to human GCK (GB:U07349), 49.2% to human KHS1 (GB:U77129), and 44.2% to human HPK1 (GB:U66464).
  • the activation loop of human STLK2 catalytic domain is identical to that of human SOK-1 and MST3 including the presence of four potential threonine phosphorylation sites that could serve an autoregulatory role on kinase activity.
  • the 142 amino acid C-terminal domain of human STLK2 is most related to human SOK-1 (X99325), MST3 (GB:AF024636), and C. elegans yk32b11.5 (GB:U53153), sharing 39.9%, 39.9%, and 33.3% amino acid identity, respectively.
  • This C-terminal domain shares some significant amino acid similarity to the C-terminal domains of the related human STLK3 (SEQ ID NO:6) and STLK4 (SEQ ID NO:7).
  • the C-terminus of the related human SOK-1 (GB:X99325) kinase has been shown to be inhibitory to the catalytic activity of this kinase (Pombo, C. M., Bonventre, J. V., Molnar, A., Kyriakis, J. and Force, T. EMBO J. 15, 4537-4546 (1996)).
  • the C-terminus of human STLK2 may also function as an inhibitory domain for its kinase.
  • the 3030 bp human STLK3 nucleotide sequence of the partial cDNA clone encodes a polypeptide of 516 amino acids (SEQ ID NO:6) with a predicted molecular mass of 56,784 daltons. Analysis of the deduced amino acid sequence predicts STLK3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-termius is not known.
  • STLK3 contains a 31 amino acid N-terminal domain, a 277 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, followed by a 181 amino acid C-terminal domain containing a 25 amino acid insert and a 27 amino acid tail relative to the sequence of human STLK2.
  • STLK3 is most closely related to human STE20-subfamily kinases, STLK4 (SEQ ID. NO:7), MST3 (GB:AF024636), SOK-1 (GB:X99325) and STLK2 (SEQ ID NO:5) sharing 71.1%, 37.6%, 38.1%, and 38.4% amino acid identity respectively.
  • the 277 amino acid catalytic domain of human STLK3 is most related to human STLK4 (SEQ ID NO:7), SOK-1 (GB:X99325), MST3 (GB:AF024636), and STLK2 (SEQ ID NO:5) sharing 88.2%, 49.2%, 49%, and 49% amino acid identity, respectively. It also shares strong homology to other STKs from lower organisms including 51.7% to A. thaliana (GB: AC002343), 43.1% to A. thaliana (GB: Z97336), 42.1% to A. thaliana (GB: U96613), and 43.3% to C. elegans (GB: U53153).
  • the activation loop of the human STLK3 catalytic domain conserves three potential threonine phosphorylation sites with other members of the STLK-subfamily of STE20-related kinases (human STE20, MST3, STLK2, STLK4) that could serve an autoregulatory role on kinase activity.
  • the 181 amino acid C-terminal domain of human STLK3 shares 55.5% amino acid identity to human STLK4 (SEQ ID NO:7), and is 100% identical to a partial human cDNA DCHT (GB:AF017635).
  • the C-terminal domain of human STLK3 contains a 26 amino acid insert relative to human STE20. A similar (87.5% amino acid identity) 26 amino acid insert is also present in human STLK4.
  • the 3857 bp human STLK4 nucleotide sequence of the partial cDNA clone encodes a polypeptide of 414 amino acids (SEQ ID NO:7) with a predicted molecular mass of 45,451 daltons. Analysis of the deduced amino acid sequence predicts STLK4 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-terminus is not known.
  • the partial STLK4 protein sequence contains a 178 amino acid catalytic domain corresponding to the C-terminal motifs VI-XI of a serine/threonine kinase, followed by a 236 amino acid C-terminal domain containing two inserts of 25 and 41 amino acids each, relative to the sequence of human STLK2.
  • STLK4 is most closely related to human STE20-subfamily kinases, STLK3 (SEQ ID. NO 6), MST3 (GB:AF024636), STLK2 (SEQ ID NO:5), and SOK-1 (GB:X99325) sharing 71.0%, 46.8%, 43.9%, and 37.7% amino acid identity, respectively.
  • the 178 amino acid catalytic domain of human STLK4 is most related to human STLK3 (SEQ ID NO. 7), SOK-1 (GB:X99325), MST3 (GB:AF024636), STLK2 (SEQ ID NO:5), and MST1 (GB:U18297), sharing 88.2%, 54.2%, 54.0%, 53.7 and 45.7% amino acid identity, respectively. It also shares strong homology to other STKs from lower organisms including 56.9% to A. thaliana (GB: AC002343), 52.5% to C. elegans (GB: U53153), 46.2% to A. thaliana (GB: Z97336) and 45.7% to A. thaliana (GB: U96613).
  • the activation loop of the human STLK4 catalytic domain conserves three potential threonine phosphorylation sites with other members of the STLK-subfamily of STE20-related kinases (human STE20, MST3, STLK2 and STLK3) that could serve an autoregulatory role on kinase activity.
  • the 236 amino acid C-terminal domain of human STLK4 shares 58.1% amino acid identity to both human STLK3 (SEQ ID NO:6) and to a partial human cDNA, DCHT (GB:AF017635).
  • the C-terminal domain of human STLK4 contains a 25 amino acid insert relative to human SOK-1 and shares 87.5% amino acid identity to an insert present in human STLK3.
  • the full-length 2110 bp human STLK5 cDNA encodes a polypeptide of 373 amino acids (SEQ ID NO:97) with a predicted molecular mass of 41,700 daltons. Analysis of the deduced amino acid sequence predicts STLK5 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and transmembrane domain. STLK5 contains a 10 amino acid N-terminal domain, a 311 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, and a 52 amino acid C-terminal domain.
  • STLK5 is most closely related to the human STE20-subfamily kinases STLK6 (SEQ ID No. 99) and SPAK (AFO99989), sharing 51% and 33% amino acid identity, respectively, over its full extent. It also shares significant homology to database entries from Arabidopsis thaliana (GB:AC002343) and C. elegans (GB:AL023843, GB:AL023843).
  • the 311 amino acid catalytic domain of human STLK5 shares 51% and 34% identity to STLK6 and SPAK, respectively.
  • the catalytic domain of STLK5 contains a 45 amino acid insert between kinase subdomains X and XI relative to human STE20.
  • Multiple human EST fragments as well as a murine EST contain this insert providing evidence that this region is an integral part of STLK5.
  • the 2,001 bp human STLK6 nucleotide sequence of the complete cDNA encodes a polypeptide of 418 amino acids (SEQ ID NO:99) with a predicted molecular mass of 47,025 daltons. Analysis of the deduced amino acid sequence predicts STLK6 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and transmembrane domain. STLK6 contains a 57 amino acid N-terminal domain, a 312 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, followed by a 49 amino acid C-terminal domain.
  • STLK6 is most closely related to human STE20-subfamily kinases STLK5 (SEQ ID NO:97), STLK7 (SEQ ID NO:101), and SPAK (AFO99989), sharing 50%, 35%, and 30% amino acid identity over its full extent. It also shares significant homology to database entries from Arabidopsis thaliana (GB:AC002343) and C. elegans (GB:U53153).
  • the 311 bp human STLK7 nucleotide sequence of the partial cDNA encodes a polypeptide of 103 amino acids (SEQ ID NO: 101). Analysis of the deduced amino acid sequence predicts STLK7 to be an internal fragment of an intracellular STE20-family kinase. This sequence lacks the N- and C-terminal portions of STLK7 and contains only the N-terminal 103 amino acids of the predicted catalytic domain.
  • Human STLK7 is most closely related to human STE20-subfamily kinases SPAK (AFO99989), STLK5 (SEQ ID NO:97), and STLK6 (SEQ ID NO:99), sharing 86%, 38%, and 35% amino acid identity within this region of the kinase domain. It also shares significant homology to database entries from Arabidopsis thaliana (GB:AC002343) and Drosophila melanogaster (GB:AF006640).
  • the 3798 bp human ZC1 nucleotide sequence encodes a polypeptide of 1239 amino acids (SEQ ID NO: 13) with a predicted molecular mass of 142,140 daltons. Analysis of the deduced amino acid sequence predicts ZC1 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • the full-length ZC1 protein contains a 22 amino acid N-terminus, a 267 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 237 amino acid region predicted to form a coiled-coil structure, a 114 amino acid proline-rich region, a 256 amino acid spacer region, followed by a 343 amino acid C-terminal domain containing a potential Rab/Rho-binding region.
  • ZC1 is most closely related to the human STE20-subfamily kinases ZC2 (SEQ ID NO:14), ZC3 (SEQ ID NO:15), and ZC4 (SEQ ID NO:16), sharing 61.7%, 60.9%, and 43.8% amino acid identity, respectively.
  • ZC1 also shares 45.5% amino acid identity to a C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029).
  • ZC1 exhibits 90.0% amino acid homology to murine NIK (GB:U88984), suggesting it may be the human orthologue of this STK.
  • the 22 amino acid N-terminal domain of human ZC1 is 58.8% identical to the C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029), and 100% identical to murine NIK (GB: U88984).
  • Human ZC1 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation.
  • a Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • the 267 amino acid catalytic domain of human ZC1 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15), ZC2 (SEQ ID NO: 14), KHS2 (SEQ ID NO:18), SOK-1 (GB:X99325), GCK (GB:U07349), and GEK2 (SEQ ID NO:107), and to the C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029) sharing 90.6%, 90.2%, 50.6%, 47.4%, 45.4%, 42.5% and 82.6% amino acid identity, respectively.
  • ZC1 kinase domain shares 98.1% identity to murine NIK (GB:U88984).
  • ZC1 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • ZC1 Immediately C-terminal to the kinase domain of human ZC1 is a 237 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (Lupas, A. Meth. Enzymol. 266, 513-525 (1996)). This region of ZC1 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15), ZC2 (SEQ ID NO: 14), and GEK2 (SEQ ID NO: 107), as well as to human PITSLRE (GB:U04824) sharing 65.5%, 65.4%, 25.3%, and 29.0% amino acid identity, respectively.
  • the ZC1 coiled-coil domain also shares 90.6% amino acid homology to murine NIK.
  • the C. elegans homologue ZC504.4 shares 32.2% sequence identity over this region.
  • the 114 amino acid proline-rich region of human ZC1 is most related to human STE20-subfamily kinases, ZC2 (SEQ ID NO: 14) and ZC3 (SEQ ID NO: 15), sharing 35.8%, and 24.9%, respectively.
  • the ZC1 proline-rich domain shares 36.4% amino acid homology to murine NIK (GB:U88984).
  • Three potential “PxxP” SEQ ID NO: 148) SH3 domain-binding motifs (I, II and III) are found within the proline-rich region of human ZC1. Motif I is conserved in human ZC1 and C. elegans ZC504.4 (GB:Z50029).
  • Motif II is conserved in ZC1, ZC2, ZC3, ZC4 and C. elegans ZC504.4.
  • Motif III is conserved in ZC1, ZC2, ZC3 and ZC4.
  • Motifs II and III of murine NIK have been shown to bind the SH3 motif of the adaptor molecule Nck (Su, Y-C. et al, EMBO J. 16, 1279-1290 (1997)). From this evidence, human ZC1 may have the potential to bind to Nck or other SH3 or WW domain-containing proteins and participate in growth factor-induced signaling pathways.
  • the 256 amino acid spacer region of human ZC1 is most related to human STE20-subfamily kinases, ZC2 (SEQ ID NO: 14) and ZC3 (SEQ ID NO: 15), as well as to human PITSLRE (GB:U04824), sharing 59.9%, 33.1%, 29.6%, and 26.4% amino acid identity, respectively. It also shares 59.9% amino acid homology to murine NIK.
  • the C. elegans homologue ZC504.4 has only limited sequence similarity in this spacer region.
  • the 343 amino acid C-terminal of human ZC1 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15), ZC2 (SEQ ID NO: 14), and ZC4 (SEQ ID NO:16), sharing 89.2%, 88.9%, and 42.3%, amino acid identity, respectively.
  • the ZC1 C-terminal domain also shares 98.8% amino acid identity to murine NIK.
  • the C. elegans homologue ZC504.4 also shares 68.7% amino acid identity with the C-tail of human ZC1.
  • GCK is a STE20-family kinase whose C-terminal domain has been shown to bind the small G-protein Rab8 (Ren, M. et al., Proc. Natl. Acad. Sci. 93, 5151-5155 (1996)).
  • Citron is a non-kinase Rho-binding protein (Madaule, P. et al., FEBS Lett. 377, 243-238 (1995)).
  • the 4055 bp human ZC2 nucleotide sequence of the partial cDNA encodes a polypeptide of 1297 amino acids (SEQ ID NO:14) with a predicted molecular mass of 147,785 daltons. Analysis of the deduced amino acid sequence predicts ZC2 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-terminus is not known.
  • the N-terminally truncated ZC2 protein contains a 255 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 187 amino acid region predicted to form a coiled-coil structure, a 184 amino acid proline-rich region, a 328 amino acid spacer region, followed by a 343 amino acid C-terminal domain containing a potential Rab/Rho-binding region.
  • ZC2 is most closely related to the human STE20-subfamily kinases ZC3 (SEQ ID NO:15), ZC1 (SEQ ID NO:13), and ZC4 (SEQ ID NO:16), sharing 88.3%, 61.7%, and 41.9% amino acid identity, respectively, and shares 41.7% amino acid identity to a C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029).
  • ZC2 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • ZC2 Immediately C-terminal to the kinase domain of human ZC2 is a 187 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (supra). This region of ZC2 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and GEK2 (SEQ ID NO:107), as well as to human PITSLRE (GB:U04824), sharing 65.8%, 61.5%, 29.7% and 29.6% amino acid identity, respectively. The C. elegans homologue ZC504.4 shares 30.8% sequence identity over this region. Human ZC2 lacks the potential leucine zipper found in ZC1 as a consequence of a 29 amino acid deletion relative to ZC1 and ZC3.
  • the 184 amino acid proline-rich region of human ZC2 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15) and ZC1 (SEQ ID NO: 13), sharing 35.9% and 28.6%, amino acid identity, respectively.
  • Significant homology is also evident to the murine WW domain-binding protein WBP7 (GB:U92455), and to the human SH3 domain-binding protein 3BP-1 (GB:X87671), with 27.7% and 25.3% amino acid identity, respectively.
  • ZC2 contains two of the potential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (II and III) found within the proline-rich region of human ZC1. Motif II is conserved in ZC1, ZC3, ZC4 and C. elegans ZC504.4, and Motif III is conserved in ZC1, ZC3 and ZC4. Motifs II and III of murine NIK have been shown to bind the SH3 motif of the adaptor molecule Nck. From this evidence, human ZC1 may have the potential to bind to Nck or other SH3 or WW domain-containing proteins, and to participate in growth factor-induced signaling pathways.
  • the 328 amino acid spacer region of human ZC2 is most related to human STE20-subfamily kinases ZC1 (SEQ ID NO:13) and ZC3 (SEQ ID NO:15), and to murine NIK (GB:U88984), sharing 31.6%, 26.9% and 25.9% amino acid identity, respectively.
  • the C. elegans homologue ZC504.4 has only limited sequence similarity in this spacer region.
  • the 343 amino acid C-terminal of human ZC2 is most related to human STE20-subfamily kinases ZC1 (SEQ ID NO: 13), ZC3 (SEQ ID NO: 15) and ZC4 (SEQ ID NO:16), and to murine NIK (GB:U88984), sharing 88.9%, 88.3%, 41.9%, and 88.0%, amino acid identity, respectively.
  • the C. elegans homologue, ZC504.4 also shares 67.2% amino acid identity with the C-tail of human ZC2.
  • a lower, yet significant, homology is also evident to human GCK (GB:U07349), murine citron (GB:U07349), and the S. cerevisiae ROM2 protein (GB:U19103), a Rho1 GDP/GTP exchange factor, with 22.3%, 22.2% and 21.9% amino acid identity, respectively.
  • the 4133 bp human ZC3 nucleotide sequence of the partial cDNA encodes a polypeptide of 1326 amino acids (SEQ ID NO: 15) with a predicted molecular mass of 149,906 daltons. Analysis of the deduced amino acid sequence predicts ZC3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-termius is not known.
  • the N-terminally truncated ZC3 protein contains a 255 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase: a 221 amino acid region predicted to form a coiled-coil structure, a 204 amino acid proline-rich region, and a 303 amino acid spacer region followed by a 343 amino acid C-terminal domain containing a potential Rab/Rho-binding region.
  • ZC3 is most closely related to the human STE20-subfamily kinases ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14), and ZC4 (SEQ ID NO:16), sharing 62.0%, 61.0%, and 42.5% amino acid identity, respectively and shares 46.7% amino acid identity to a C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029).
  • the 255 amino acid catalytic domain of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO: 13), ZC2 (SEQ ID NO: 14), SOK-1 (GB:X99325), KHS2 (SEQ ID NO:18), GCK (GB:U07349), SULU1 (SEQ ID NO:22), and GEK2 (SEQ ID NO: 107), and to the C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029) sharing 90.6%, 89.3%, 49.0%, 48.3%, 45.0%, 43.1%, 42.3% and 76.7% amino acid identity, respectively.
  • ZC1 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • ZC3 Immediately C-terminal to the kinase domain of human ZC3 is a 221 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (supra). This region of ZC3 is most homologous to human STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14), and GEK2 (SEQ ID NO:107), sharing 66.9%, 61.5%, and 27.5% identity, as well as to rat PLC-beta (GB:A45493) and human PITSLRE (GB:H54024) sharing 29.6% and 25.9% amino acid identity, respectively.
  • the C. elegans homologue ZC504.4 shares 26.8% sequence identity over this region.
  • the 204 amino acid proline-rich region of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO: 13) and ZC2 (SEQ ID NO: 14), sharing 66.9% and 61.5% amino acid identity, respectively.
  • ZC3 contains two of the potential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (II and III) found within the proline-rich region of human ZC1. Motif II is conserved in ZC1, ZC2, ZC4 and C. elegans ZC504.4; Motif III is conserved in ZC1, ZC2 and ZC4. Motifs II and III of murine NIK have been shown to bind the SH3 motif of the adaptor molecule Nck. From this evidence, human ZC3 may have the potential to bind to Nck or other SH3 or WW domain-containing proteins and participate in growth factor-induced signaling pathways.
  • the 303 amino acid acid spacer region of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO: 13) and ZC2 (SEQ ID NO: 14) sharing 30.1%, and 27.1% amino acid identity, respectively.
  • the C. elegans homologue ZC504.4 lacks nearly the entire spacer region of ZC3.
  • the 343 amino acid C-terminal of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14) and ZC4 (SEQ ID NO: 16), sharing 89.2%, 88.9%, and 42.5%, amino acid identity, respectively.
  • the C. elegans homologue ZC504.4 also shares 67.2% amino acid identity with the C-tail of human ZC3.
  • a lower, yet significant, homology is also evident to human GCK (GB:U07349), as well as to the non-kinases murine citron (GB:U07349) and the S. cerevisiae ROM2 protein (GB:U 19103), a Rho1 GDP/GTP exchange factor, with 21.6%, 32.4% and 22.9% amino acid identity, respectively.
  • the 3,684 bp human ZC4 nucleotide sequence of the complete cDNA encodes a polypeptide of 1,227 amino acids (SEQ ID NO:105) with a predicted molecular mass of 138,205 Daltons. Analysis of the deduced amino acid sequence predicts ZC4 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and a transmembrane domain.
  • the full-length ZC4 protein contains a 25 amino acid N-terminus, a 265 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 108 amino acid region predicted to form a coiled-coil structure, a 231 amino acid proline-rich region, a 40 amino acid region predicted to form a coiled-coil structure spacer region, a 204 amino acid spacer region (domain B), followed by a 355 amino acid C-terminal domain containing a potential Rab/Rho-binding region (domain C).
  • ZC4 is most closely related to human ZC1 (SEQ ID NO:13, also known as human HGK, human KIAA0687, murine NIK, human AC005035, human NIK, and C. elegans MIG-15), ZC2 (SEQ ID NO:14, similar to partial sequence human KIAA0551), and ZC3 (SEQ ID NO:15).
  • An assembled genomic fragment in the database (Z83850) is identical to ZC4, except for inappropriate identification of the exon boundaries. (Abo et al. (1998) EMBO J. 17: 6527-6540.)
  • the 265 amino acid catalytic domain of human ZC4 is most related to human ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO:14), sharing 63%, 64% and 62% amino acid identity, respectively.
  • the 231 amino acid proline-rich region of human ZC4 does not reveal any significant homologies to the protein database.
  • This region of ZC4 contains two “PxxP” (SEQ ID NO: 148) motifs that could potentially bind to proteins containing SH3 or WW domains and allow ZC4 to participate in growth factor activated signaling pathways.
  • PxxP SEQ ID NO: 148
  • motifs that could potentially bind to proteins containing SH3 or WW domains and allow ZC4 to participate in growth factor activated signaling pathways.
  • a region predicted to form a leucine zipper (Leu-X6-Leu-X6-Leu-X6-Leu-X20-Leu-X6-Leu) (SEQ ID NO: 149) which may serve as a dimerization interface.
  • the ZC STE20 subfamily kinases (ZC1, ZC2 and ZC3) have similarly located “PxxP” (SEQ ID NO: 148) motifs and potential Leu zippers.
  • human ZC4 Immediately C-terminal to the proline-rich region of human ZC4 is a 40 amino acid region also predicted to form a coiled-coil structure based on the Lupas algorithm. This region of human ZC4 does not reveal any significant homologies to the protein database.
  • the 355 amino acid C-terminal of human ZC4 is most related to human ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO:14), sharing 43%, 42% and 42% amino acid identity, respectively.
  • the 4023 bp human KHS2 nucleotide sequence encodes a polypeptide of 894 amino acids (SEQ ID NO: 18) with a predicted molecular mass of 101,327 daltons. Analysis of the deduced amino acid sequence predicts KHS2 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • the full-length KHS2 protein contains a 13 amino acid N-terminus, a 260 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 73 amino acid spacer region, a 188 proline-rich region, followed by a 360 amino acid C-terminal domain containing a potential Rab/Rho-binding site.
  • KHS2 is most closely related to the human STE20-subfamily kinases KHS1 (GB:U177129), GCK (GB:U07349), and HPK1 (GB:U07349), sharing 65.5%, 51.9%, and 44.9% amino acid identity, respectively and shares 38.5% amino acid identity to a C. elegans STK (GB:U55363).
  • KHS2 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, SULU1, SULU3, PAK4 and PAK5.
  • KHS2 The 73 amino acid acid spacer region of human KHS2 is most related to human STE20-subfamily kinases, KHS1 (GB:U177129), HPK1 (GB:U66464) and GCK (GB:U07349), sharing 60.3%, 43.5% and 44.0%, amino acid identity, respectively.
  • the 188 amino acid proline-rich region of human KHS2 is most related to human STE20-subfamily kinases, HPK1 (GB:U66464), GCK (GB:U07349) and KHS1 (GB:U177129), sharing 33.3%, 31.9% and 31.4%, amino acid identity, respectively.
  • SH3 domain-binding motifs (I and II) are found within the proline-rich region of human KHS2. Motif I is conserved with human KHS1 and HPK1; motif II is conserved with GCK and KHS2. A 192 amino acid region of human HPK1 containing motif II has been shown to bind to the C-terminal SH3 motif of the adaptor molecule Grb2 (Anafi, M et al, J. Biol. Chem. J. 272, 27804-27811 (1997)). Human KHS2 may bind SH3 or WW domain-containing proteins through this proline-rich region.
  • the 360 amino acid C-terminal of human KHS2 is most related to KHS1 (GB:U177129), GCK (GB:U07349) and HPK1 (GB:U66464), and to the C. elegans kinase (GB:U55363), sharing 74.9%, 54.8%, 42.9%, and 31.0%, amino acid identity, respectively.
  • GCK is a STE20-family kinase whose C-terminal domain has been shown to bind the small G-protein Rab8 (Ren, M. et al., Proc. Natl. Acad. Sci. 93, 5151-5155 (1996)).
  • the 4196 bp human SULU1 nucleotide sequence encodes a polypeptide of 898 amino acids (SEQ ID NO:22) with a predicted molecular mass of 105,402 daltons. Analysis of the deduced amino acid sequence predicts SULU1 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • the full-length SULU1 protein contains a 21 amino acid N-terminus, a 256 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 150 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure, a 114 amino acid spacer region and a 147 amino acid C-terminal domain predicted to form a coiled-coil structure.
  • SULU1 is most closely related to the STE20-subfamily kinases murine SULU3 (SEQ ID NO:24), human SULU3 (SEQ ID NO:23), and to the C. elegans kinase SULU (GB:U11280), sharing 68.9%, 72.2% and 38.2% amino acid identity, respectively.
  • the 21 amino acid N-terminal domain of human SULU1 is most related to murine SULU3 (SEQ ID NO:24) and to the C. elegans kinase SULU (GB:U11280), sharing 86.3% and 62.3% amino acid identity.
  • Human SULU1 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristoylation.
  • a Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • the 256 amino acid catalytic domain of human SULU1 is most related to murine SULU3 (SEQ ID NO:24), and to human SOK-1 (GB:X99325), STLK2 (SEQ ID NO:5), MST1 (GB:U18297), PAK1 (GB:U24152), ZC2 (SEQ ID NO:14), and KHS2 (SEQ ID NO:18) sharing 86.3%, 48.1%, 46.9%, 45.2%, 43.3%, 43.1% and 42.0% amino acid identity, respectively.
  • the C. elegans SULU STK (GB:U11280) shares 62.3% sequence identity over this region.
  • SULU1 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU3, PAK4 and PAK5.
  • the 150 amino acid spacer region of human SULU1 is most related to human SULU3 (SEQ ID NO:23) and to the C. elegans kinase (GB:U11280), sharing 53.5% and 10.4% amino acid identity, respectively.
  • SULU1 Immediately C-terminal to the spacer region of human SULU1 is a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of SULU1 is most related to SULU3 (SEQ ID NO:23), the C. elegans SULU kinase (GB:U11280), GEK 2 (SEQ ID NO:107) and ZC1 (SEQ ID NO:13), sharing 68.6%,26.8%,23.2%, and 22.8% amino acid identity, respectively.
  • SEQ ID NO:23 the C. elegans SULU kinase
  • GEK 2 SEQ ID NO:107
  • ZC1 ZC1
  • the 114 amino acid spacer region human SULU1 is most related to human SULU3 (SEQ ID NO:24) with 73.7% amino acid sequence identity. A lower, yet significant, homology is also evident to murine PITSLRE (GB:U04824) and DLK (GB:A55318), human ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107), as well as to the C. elegans SULU STK (GB:U11280), sharing 39.7%, 35.4%, 29.5%, 23.6% and 37.6% amino acid identity, respectively.
  • SULU1 Immediately C-terminal to the second spacer region of human SULU1 is a 147 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of SULU1 is most related to human SULU3 (SEQ ID NO:24), ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107), as well as to the C. elegans SULU STK (GB:U11280), sharing 73.3%, 28.4%, 26.1% and 39.5%, amino acid identity, respectively.
  • the 3824 bp partial cDNA human SULU3 nucleotide sequence encodes a polypeptide of 786 amino acids (SEQ ID NO:23) with a predicted molecular mass of 92,037 daltons. Analysis of the deduced amino acid sequence predicts SULU3 to be an intracellular serine/threonine kinase lacking a transmembrane domain.
  • the N-terminally truncated human SULU3 protein contains a 66 amino acid partial catalytic domain followed by a 149 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure, a second spacer region of 114 amino acids, a 247 amino acid C-terminal region predicted to form a second coiled-coil structure and a 100 amino acid C-terminal tail.
  • Human SULU3 is most closely related murine SULU3 (SEQ ID NO:24), human SULU1 (SEQ ID NO:22), and to the C. elegans SULU kinase (GB:U 11280), sharing 66.3%, 68.9% and 32.9% amino acid identity, respectively.
  • the high sequence homology between murine and human SULU3 suggests that these two proteins are orthologs of each other.
  • the 66 amino acid partial catalytic domain of human SULU3 is most related to murine SULU3 (SEQ ID NO:24), and to the human STE20 subfamily kinases ZC1 (SEQ ID NO:13), STE20 (GB:X99325), KHS1(GB:U177129) and GEK 2 (SEQ ID NO: 107), as well as to the C. elegans SULU kinase (GB:U11280), sharing 83.3%, 47.0%, 45.5%, 43.5%,41.8% and 55.6% amino acid identity, respectively.
  • the 149 amino acid spacer region of human SULU3 is most related to murine SULU3 (SEQ ID NO:24), human STE20 (GB:X99325), MST1 (GB:U18297), and to the C. elegans SULU kinase (GB:U11280) sharing 98.7%, 21.9% and 21.8% amino acid identity, respectively.
  • SULU3 Immediately C-terminal to the first spacer region of human SULU3 is a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of SULU3 is most related to murine SULU3 (SEQ ID NO:24), and to human SULU1 (SEQ ID NO:22), ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107), as well as to the C. elegans SULU kinase (GB:U11280), sharing 99.5%, 68.6%, 27.4% and 22.5% amino acid identity, respectively.
  • SEQ ID NO:24 murine SULU3
  • human SULU1 SEQ ID NO:22
  • ZC1 SEQ ID NO:13
  • GEK 2 SEQ ID NO:107
  • the 114 amino acid second spacer region of human SULU3 is most related to murine SULU3 (SEQ ID NO:24), and to human SULU1 (SEQ ID NO:22) GEK 2 (SEQ ID NO:107), and ZC1 (SEQ ID NO:13), as well as to the C. elegans SULU kinase (GB:U11280), sharing 99.1%, 73.7%, 24.6%,24.1% and 41.2% amino acid identity, respectively.
  • SULU3 Immediately C-terminal to the second spacer region of human SULU3 is a 247 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (supra). This region of SULU3 is most related to human SULU1 (SEQ ID NO:22) and ZC1 (SEQ ID NO:13) as well as to rat PKN-(GB:D26180) murine pl60 ROCK1 (GB:U58512), and the C. elegans SULU kinase (GB:U11280), sharing 73.7%, 26.7%, 24.0% and 21.0% amino acid identity, respectively.
  • the 100 amino acid C-tail of human SULU3 is most related to a human prion protein (GB:L38993), with 45.0% amino acid identity.
  • the 2249 bp murine, partial cDNA SULU3 nucleotide sequence encodes a polypeptide of 748 amino acids (SEQ ID NO:24) with a predicted molecular mass of 87,520 daltons. Analysis of the deduced amino acid sequence predicts SULU3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • the partial murine SULU3 protein contains a 25 amino acid N-terminus, a 248 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 149 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure, and a 116 amino acid spacer region.
  • Murine SULU3 is most closely related to human SULU3 (SEQ ID NO:23) and SULU1 (SEQ ID NO:22), as well as to the C. elegans SULU kinase (GB:U112 80), sharing 97.0%, 72.3% and 38.4% amino acid identity, respectively.
  • the high sequence homology between murine and human SULU3 suggests that these two proteins are orthologs.
  • the 25 amino acid N-terminal domain of murine SULU3 is most related to human SULU1 (SEQ ID NO:22) and to the C. elegans SULU kinase (GB:U11280), sharing 70.0% and 44.4% amino acid identity, respectively.
  • Murine SULU3 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristoylation.
  • a Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • Murine SULU3 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • the 149 amino acid spacer of murine SULU3 is most related to human SULU3 (SEQ ID NO:23), SULU1 (SEQ ID NO:22), and STE20 (GB:X99325), as well as to the C. elegans SULU (GB:U11280) and the S. cerevisiae STE20 (GB:L04655) kinases, sharing 98.7%, 53.4%, 21.9%, 59.4% and 21.9% amino acid identity, respectively.
  • murine SULU3 Immediately C-terminal to the spacer region of murine SULU3 is a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of murine SULU3 is most related to human SULU3 (SEQ ID NO:23), ZC1 (SEQ ID NO:13), and GEK 2 (SEQ ID NO:107), as well as to the C. elegans SULU kinase (GB:U11280), sharing 99.5%, 27.4%, 22.5% and 29.2% amino acid identity, respectively.
  • the 116 amino acid C-terminal spacer region of murine SULU3 is most related to human SULU3 (SEQ ID NO:23), GEK 2 (SEQ ID NO:107), and ZC1 (SEQ ID NO: 13), well as to the C. elegans SULU kinase (GB:U11280), sharing 98.3%, 24.6%, 24.1% and 40.5% amino acid identity, respectively.
  • the 2249 bp murine SULU3 and the 3824 bp human SULU3 cDNAs contain a 1620 nucleotide overlap (541 amino acids) with 90% and 98% DNA and amino acid sequence identity, respectively. Owing to the high degree of sequence identity in this extended overlap, we propose that these are functional orthologues of a single gene.
  • the combined murine/human 4492 bp SULU3 sequence encodes a polypeptide of 1001 amino acids (SEQ ID NO:31) with a predicted molecular mass of 116,069 daltons. Analysis of the deduced amino acid sequence predicts SULU3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • SULU3 contains a 25 amino acid N-terminus, a 248 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 149 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure and a second spacer region of 114 amino acids, a 247 amino acid C-terminal region predicted to form a second coiled-coil structure and a 100 amino acid C-terminal tail.
  • the murine SULU3 clone lacks the region from the second C-terminal coiled-coil to the C-terminus, whereas the human clone lacks the N-terminal domain, and all but 66 amino acids of the 248 amino acid kinase domain.
  • SULU3 is most closely related to SULU1 (SEQ ID NO:22) and the C. elegans SULU kinase (GB:U11280) sharing 72.3% and 38.4% amino acid identity, respectively.
  • the 25 amino acid N-terminal domain of SULU3 is most related to human SULU1 (SEQ ID NO:22) and to the C. elegans SULU kinase (GB:U11280), sharing 70.0% and 44.4% amino acid identity, respectively.
  • SULU3 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation.
  • a Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • the 248 amino acid catalytic domain of SULU3 is most related to human SULU1 (SEQ ID NO:22), SOK-1 (GB:X99325), ZC1 (SEQ ID NO:13), KHS1 (GB:U77129) and the C. elegans SULU kinase (GB:U11280), sharing 86.7%, 46.6%, 43.3%, 42.0% and 59.4% amino acid identity, respectively.
  • SULU3 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, PAK4 and PAK5.
  • the 149 amino acid spacer of SULU3 is most related to SULU1 (SEQ ID NO:22) and SOK-1 (GB:X99325), and to the C. elegans SULU (GB:U11280), and S. cerevisiae STE20 (GB:L04655) kinases, sharing 53.4%, 21.9%, 59.4% and 21.9% amino acid identity, respectively.
  • a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region is most related to ZC1 (SEQ ID NO:13), GEK 2 (SEQ ID NO:107), and the C. elegans SULU kinase (GB:U11280), sharing 27.4% 22.5% and 29.2% amino acid identity, respectively.
  • the 114 amino acid spacer region of SULU3 is most related to human SULU1 (SEQ ID NO:22), GEK 2 (SEQ ID NO:107), ZC1 (SEQ ID NO:13), and to the C. elegans SULU kinase (GB:U11280), sharing 73.7%, 24.6%, 24.1% and 41.2% amino acid identity, respectively.
  • SULU3 Immediately C-terminal to the second spacer region of SULU3 is a 247 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm.
  • This region of SULU3 is most related to human SULU1 (SEQ ID NO:22) and ZC1 (SEQ ID NO:13), as well as to rat PKN (GB:D26180), murine pl60 ROCK1 (GB:U58512) and the C. elegans SULU kinase (GB:U11280), sharing 73.7%, 26.7%, 24.0%, 21.0% and 37.6% amino acid identity, respectively.
  • the 100 amino acid C-tail of SULU3 is most related to a human prion protein (GB:L38993) with 45.0% amino acid identity.
  • the 2926 bp human GEK2 nucleotide sequence of the complete cDNA encodes a polypeptide of 968 amino acids (SEQ ID NO: 107) with a predicted molecular mass of 112,120 daltons. Analysis of the deduced amino acid sequence predicts GEK2 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • the complete GEK2 protein contains a 33 amino acid N-terminus, a 261 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 43 amino acid spacer region, a 135 amino acid proline-rich region, a 252 amino acid region predicted to form a coiled-coil structure followed by a 244 amino acid region also predicted to form a coiled-coil structure.
  • GEK2 is most closely related to rat AT1-46 (GB:U33472) (a partial cDNA that extends from the middle of the first potential coiled-coil domain of GEK2 to the C-terminus), murine LOK (GB:D89728), Xenopus laevis polo-like kinase 1 (GB:AF100165), and human SLK (GB:AB002804), sharing 91.3%, 88.5%, 65.0%, and 44.7% amino acid identity, respectively.
  • the high sequence homology between human GEK2, murine LOK and rat AT 1-46 suggests that human GEK2 is a highly related protein to the rodent forms, or alternatively, its orthologue. Recently, a full-length version of GEK2 was reported (STK10 or human LOK AB015718).
  • the 968 amino acid sequence is 99% identical to GEK2 (SEQ ID NO:107).
  • the 33 amino acid N-terminal domain of human GEK2 is most related to murine LOK (GB:D89728) and to human SLK (GB:AB002804), sharing 100% and 54.5% amino acid identity, respectively.
  • Human GEK2 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation.
  • a Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • the 261 amino acid catalytic domain of human GEK2 is most related to murine LOK (GB:D89728), rat AT1-46 (GB:D89728) and human SLK (GB:AB002804) as well as to a C. elegans kinase (GB:Z81460), sharing 97.7%, 90.8%, 54.5% and 55.9% amino acid identity, respectively.
  • GEK2 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • the 43 amino acid spacer region of human GEK2 is most related to murine LOK (GB:D89728) and to human SLK, sharing 83.7% and 77.6% amino acid identity, respectively.
  • the 135 amino acid proline-rich region of human GEK2 is most related to murine LOK (GB:D89728) with 66.2% amino acid identity, respectively.
  • a potential “PxxP” SEQ ID NO: 148) SH3-binding domain conserved with murine LOK.
  • human GEK2 Immediately C-terminal to the proline-rich region of human GEK2 is a 252 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of human GEK2 is most related to rat AT1-46 (GB:D89728), murine LOK (GB:D89728) and human SLK (GB:AB002804), and ZC2 (SEQ ID NO:14), sharing 90.8%, 86.9%, 42.2%, and 29.7% amino acid identity, respectively.
  • human GEK2 Immediately C-terminal to the predicted coiled-coil structure of human GEK2 is a second potential coiled-coil structure of 244 amino acids predicted based on the Lupas algorithm.
  • This region of human GEK2 is most related to rat AT1-46 (GB:D89728) and murine LOK (GB:D89728) as well as to human SLK (GB:AB002804) and ZC1 (SEQ ID NO:13), sharing 91.8%, 92.6%, 70.4% and 26.7% amino acid identity, respectively.
  • the C. elegans kinase (GB:Z81460) shares 31.5% amino acid sequence identity over this region.
  • the 3604 bp human PAK4 nucleotide sequence encodes a polypeptide of 681 amino acids (SEQ ID NO:29) with a predicted molecular mass of 74,875 daltons. Analysis of the deduced amino acid sequence predicts PAK4 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • the full-length PAK4 protein contains a 51 amino acid N-terminus predicted to contain a rac-binding motif, a 173 amino acid insert relative to the known mammalian PAK proteins, a 169 amino acid spacer region, a 265 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase and a 23 amino acid C-terminal tail.
  • PAK4 is most closely related to human PAK5 (SEQ ID NO:30), PAK1 (GB: U24152), and PAK65 (GB:U25975), as well as to a C. elegans kinase (GB: Z74029), sharing 76.8%, 49.5%, 49.8%, and 34.6% amino acid identity, respectively.
  • the 51 amino acid N-terminal domain of human PAK4 is most related to human PAK1 (GB:U24152), and PAK65 (GB:U25975), as well as to a C. elegans kinase (GB: Z74029), sharing 50.0%, 50.0% and 49.0% amino acid identity, respectively.
  • PAK1 The equivalent spacer region in PAK1 binds to the guanine nucleotide exchange factor PIX (Manser, E. et al (1998) Molecular Cell, 1, 183-192). Since PAK4 differs substantially from PAK1 over this region, the spacer domain of PAK4 may differ in its guanine nucleotide exchange factor binding specificity, relative to PAK1.
  • PAK4 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3 and PAK5.
  • the 23 amino acid C-tail of human PAK4 contains a sequence that is homologous to a G-protein beta subunit binding site (Leeuw, T. et al. Nature, 391, 191-195 (1998)). PAK4 has, therefore, the potential to be activated by both Cdc42-as well as G-protein-dependant pathways.
  • the 2,806 bp human PAK5 nucleotide sequence of the complete cDNA encodes a polypeptide of 591 amino acids (SEQ ID NO:103) with a predicted molecular mass of 64,071 Daltons. Analysis of the deduced amino acid sequence predicts PAK5 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and transmembrane domain.
  • the full-length PAK5 protein contains a 52 amino acid N-terminus predicted to contain a p21 (small G-protein) binding domain (PDB or CRIB), a 121 amino acid insert relative to the known mammalian PAK proteins, a 134 amino spacer region, a 265 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase and a 19 amino acid C-terminal tail.
  • PDB or CRIB small G-protein binding domain
  • PAK5 is most closely related to Human PAK4 (SEQ ID NO:29), Drosophila melanogaster PAK (also known as “mushroom bodies tiny”) (AJ01578), C45B11.1b from C. elegans (Z74029), and human PAK3 (Q13177) sharing 48% (327/674 aa), 50% (330/651 aa), 43% (234/435 aa excluding gap), and 47% (190/405 aa excluding gap) amino acid identity, respectively. Recently, a full length version of PAK5 was reported (PAK4 AF005046) whose 591 amino acid sequence is identical to PAK5 (SEQ ID NO:103). (Abo, et al. (1998) EMBO J. 17:6527-6540).
  • the 52 amino acid N-terminal domain of human PAK5 is most related to human PAK4 (SEQ ID NO:29), Drosophila melanogaster PAK (AJ011578), C45B11.b from C. elegans (Z74029), and human PAK3 (Q13177), sharing 65%, 57%, 54%, and 53% amino acid identity, respectively.
  • the 121 amino acid insert of human PAK5 shares 43% amino acid identity with a similar domain from PAK4 (SEQ ID NO:29), but that is absent from other known PAKs.
  • PAK1 The equivalent spacer region in PAK1 binds to the guanine nucleotide exchange factor PIX (Manser, E. et al (1998) Molecular Cell, 1, 183-192 hereby incorporated by reference herein in its entirety including any drawings, figures, or tables.). Since PAK5 differs substantially from PAK1 over this region, the spacer domain of PAK5 may differ in its guanine nucleotide exchange factor binding specificity, relative to PAK1.
  • the 265 amino acid catalytic domain of human PAK5 is most related to human PAK4 (SEQ ID NO:29), Drosophila melanogaster PAK (AJ011578), C45B11.1b from C. elegans (Z74029), and human PAK3 (Q13177), sharing 78%, 80%, 61%, and 55% amino acid identity, respectively.
  • PAK5 also contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3 and PAK4.
  • PAK5 has, therefore, the potential to be activated by both, Cdc42 as well as G-protein-dependant pathways.
  • the present invention relates to an antibody having binding affinity to a kinase of the invention.
  • the polypeptide may have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or a functional derivative thereof, or at least 9 contiguous amino acids thereof (preferably, at least 20, 30, 35, or 40 or more contiguous amino acids thereof).
  • the present invention also relates to an antibody having specific binding affinity to a kinase of the invention.
  • an antibody may be isolated by comparing its binding affinity to a kinase of the invention with its binding affinity to other polypeptides.
  • Those which bind selectively to a kinase of the invention would be chosen for use in methods requiring a distinction between a kinase of the invention and other polypeptides.
  • Such methods could include, but should not be limited to, the analysis of altered kinase expression in tissue containing other polypeptides.
  • the STE20-Related kinases of the present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction.
  • the kinases of the present invention can be used to produce antibodies or hybridomas.
  • One skilled in the art will recognize that if an antibody is desired, such a peptide could be generated as described herein and used as an immunogen.
  • the antibodies of the present invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting.
  • the present invention also relates to a hybridoma which produces the above-described monoclonal antibody, or binding fragment thereof.
  • a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
  • the polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity.
  • Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or ⁇ -galactosidase
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0-Ag14 myeloma cells
  • Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124, 1988).
  • Hybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell, “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology”, supra, 1984).
  • antibody-containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
  • the above-described antibodies may be detectably labeled.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic atoms, and the like.
  • the above-described antibodies may also be immobilized on a solid support.
  • solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10, 1986; Jacoby et al., Meth. Enzym. 34, Academic Press, N.Y., 1974).
  • the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
  • Anti-peptide peptides can be generated by replacing the basic amino acid residues found in the peptide sequences of the kinases of the invention with acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.
  • the present invention also encompasses a method of detecting a STE20-related kinase polypeptide in a sample, comprising: (a) contacting the sample with an above-described antibody, under conditions such that immunocomplexes form, and (b) detecting the presence of said antibody bound to the polypeptide.
  • the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample. Altered levels of a kinase of the invention in a sample as compared to normal levels may indicate disease.
  • Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay.
  • immunological assay formats such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays
  • Examples of such assays can be found in Chard (“An Introduction to Radioimmunoassay and Related Techniques” Elsevier Science Publishers, Amsterdam, The Netherlands, 1986), Bullock et al.
  • the immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine.
  • the test samples used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is testable with the system utilized.
  • kits contains all the necessary reagents to carry out the previously described methods of detection.
  • the kit may comprise: (i) a first container means containing an above-described antibody, and (ii) second container means containing a conjugate comprising a binding partner of the antibody and a label.
  • the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies.
  • detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • the compartmentalized kit may be as described above for nucleic acid probe kits.
  • the antibodies described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
  • the present invention also relates to a method of detecting a compound capable of binding to a STE20-related kinase of the invention comprising incubating the compound with a kinase of the invention and detecting the presence of the compound bound to the kinase.
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts.
  • the present invention also relates to a method of detecting an agonist or antagonist of kinase activity or kinase binding partner activity comprising incubating cells that produce a kinase of the invention in the presence of a compound and detecting changes in the level of kinase activity or kinase binding partner activity.
  • the compounds thus identified would produce a change in activity indicative of the presence of the compound.
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts. Once the compound is identified it can be isolated using techniques well known in the art.
  • the present invention also encompasses a method of agonizing (stimulating) or antagonizing kinase associated activity in a mammal comprising administering to said mammal an agonist or antagonist to a kinase of the invention in an amount sufficient to effect said agonism or antagonism.
  • a method of treating diseases in a mammal with an agonist or antagonist of STE20-related kinase activity comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize STE20-related kinase associated functions is also encompassed in the present application.
  • indolinone compounds form classes of acid resistant and membrane permeable organic molecules.
  • WO 96/22976 published Aug. 1, 1996 by Ballinari et al. describes hydrosoluble indolinone compounds that harbor tetralin, naphthalene, quinoline, and indole substituents fused to the oxindole ring. These bicyclic substituents are in turn substituted with polar moieties including hydroxylated alkyl, phosphate, and ether moieties.
  • U.S. patent application Ser. No. 08/702,232 filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al.
  • Other examples of substances capable of modulating kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines.
  • the quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well known compounds such as those described in the literature.
  • representative publications describing quinazolines include Barker et al., EPO Publication No. 0 520 722 A1; Jones et al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No. 4,757,072; Kaul and Vougioukas, U.S. Pat. No.
  • oxindolinones such as those described in U.S. patent application Ser. No. 08/702,232 filed Aug. 23, 1996, incorporated herein by reference in its entirety, including any drawings.
  • STLK2, STLK4, STLK5, STLK6 and STLK7 belong to an expanding family of intracellular STKs that have varying degrees of sequence homology to SOK-1, a kinase implicated in oxidative stress agents (Pombo, CM et al, EMBO J. (17) 4537-4546, 1996).
  • Our data shows that STLK2 is expressed highly in hematopoietic cells. Therefore, STLK2 may participate in the oxidative response pathway during inflammation.
  • STLK2 could also be a possible component in the signaling pathways leading to T cell activation. High levels of STLK2 in several tumor cell lines could also imply that STLK2 might be involved in tumorigenesis.
  • STLK2 is most closely related to two human STE20-subfamily kinases: MST3 and SOK-1.
  • MST3 is a 52,000 daltons cytoplasmic kinase that is ubiquitously expressed with its highest levels of expression found in heart, skeletal muscle and pancreas. The serine/threonine kinase activity of MST3 is activated by phosphorylation. Unlike SOK-1, MST3 prefers Mn ++ over Mg ++ and can use both GTP and ATP as phosphate donors. MST3 may undergo dimerization. No agonists have yet been identified that activate MST3. The downstream signaling mechanism of this kinase is unknown (Schinkmann, K and Blenis, J. (1997) J. Biol. Chem. 272, 28695-28703).
  • SOK-1 is a 50,000 daltons cytoplasmic kinase expressed predominantly in testis, large intestine, brain and stomach and to a lesser extent in heart and lung. SOK-1 is also expressed in the germinal center B-cell line (RAMOS) and in a mature B cell line (HS Sultan). The serine/threonine kinase activity of SOK-1 is activated by phosphorylation. The C-terminus of SOK-1 has been shown to be inhibitory to the catalytic activity of this kinase. The only agonists known to activate SOK-1 are oxidant agents, like H 2 O 2 and menadione, a quinone that is a potent intracellular generator of reactive oxygen species (Pombo, C.
  • SOK-1 is also activated by chemical anoxia through the generation of reactive oxygen species and release of calcium into the cytoplasm from intracellular stores. SOK-1, therefore, may play an important role in ischemia, the cause of myocardial infarction, stroke and acute renal failure (Pombo, C. M. et al. J. Biol. Chem. 272, 29372-29379 (1997)).
  • SAPKs stress-activated protein kinases
  • SOK-1 does not activate any of the four MAP kinase pathways, SAPKs, p38, ERK-1 or MEK-5/ERK-5 (Pombo, C. M. et al. EMBO J. 15, 4537-4546). The downstream signaling mechanism of this kinase remains unknown.
  • STLK2 is expressed in a wide variety of immune cell types and tissues including thymus, dendrocytes, mast cells, monocytes, B cells (primary, Jurkat, RPMI, SR), T cells (CD8/CD4+, TH1, TH2, CEM, MOLT4) and megakaryocytes (K562), whereas STLK3 is restricted to thymus and STLK4 is predominately expressed in thymus, T cells (CD4/CD8+, TH1, CEM) and B cells (Jurkat, RPMI).
  • these STKs might participate in the oxidative response pathway during inflammation, reperfusion injury (stroke, surgery, shock), TNF ⁇ -mediated signaling, insulin desensitization, atherogenesis, vascular injury, T or B cell costimulation, or alternatively, participate in other MAPK-related signal transduction processes.
  • STLK5 is more distantly related to this STE20-subfamily including SOK-1 and STLK2, STLK3 and STLK4. STLK5, may therefore mediate a signaling pathway that is distinct from the oxidative stress response pathway.
  • ZC1 is a good candidate for any disease in which tyrosine kinase, cytokine, or heterotrimeric G-protein coupled receptors have been implicated.
  • the mouse homologue binds to NCK, and is recruited to activated PDGF (Su et al., EMBO 16: 1279-1290, 1997).
  • the Drosophila homolog has been shown to bind to TRAF2, implicating it in TNF- ⁇ signaling (Liu et al., (1999) Curr. Biol. 9:101-104, 1999)). While ZC1 does not contain the exact NCK- and TRAF2-binding domains, it is likely to bind to related proteins.
  • ZC1 has very broad over-expression in many tumor types, suggesting that it may be involved in cellular growth, transformation, or tumor progression.
  • a truncated form of ZC1 containing only the C-terminal putative MEKK1-binding domain was found to reduce the number of foci generated by H-Ras-V12 in Rat Intestinal Epithelial cells (RIE-1). These data indicate that ZC1 may play a role in the ability for these cells to overcome contact inhibition and anchorage-dependent growth.
  • the association of the ZC1 family members msn and NIK with TRAF2 may indicate a role for this kinase in cell survival and/or in apoptosis.
  • the ZC1 family contains a highly conserved domain that in the mouse homolog, NIK, has been shown to bind to MEKK1 (Mitogen-activated/Extracellular-regulated Kinase Kinase 1) (Su et al., (1997) EMBO 16(6): 1279-90).
  • MEKK1 is involved in cell survival and/or apoptosis in several systems (Schlesinger et al., Front. Biosci.3:D1181-6, 1998).
  • MEKK1 appears to be upstream of either the ERK1/MAPK or the JNK/SAPK pathway [Schlesinger et al., (1998 Front. Biosci. 3:D1181-6).
  • ZC1 shares a high degree of homology with these other family members in both the kinase domain and the “MEKK”-binding domains, yet it differs in the intervening region, which contains several putative binding domains for upstream signaling adapter molecules (e.g. NCK, TRAF2). Unlike the other family members, ZC1 does not appear to activate the JNK pathway in 293T cells as seen by its ability to induce expression of either a JUN or ATF2-driven luciferase gene. Upon co-transfection into these cells with HA-tagged JNK, modest activation of JNK was detected. ZC1 also modestly activated co-transfected ERK1.
  • upstream signaling adapter molecules e.g. NCK, TRAF2
  • ZC1 profoundly inhibits ERK1 kinase expression in co-transfection assays. This effect is dependent on ZC1 kinase activity, occurring with the wild-type and the kinase domain alone, but not with the kinase-dead mutant even though all three forms of ZC1 are expressed at similar levels. This may suggest a role for this kinase in transcriptional or post-transcriptional regulation.
  • ZC1 may be an important component in the signaling pathways mediated by the co-stimulatory receptor CD28 in T cells and/or by the pro-inflammatory cytokine TNF ⁇ , since co-transfection of the wild-type ZC1 activated the RE/AP-luciferase and NF ⁇ B-luciferase reporter genes. While our data showed that ZC1 strongly activates NF ⁇ B in T-cells, no activation of NF ⁇ B driven luciferase was detectable in NIH 3T3 cells. A recent paper (J. Biol. Chem. 274:2118-25; 1999.) has shown that a human ZC1 splicing isoform, HGK, is involved in the TNF ⁇ -signaling pathways.
  • ZC1 could be a therapeutic target for immunological diseases which include but are not limited to: rheumatoid arthritus, chronic inflammatory bowel diseases (ie Crohn's disease), chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, and autoimmunity as well as organ transplantation and cardiovascular diseases.
  • immunological diseases include but are not limited to: rheumatoid arthritus, chronic inflammatory bowel diseases (ie Crohn's disease), chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, and autoimmunity as well as organ transplantation and cardiovascular diseases.
  • ZC1 appears to be the human orthologue of murine NIK and possibly an orthologue of a C. elegans STE20-subfamily kinase encoded by the ZC504.4 cosmid.
  • Murine NIK is a 140,000 daltons kinase that is most highly expressed in brain and heart. NIK interacts with the SH3 domains of the adaptor molecule Nck through its proline-rich regions found in the C-terminal extra-catalytic region. The specific regions that mediate this interaction are two PxxP (SEQ ID NO: 148) motifs that are nearly uniformly conserved between NIK, ZC1,2,3 and the C. elegans STE20 ZC504.4 kinase. In addition, NIK binds MEKK1 through its 719 amino acid C-terminal (Su, Y-C. et al. (1997) EMBO J. 16, 1279-1290).
  • MEKK1 is a membrane-associated kinase responsible for activating MKK4 (also known as SEKI), which in turn activates SAPK (Yan, M et al. (1994) Nature, 372, 798-800).
  • NIK may function as a kinase that links growth factor activated pathways and the stress-response pathway mediated by SAPKs.
  • activation of growth factor receptors leads to receptor tyrosine phosphorylation, Nck binding to the phosphorylated tyrosines via its SH2 domain, NIK redistribution to a membrane compartment via binding to the SH3 domain of Nck, and juxtaposition to the membrane-associated MEKK1.
  • the NIK-MEKK1 interaction would, in this fashion, turn on the SAPK pathway in response to growth factor stimulation (Su, Y-C. et al. (1997) EMBO J. 16, 1279-1290).
  • the ZC kinases also display strong homology at their C-termini to protein domains that bind small GTPase proteins such as Rab, Rho and Rac.
  • the C-termini of ZC1 is 36.2% identical to citron, a murine Rho-binding protein, and 23.1% identical to the rab-binding region of GC kinase. This suggests that, in addition to adaptor molecules, small GTPase proteins may also mediate membrane association and activation of the ZC kinases.
  • the presence of a potential coiled-coil region located immediately C-terminal to the catalytic region strongly suggests that the ZC kinases may also be subject to regulation via homo or heterodimerization events.
  • the C. elegans STE20 ZC504.4 kinase is the product of the mig-15 gene.
  • the product of this gene has been implicated in several developmental processes such as epidermal development, Q neuroblast migrations and muscle arm targeting in the developing worm (Zhu, X. and Hedgecock E. (1997) Worm Breeder's Gazette 14, 76).
  • the high level of sequence conservation between the ZC kinases and the ZC504.4 C. elegans kinase will make C. elegans a valuable model organism to study, through epistatic analysis, the signaling properties of the human ZC kinases.
  • KHS1 kinase homologous to SPS1/STE20
  • SPS1/STE20 is a 100,000 dalton cytoplasmic STK that is expressed ubiquitously.
  • KHS1 has been implicated in the mechanism of SAPK activation in response to inflammatory cytokines such as TNF ⁇ as well as to ultraviolight light, which also uses the TNF signaling pathway.
  • TNF ⁇ binding to its receptors results in the sequential association with the receptor C-tail of multiple signaling molecules including TNFR1-associated death domain protein (TRADD), Fas-associated death domain protein (FADD or MORT1), TNFR-associated factor 2 (TRAF2), and the STK RIP (receptor interacting protein).
  • TRADD TNFR1-associated death domain protein
  • FADD or MORT1 Fas-associated death domain protein
  • TRAF2 TNFR-associated factor 2
  • STK RIP receptor interacting protein
  • the TRADD-TRAF2 interaction is mediated by a conserved region present at the C-terminus of TRAF2, the TRAF domain.
  • Activation of the NF ⁇ B and SAPK pathways is mediated by the ring finger motif present at the N-terminus of TRAF2 (Curr. Opinion in Cell. Biol. (1997) 9:247-251).
  • KHS1 is activated by TNF ⁇ stimulation in a TRAF2-dependant manner and inhibition of KHS1 blocks TNF ⁇ -induced SAPK activation but not NF ⁇ B activation.
  • the mechanism by which TRAF2 activates KHS1 is not known. Cotransfection of TRAF2- and KHS1-expressing constructs in 293T cells failed to reveal a direct association between these two molecules.
  • KHS1 activates the SAPK pathway by a direct association with the constitutively active kinase MEKK1.
  • MEKK1 subsequently activates SEK1, which in turn activates SAPK.
  • MAPK MAPK
  • SEK1 p38 kinase pathway
  • MAPK MAPK
  • p38 kinase pathways are activated by KHS1 (Shi, C-S and Kehrl. J. H. (1997) J. Biol. Chem. 272, 32102-32107).
  • downstream signaling of KHS1 requires its conserved C-terminus (Diener, K. et al (1997) Proc. Natl. Acad. Sci. 94, 9687-9692).
  • GCK germinal center kinase
  • GCK is a constitutively active 97,000 dalton STK that is broadly expressed. GCK may participate in B-cell differentiation since its expression is localized to the germinal center within lymphoid follicles. GCK activates the SAPK pathway in response to TNF ⁇ via activation of SEK1. The upstream activators of GCK in response to cytokines as well as the immediate downstream target of this kinase are unknown. The C-terminus of GCK is sufficient to activate SEK1 (Pombo, C. M. et al (1995) Nature, 377, 750-754).
  • the murine orthologue of GCK, rab8ip (rab8-interacting protein), is a 97,000 dalton protein that fractionates with both the soluble cytoplasmic fraction as well as with a salt-sensitive fraction associated with the basolateral membrane of the trans-Golgi region in polarized MDCK epithelial cells.
  • the C-terminus of rab8ip binds to rab8, a small GTP-binding protein required for vesicular transport from the Golgi apparatus (Ren, M. et al. (1996) Proc. Natl. Acad. Sci. 93, 5151-5155).
  • GCK may also promote the rab-dependent release of secretory proteins in response to TNF ⁇ (Buccione, R. et al (1995) Mol. Bio. Cell 6, 291).
  • HPK1 hematopoietic protein kinase
  • HPK1 hematopoietic protein kinase
  • MEKK1 Human, M. et al (1996) Genes and Dev. 10:2251-2264
  • MLK-3 ubiquitously expressed mixed-lineage kinase MLK-3
  • This function of HPK1 requires, in contrast to GCK, both its kinase domain as well as its C-terminus.
  • the upstream activators of HPK1 remain unknown.
  • HPK1 also plays a key role as a mediator of transforming growth factor- ⁇ -(TGF ⁇ ) signaling.
  • HPK1 activates the TGFb-activated kinase (TAK), which in turn stimulates the SAPK pathway by phosphorylating SEK1 (Wang W. et al (1997) J. Biol. Chem. 272:22771-22775).
  • TGFb-activated kinase TGFb-activated kinase
  • KHS2 is expressed in thymus, dendrocytes and monocytes. KHS2 could have a complementary function to that of KHS1 as a mediator of SAPK activation in the cellular response to inflammatory cytokines. KHS2 could have the potential to interact directly with TRAF2 since a STK with the predicted molecular weight of KHS2 (approximately 101,000 daltons) is found in the TNFR-TRAF2 complex upon TNF ⁇ stimulation (VanArsdale, T. and Ware, C. F. (1994) J. Immunol. 153, 3043-3050).
  • KHS2 in addition to having a potential role in the TRAF2-dependant TNF ⁇ cytokine response, could also mediate signaling events that utilize small GTPase proteins.
  • the binding of a small GTPase protein to the C-terminus of KHS2 may be required for its potential TRAF2-dependant signaling to a downstream kinase such as MEKK1.
  • xPlkk1 as the activator of Plx1 (the Xenopus Polo kinase).
  • Plx1 the Xenopus Polo kinase
  • the STK Plkk1 can phosphorylate and activate Plx1 STK (the mammalian Polo kinase or PLK).
  • Plx1 STK the mammalian Polo kinase or PLK.
  • a dominant-negative (kinase-dead) form of xPlkk1 prevents Plx1 activation and delays germinal vesicle breakdown. Yet another unidentified kinase is probably responsible for xPlkk1 activation during mitosis.
  • GEK2 might represent the human homologue for xPlkk1. Based on this, GEK2 might be upstream of PLK in mammalian cells.
  • SULU1 based on the phage display screen results using the SULU1 coiled-coil2 domain as bait, SULU1 might also interact in vivo with GEK2 and therefore regulate GEK2 (and/or SLK through the coiled-coil domain) activation leading to PLK activation and mitosis.
  • TAO1 A recently cloned STE20-subfamily kinase, rat TAO1, is most likely the rodent orthologue of human SULU3 (Hutchinson, M. et al. J. Biol. Chem 273:28625-28632, 1998). TAO1 activates MEK3, 4 and 6 in vitro, while in transfected cells it associates and activates only MEK3, resulting in phosphorylation and activation of p38. These results implicate TAO1 (SULU3) in the regulation of the p38 containing stress-responsive MAP kinase pathway.
  • Human SULU1 is weakly expressed in hematopoietic sources whereas SULU3 is found in B-cells and TH1-restricted T cells.
  • These mammalian SULU STKs display strong homology to the C. elegans SULU kinase. The role that this kinase plays in nematode development is unknown.
  • the strong sequence homology between the catalytic domain of mammalian SULU kinases and other STE20-subfamily kinases such as SOK-1 (human STE20) and KHS2 suggests that the mammalian kinases may participate in the stress-response pathway.
  • the potential coiled-coil domains found at the C-terminus of the SULU kinases may play a role in the regulation of this kinase.
  • Murine LOK lymphocyte-oriented kinase
  • STK serum-derived neuropeptide
  • LOK does not activate any of the known MAPK pathways (ERK, JNK and p38) nor the NF ⁇ B pathway.
  • the upstream signaling elements of LOK as well as the extracellular stimuli that utilize this kinase to elicit a biological response are also unknown (Kuramochi, S. et al (1997) J. Biol. Chem. 272: 22679-22684).
  • Human GEK2 is highly related to murine LOK, but based on sequence divergence in the non-catalytic domain, it appears to be a distinct member of this STE20-subfamily. GEK2 may signal through a pathway that remains to be defined. The presence of potential coiled-coil regions at the C-terminus of GEK2 could play a key role in regulating the functions of this kinase.
  • PAK activated protein kinases
  • STE20 The p21 activated protein kinases (PAK) are a closely related subgroup of the STE20 family of serine/threonine kinases. Extensive genetic and biochemical analysis of the budding yeast STE20 has shown the critical role this serine/threonine kinase plays at the juncture of several important intracellular pathways required to appropriately respond to extracellular signals. STE20 links the transcriptional response by mediating the activation of the appropriate downstream MAPK pathway as well as coupling changes in cellular morphology via its control of the actin cytoskeleton.
  • PAK subgroup A hallmark of the PAK subgroup is their small G protein-binding domain (PBD) that confers G protein-dependent activation upon this group of kinases. Via the PBD, PAKs bind to activated small G proteins resulting in the derepression of the PAK's intrinsic kinase activity.
  • PBD small G protein-binding domain
  • PAK1 a 68 kD protein whose expression is restricted expression to brain, muscle, and spleen
  • PAK2 PAK1, PAK65
  • PAK3 a 65kD protein whose expression is restricted to the brain.
  • the mammalian PAKs (1,2, and 3) have been shown to respond to extracellular signals (growth factors, mitogens, cytokines and a variety of cellular stresses) (Bagrodia, et al. (1995). J. Biol. Chem. 270: 22731-22737; Zhang, S., et al. (1995). J. Biol.
  • PAK p21-activated kinases
  • Nck The adaptor molecule, Nck, is constitutively bound via its SH3 domain to the proline-rich motif in the N-terminal portion of PAK1. Binding of the Nck-PAK complex to activated growth factor receptors in response to growth factor stimulation provides a mechanism to link growth factor-stimulated and stress-response pathways (Galisteo, M. et al. (1996) J. Biol. Chem. 271:20997-21000).
  • the PBD found at the N-terminus of PAK1 is responsible for its high-affinity interaction with the GTP-bound forms of Cdc42 and Rac (Burbelo, P. et al. (1995) J. Biol. Chem. 270:29071-29074).
  • the exact mechanism through which the small GTPases activate PAKs may involve, in part, association of the kinase with activated growth factor receptors through guanine nucleotide exchange factors (GEFs). GEFs activate small GTPases by catalyzing the formation of their GTP-bound state, thereby promoting their association with, and activation of, PAKs.
  • GEFs guanine nucleotide exchange factors
  • PAKs all conserve an N-terminal extracatalytic motif responsible for a high-affinity interaction with the GEF, PIX.
  • the PAK-Cdc42 interaction and subsequent PAKs occurs as a PIX/PAK complex (Manser, E. et al. (1998) Molecular Cell, 1, 183-192).
  • PAK signaling stimulated by heterotrimeric G proteins is mediated through the interaction between a short conserved amino acid region located at the C-terminus of PAK1 with the G-protein ⁇ -subunit (Leeuw, T. et al.(1998) Nature, 391: 191-195).
  • PAKs are involved in mediating the activation of stress-activated protein kinase pathways (JNK and to lesser extent p38). PAKs are also potential mediators in the crosstalk between the pathways regulated by the Rho family of small G proteins and the signaling pathways directly downstream of Ras leading to the activation of the ERK pathway (Bagrodia, et al. (1995). J. Biol. Chem. 270: 22731-22737; Zhang, S., et al. (1995). J. Biol. Chem. 270: 23934-23936; Brown, J., et al. (1996) Curr Biol. 6:598-60596; Frost, J., et al. (1996). Mol. Cell. Biol. 16: 3707-3713).
  • PAK1 has been implicated in phosphorylating a regulatory site in MEK1 that is necessary for MEK1's ability to interact with Raf1 (Frost, et al. (1997) EMBO J. 16:6426-6438).
  • PAK3 has been shown to phosphorylate Raf1 on a site that is important for Raf1 activity (King, A., et al. (1998). Nature 396: 180-183).
  • PAKs play an important role in controlling morphological changes in cell shape mediated by the actin cytoskeleton. Such morphological changes are required for cellular functions ranging from cell division and proliferation to cell motility and vesicle transport. PAK activity has been implicated in the localized assembly (leading edge) and disassembly (retracting edge) of focal adhesions necessary for cell motility (Frost J. et al (1998) J. Biol. Chem. 273:28191-28198).
  • PAK2 may have a role in the morphological changes induced during apoptosis (Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. (Rudel, T. (1997) Science. 276:1571-4)), and PAK1 maybe important in preventing apoptosis (Faure S, et al. (1997) EMBO J. (1997) 16:5550-61). In addition to overcoming mitogen- and anchorage-independent growth, tumor cells need to escape the programmed cell death that accompanies deregulated cell growth. Thus, inhibition of PAKs may be effective in triggering apoptosis in tumors.
  • PAK1 and PAK2 A direct requirement for PAKs in the transformation of mammalian cells has been shown for PAK1 and PAK2.
  • Kinase-dead alleles of PAK1 block ras transformation of RAT1 and Schwann cells (Tang, Y., et al. (1997) Mol. Cell. Biol. 17, 4454-4464).
  • Dominant-negative alleles of PAK2 have been shown to interfere with ras-mediated transformation of mammalian cells (Osada, S., (1997) FEBS Lett 404:227-233)
  • PAK3 has been implicated in nonsyndromic X-linked mental retardation suggesting a role for PAK3 in cognitive function (Allen, K. et al. (1998) Nat. Genet. 20: 25-30). PAK1 has been implicated in neurite outgrowth in PC12 cells (Daniels, R. et al. (1998) EMBO J. 17: 754-764; Nikolic, M. et al. (1998) Nature 395:194-198).
  • PAK-like STKs may also play a role in AIDS pathogenesis since the myristoylated 27kD membrane-associated HIV Nef gene product directly interacts with and activates these kinases via cdc42 and Rac.
  • the Nef-mediated activation of PAK-like STKs correlates with the induction of high viral titers and the development of AIDS in infected hosts (Cullen, B. R. (1996) Curr. Biol. 6:1557-1559).
  • PAK4 is expressed in thymus, dendrocytes, mast cells, monocytes, as well as in T cells (TH2-restricted cells and MOLT4) and the B cell line RPMI.
  • PAK5 is found in mast cells and in the T cell line MOLT4.
  • PAK4 and PAK5 share with the known PAKs a potential cdc42-binding motif at their N-termini. Both PAK4 and PAK5 display sequence homology in their C-termini to a motif responsible for an interaction between PAK1 and the ⁇ -subunit of heterotrimic G-proteins (amino acid residues 665-676 in PAK 4, and amino acid residues 386-398 in PAK5). Consequently, PAK4, and possibly PAK5, could mediate signaling events originating from growth factors as well as from ligands that stimulate G-protein-linked receptors.
  • PAK4 conserves a leucine (leu 44), that when mutated to a phenylalanine renders the kinase activity of human PAK1 constitutively active, bypassing its cdc42-binding requirement for activation (Brown J. et al (1996) Current Biol. 6:598-605).
  • PAK5 contains an isoleucine at the equivalent position. Therefore, the mechanism by which cdc42 potentially activates human PAK1, PAK4, and possibly PAK5, may be very similar.
  • PAK4 and PAK5 however, lack the PIX-binding motif, and consequently cdc42-activating GEFs other than PIX (for example Db1 and Cool) must be responsible for the activation of these kinases.
  • PAK4 and PAK5 may be activated by another GTPase, such as Rac1 which uses the Tiam1 GEF for its activation to the GTP-bound state.
  • PAK4 and PAK5 also lack the PxxP (SEQ ID NO: 148) motif responsible for the Nck-PAK1 association. Between the PBD or cdc42-binding N-terminal motifs and the putative GEF-binding regions, PAK4 and PAK5 have long insertions (185 and 123 amino acids for PAK4 and PAK5, respectively) relative to PAK1. This region probably confers different binding characteristics to adaptor molecules and/or GEFs from those exhibited by known mammalian PAKs.
  • PAKs have been shown to be upstream in pathways leading to activation of both the JNK (Bagrodia, S., et al. (1995) J. Biol. Chem. 270: 22731-22737) and ERK kinase pathways (Brown, J., et al. (1996). Curr Biol. 6:598-605).
  • PAK1 was shown to synergize with ras in activation of the ERK pathway through phosphorylation of MEK1 (Frost, J. et al. (1997). EMBO J. 16:6426-6438).
  • MEK1 serves as an in vitro substrate for PAK4, suggesting a potential role for PAK4 in the activation of the ERK pathway and mitogenesis.
  • PAK5 may also have a mitogenic role, and be linked to cancer, based on its expression profile (elevated RNA and protein levels in a wide variety of tumor cell lines), its interaction with cdc42 via its PBD, and the ability of a kinase-dead allele (Lys350, 351 Ala) to block ras transformation of NIH3T3 cells.
  • a screen for small molecule inhibitors of PAK5 kinase activity may yield compounds with therapeutic potential for intervention in cancer derived from a wide variety of tissue types.
  • PAK4 and PAK5 may also play a role in HIV pathogenesis as potential mediators of Nef signaling, since none of the known PAKs correspond to the PAK-like kinase shown to interact with, and be activated by, the HIV nef protein (Lu, X. et al. (1996) Current Biology 6:1677-1684)
  • the 3′ untranslated region of PAK4 contains a CA repeat that is prone to undergo expansion.
  • CA dinucleotide repeat instability has been associated with disease (Toren, M. Z. et al (1998) Am. J. Hematol. 57: 148-152), and expansion of such repeat in the 3′ untranslated region of PAK4 could implicate this kinase in as yet unknown pathologies.
  • STLK3, STLK5, STLK6 and STLK7 may play an important role as mediators of the immune response.
  • they are targets for the development of specific small molecule inhibitors to treat immunological diseases, including, but not limited to, rheumatoid arthritis, chronic inflammatory bowel diseases (e.g. Crohn's disease), chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis and autoimmunity, as well as in organ transplantation.
  • Other diseases include cardiovascular diseases.
  • the human STLKs may also play an important role in cell growth regulation. Thus, they are targets for developing small molecule kinase inhibitors for the treatment of cancer and metastases.
  • STLK5 maps to a chromosomal region frequently amplified in a variety of tumors including those from non-small cell lung cancer, breast cancer and peripheral nerve tumors. This suggests that STLK5 could play a role in the development, maintenance, or progression of human tumors.
  • ZC1 may be a component of the CD28-signaling pathway and therefore important in T cell activation.
  • ZC1 as well as other ZC subfamily kinases are targets for the development of specific small molecule inhibitors to treat immunological diseases, including, but not limited to, rheumatoid arthritis, chronic inflammatory bowel diseases (e.g. Crohn's disease), chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis and autoimmunity, as well as organ transplantation.
  • Other diseases include cardiovascular diseases.
  • ZC1 and ZC2 are also implicated in cell growth regulation.
  • ZC subfamily kinases are targets for developing small molecule inhibitors for the treatment of cancer and metastases.
  • ZC2 maps to a chromosomal region frequently amplified in a variety of tumors including those from non-small cell lung cancer, small cell lung cancer, and cervical cancer. This suggests that ZC2 could play a role in the development, maintenance, or progression of human tumors.
  • PAK5 has a role in cancer based on its expression profile (elevated RNA and protein levels in wide variety of tumor lines), its interaction with Cdc42 via its PBD, and the ability of the kinase-dead allele of PAK5 (Lys350, 351Ala) to block ras transformation of NIH3T3 cells.
  • a screen for small molecule inhibitors of PAK5 kinase activity may yield compounds with therapeutic potential for intervention in cancers and metastases derived from a wide range of tissue types.
  • PAK5 maps to a chromosomal region frequently amplified in a variety of tumors including those from non-small cell lung cancer, and small cell lung cancer.
  • DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442, 1985).
  • Embryos can be infected with viruses, especially retroviruses, modified to carry inorganic-ion receptor nucleotide sequences of the invention.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc.
  • transgenic mouse female mice are induced to superovulate. Females are placed with males, and the mated females are sacrificed by CO 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice (Hammer et al., Cell 63:1099-1112, 1990).
  • a clone containing the sequence(s) of the invention is co-transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention.
  • Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E. J. Robertson, supra).
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination (Capecchi, Science 244: 1288-1292, 1989).
  • Methods for positive selection of the recombination event (i.e., neo resistance) and dual positive-negative selection (i.e., neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et al. (Nature 338: 153-156, 1989), the teachings of which are incorporated herein in their entirety including any drawings.
  • the final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females.
  • the resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene.
  • Procedures for the production of non-rodent mammals and other animals have been discussed by others (Houdebine and Chourrout, supra; Pursel et al., Science 244:1281-1288, 1989; and Simms et al., Bio/Technology 6:179-183, 1988).
  • the invention provides transgenic, nonhuman mammals containing a transgene encoding a kinase of the invention or a gene effecting the expression of the kinase.
  • Such transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introduction of a kinase, or regulating the expression of a kinase (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes).
  • a “transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats.
  • the transgenic DNA may encode human STE20-related kinases. Native expression in an animal may be reduced by providing an amount of anti-sense RNA or DNA effective to reduce expression of the receptor.
  • STE20-related kinases or their genetic sequences will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460, 1992). Miller states that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results. The basic science of gene therapy is described in Mulligan (Science 260:926-931, 1993).
  • an expression vector containing STE20-related kinase coding sequence is inserted into cells, the cells are grown in vitro and then infused in large numbers into patients.
  • a DNA segment containing a promoter of choice (for example a strong promoter) is transferred into cells containing an endogenous gene encoding kinases of the invention in such a manner that the promoter segment enhances expression of the endogenous kinase gene (for example, the promoter segment is transferred to the cell such that it becomes directly linked to the endogenous kinase gene).
  • the gene therapy may involve the use of an adenovirus containing kinase cDNA targeted to a tumor, systemic kinase increase by implantation of engineered cells, injection with kinase-encoding virus, or injection of naked kinase DNA into appropriate tissues.
  • Target cell populations may be modified by introducing altered forms of one or more components of the protein complexes in order to modulate the activity of such complexes. For example, by reducing or inhibiting a complex component activity within target cells, an abnormal signal transduction event(s) leading to a condition may be decreased, inhibited, or reversed. Deletion or missense mutants of a component, that retain the ability to interact with other components of the protein complexes but cannot function in signal transduction may be used to inhibit an abnormal, deleterious signal transduction event.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA) encoding recombinant kinase of the invention protein into the targeted cell population (e.g., tumor cells).
  • viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus.
  • recombinant viral vectors containing coding sequences can be used to construct recombinant viral vectors containing coding sequences (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., 1989).
  • recombinant nucleic acid molecules encoding protein sequences can be used as naked DNA or in a reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (e.g., Felgner et al., Nature 337:387-8, 1989).
  • gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection (Capecchi, Cell 22:479-88, 1980). Once recombinant genes are introduced into a cell, they can be recognized by the cell's normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been attempted for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with CaPO 4 and taken into cells by pinocytosis (Chen et al., Mol. Cell Biol.
  • adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells.
  • the admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene (Curiel et al., Am. J. Respir. Cell. Mol. Biol., 6:247-52, 1992).
  • Gene transfer means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene.
  • the product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA.
  • Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cytoplasm from the plasma membrane or endosome. Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription.
  • gene therapy is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient.
  • a vector having nucleic acid sequences encoding a STE20-related kinase polypeptide in which the nucleic acid sequence is expressed only in specific tissue.
  • Methods of achieving tissue-specific gene expression are set forth in International Publication No. WO 93/09236, filed Nov. 3, 1992 and published May 13, 1993.
  • nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above.
  • Gene replacement means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
  • the proper dosage depends on various factors such as the type of disease being treated, the particular composition being used, and the size and physiological condition of the patient.
  • Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC 50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
  • Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors, and major organs can be also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder. Such measurements can be carried out. For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan, and MRI. Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.
  • Toxicity studies can also be carried out by measuring the blood cell composition.
  • toxicity studies can be carried out in a suitable animal model as follows: 1) the compound is administered to mice (an untreated control mouse should also be used); 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition, and the percent of lymphocytes versus polymorphonuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present.
  • the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness.
  • Plasma levels should reflect the potency of the drug. Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.
  • RNAs were isolated using the Guanidine Salts/Phenol extraction protocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987)) from primary human tumors, normal and tumor cell lines, normal human tissues, and sorted human hematopoietic cells. These RNAs were used to generate single-stranded cDNA using the Superscript Preamplification System (GIBCO BRL, Gaithersburg, MD; Gerard, GF et al. (1989), FOCUS 11, 66) under conditions recommended by the manufacturer. A typical reaction used 10 ⁇ g total RNA with 1.5 ⁇ g oligo(dT) 12-18 in a reaction volume of 60 ⁇ L. The product was treated with RNaseH and diluted to 100 ⁇ L with H 2 O. For subsequent PCR amplification, 1-4 ⁇ L of this sscDNA was used in each reaction.
  • PCR reactions were performed using degenerate primers applied to multiple single-stranded cDNAs.
  • the primers were added at a final concentration of 5 ⁇ M each to a mixture containing 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4 ⁇ L cDNA.
  • the cycling conditions were 94° C. for 30 s, 50° C. for 1 min, and 72° C. for 1 min 45 s for 35 cycles.
  • PCR fragments migrating between 300-350 bp were isolated from 2% agarose gels using the GeneClean Kit (Bio101), and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's protocol.
  • Colonies were selected for mini plasmid DNA-preparations using Qiagen columns and the plasmid DNA was sequenced using a cycle sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA). Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S. F. et al., J. Mol. Biol. 215: 403-10).
  • the EST reports were downloaded from National Institute for Biotechnology Information. After uncompressing the files, the program ‘report2est’ was scripted to extract the following information: 1) EST names, 2) GenBank Accession numbers, 3) GenBank gi numbers, 4) Clone Id numbers, 5) the nucleotide sequences of the ESTs 6) the organism, 7) the library name, 8) the name of the lab, and 9) the institution.
  • the output of ‘report2est’ is a file in FASTA format with all of the information listed above in the first line of each entry except the sequence, which is listed in the second line of each entry. The resulting file is formatted for BLAST using ‘pressdb’ (available as part of the ncbi tool kit).
  • the program ‘makegene’ was developed. Input to this program is a query sequence and the organism/species for which a gene is to be built. An initial search of the formatted EST database described above is performed using BLAST (blastn). Any results that contain warnings, such as polyA tails or other repeat elements, are eliminated from future queries. The program ‘blast_parse_reports’ was developed to extract the FASTA header line from the search results and the output is then filtered to extract only FASTA header lines for the desired species.
  • the initial results having been filtered for warnings and species, go into a loop in which searches against the database are repeated until no new ESTs are found.
  • the loop consists of the following steps: 1) when possible the names of both ends of the ESTs are extracted from the database by searching using the ‘Clone Id’ field or the part of the ‘EST name’ field before the .r or s postscript, 2) any ESTs that have been used as queries in previous loops are removed from the current query by the program ‘subtract’, 3) the resulting list of ESTs is used to extract the sequences from the database by the program batch_parse_fasta, 4) BLAST is run against the database using each sequence, 5) the output files from BLAST containing warnings are removed, 6) the results are filtered by species, and 7) the loop is reentered if there were new ESTs found in the previous pass through the loop.
  • the ESTs chosen by ‘makegene’ are used as input for the program ‘mpd2_cluster’ (Hide, W., Burke, J, and Davison, D. U. of Houston, unpublished) which clusters overlapping sequences.
  • the programs ‘contig’ (Kerlavage, T., TIGR, unpublished), ‘gde2mult’ and ‘gde2sing’ (Smith, S. W., et al., CABIOS 10, 671-675 (1994)), are used to make an alignment and consensus sequence of the overlapping ESTs.
  • the human STLK2 cDNA sequence is composed of two overlapping EST fragments, AA191319 and W16504, that were identified using a Smith-Waterman search of the EST database with STLK1 (MST3 GB:AF024636) as a query. The complete sequence of both clones was determined and used to generate the full-length human STL2 sequence.
  • EST clone AA191319 contains a 1327 bp insert and an ORF of 1146 bp (382 amino acids).
  • EST clone W16504 contains a 2474 bp insert (not including the poly-A tail) and an ORF of 687 bp (382 amino acids).
  • the full-length human STLK2 cDNA (SEQ ID NO. 1) is 3268 bp long. AA191319 spans positions 1-1327 and W16504 positions 743-3216. The overlap between these two clones exhibits 100% sequence identity.
  • the human STLK2 cDNA constains a 1248 bp ORF flanked by a 181 bp 5′ UTR (1-181) and a 1784 bp 3′ UTR (1433-3216) that is followed by a 52 nucleotide polyadenylated region.
  • a polyadenylation signal (AATAAA) is found at positions 3193-3198.
  • sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for STLK2. Furthermore, human STLK2, and the related SOK-1 and MST3 proteins, conserve the amino acid sequence immediately following this presumed initiating methionine.
  • a Makegene analysis generated a 3037 bp contig from approximately 44 EST sequences. Since the 3′ ESTs were not commercially available, a pair of primers (5′-CACAGAAACGGTCAGATTCAC-3′(SEQ ID NO: 42) and 5′-GATCAGGGTGACATCAAGGGAC-3′(SEQ ID NO: 43)) were derived from this region to generate PCR clone 3R21-20-6 from human fetal liver sscDNA. This clone and EST AA278967 were fully sequenced to generate the full-length STLK2 cDNA sequence.
  • AA278967 is a 837 bp EST isolated by the IMAGE consortium from cDNA made from CD20+/IgD-germinal center B cells sorted from human tonsillar cells.
  • PCR clone 3R21-20-6 was isolated from human fetal sscDNA and contains a 1116 bp insert, including a 1086 bp ORF encoding the 362 C-terminal amino acids of STLK3.
  • the full-length human STLK3 cDNA (SEQ ID NO. 2) is 3030 bp long. AA278967 spans positions 1-814 and 3R21-20-6 spans positions 464-1579. The overlap between these two clones exhibits 100% sequence identity. The remaining 1452 bp of 3′ UTR is derived from an assembly of multiple unconfirmed EST fragments.
  • the near full-length human STLK3 cDNA (SEQ ID NO.2) is 3030 bp long and consists of a 1548 bp ORF flanked by a 1476 bp 3′ UTR (1550-3025) and a 5 nucleotide polyadenylated region.
  • a polyadenylation signal begins at position 3004. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. Six copies of a “GGCCCC” repeat were observed in positions 21-67.
  • the human STLK4 cDNA sequence is composed of two overlapping EST fragments, AA297759 and AA100484, that were identified using a Smith-Waterman search of the EST database with STLK1 (MST3 GB:AF024636) as a query. The complete sequence of both clones was determined and used to generate the near full-length human STLK4 sequence.
  • AA100484 is an IMAGE consortium cDNA clone isolated from the T-84 colonic epithelium cell line. It has an insert of 3694 bp and a coding region of 1146 bp (382 amino acids). A Smith-Waterman sequence alignment against the NRN database showed this EST to be 71.4% identical to the human STE20-like kinase (GB:X99325).
  • W16504 is an IMAGE consortium clone isolated from a human fetal heart cDNA library. It has an insert length of 2474 bp (not including the poly-A tail) and a coding region of 687 bp (229 amino acids). A Smith-Waterman sequence alignment of W16504 against the NRN database showed this EST to be 69.2% identical to the human STE20-like kinase (GB:X99325).
  • the full-length human STLK2 cDNA (SEQ ID NO. 1) is 3268 bp long. AA191319 spans positions 1-1327, and W16504 positions 743-3216. The overlap between these two clones is 585 bp long with 100% sequence identity.
  • AA100484 is an IMAGE consortium cDNA clone isolated from the T-84 colonic epithelium cell line. AA100484 covers the bulk of Human STLK4 with its 3694 bp, which spans positions 146-3839 of SEQ ID NO:3. A second EST, AA297759, isolated from a Jurkat T cell cDNA library, spans positions 1-271 of the human STLK4 contig. The two ESTs overlap over a 126 bp stretch that has only one nucleotide discrepancy at position 149 (G in AA297759 and T in AA100484). A T at this position was chosen for the SEQ ID NO:3 based on sequence data generated from A100484.
  • the 5′ 145 bp of human STLK4 contains three sequencing ambiguities (N's in SEQ ID NO:3) arising from sequence errors in the GenBank entry for AA297759. Three amino acid sequence ambiguities in the N-terminus of human STLK4 are present also in SEQ ID NO:7 as a consequence of the sequence inaccuracies from the EST entry.
  • the coding region of human STLK4 is 1242 bp long (2-1243), capable of encoding a 414 amino acid polypeptide, and is followed by a 2596 nucleotide 3′ UTR (1244-3839).
  • Human STLK4 ends in a polyadenylated stretch that has 18 adenines (3840-3857).
  • a polyadenylation signal (AATAAA) is found between positions 3822-3827.
  • Targeted-PCR cloning identified one rat orthologue of human STLK4, clone 135-31-19.
  • one murine orthologue of human STLK4 was recognized in the EST database as AA 117483. None of these orthologues add additional N-terminal sequence to the human STLK4.
  • the near full-length human STLK4 cDNA (SEQ ID NO.3) is 3857 bp long and consists of a 1242 bp ORF flanked by a 2596 bp 3′ UTR (1244-3839) and an 18 nucleotide polyadenylated region. Polyadenylation signals (AATAAA) begin at positions 2181 and 3822. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • a near full-length murine STLK4 cDNA is represented in the 1773 bp EST AA117438.
  • the human STLK5 cDNA sequence is composed of four overlapping sequences, A1418298, 2R96-13-1, 3R25-45-3 and R46685.
  • AI418298 is an IMAGE consortium cDNA clone with an 895 bp insert.
  • PCR clone 2R96-13-1 was isolated from human brain sscDNA using primers 5′-CTCATCTGTACACACTTCATGG(SEQ ID NO:44) and 5′-GATTCCCACACTGTAGATGTC(SEQ ID NO:45) derived from F07734. 2R96-13-1 contains a 330 bp insert and an ORF of 330 bp (110 amino acids).
  • EST clone R46685 was identified using a Smith-Waterman search of the EST database with the C-terminus of SPS_sc (GB:U33057) as query. Sequence analysis of the 1047 bp insert identified this EST to contain an ORF of 285 bp (95 amino acids) encoding the C-terminus of human STLK5.
  • PCR clone 3R25-45-3 was isolated from human fetal brain sscDNA using primers 5′-GGCCCTCGACTACATCCACCACAT(SEQ ID NO:46) and 5′-CAACGAAACTAACACAGCATAAGG(SEQ ID NO:47) derived from 2R96-13-1 and R46685, respectively.
  • 3R25-45-3 contains a 330 bp insert and an ORF of 750 bp (250 amino acids).
  • the full-length human STLK5 cDNA (SEQ ID NO:96) is 2110 bp long and consists of a 1119 bp ORF flanked by a 229 bp 5′ UTR and a 762 bp 3′ UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for STLK5.
  • STLK5 displays a 100% match over a 41 bp stretch (position 2-42, SEQ ID NO. 97) to a human CpG island repeat (Z61277).
  • Human STLK6 was first identified in the translated EST database (AA219667) as a novel serine threonine kinase.
  • the partial human STLK6 cDNA (SEQ ID NO:98) is 2,001 bp long and consists of a 1,254 bp ORF flanked by a 75 bp 5′ UTR and a 673 bp 3′ UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiating methionine, and is believed to be the translational start site for STLK6.
  • the partial human STLK7 cDNA (SEQ ID NO: 100) is 311 bp long and consists of a 309 bp ORF. Since the coding region is open throughout the 5′ and 3′ extent of this sequence, this appears to be a partial cDNA clone lacking the N-terminal start methionine and C-terminal stop codon.
  • STLK7 shares 80% sequence identity to human SPAK (AF099989) over a 167 bp region and 50% nucleotide sequence identity to SLTK7 (SEQ ID NO. 101) over 391 nucleotides.
  • the human ZC1 cDNA sequence is composed of two overlapping PCR clones, 3R25-24-2 and R65-12-2.
  • a human ZC1 clone, 125-33-5 was first identified from a PCR screen with degenerate oligos, TRK1 and TRK4, applied to sscDNA generated from human small airway epithelial cells (Clontech). Sequence analysis of the 503 bp insert identified a 501 bp ORF (167 amino acids) with the potential to encode a novel human STK related to the C. elegans ZC504.4 gene product.
  • PCR clone 3R25-24-2 was isolated from human SNB19 glioblastoma sscDNA using primers 5′-ATGGCGAACGACTCTCCCGCGAA(SEQ ID NO:48) and 5′-ACACCAAAATCAACAAGTTTCACCTC(SEQ ID NO:49) derived from the N-terminus of a murine orthologue of ZC1 (NIK, GB:U88984) and the original human ZC1 clone 125-33-5, respectively.
  • 3R25-24-2 contains a 527 bp insert and an ORF of 519 bp (173 amino acids).
  • PCR clone R65-12-2 was isolated as follows: A Smith-Waterman search of the EST database with the C. elegans ZC504.4 gene (GB:Z50029) as a query identified a human EST (W81656) whose ORF is related to the C. elegans gene and terminates in an identical residue (Trp).
  • a primer was designed 3′ to this stop codon (5′-AGTTACAAGGAATTCCAAGTTCT(SEQ ID NO:50)) and used in a PCR reaction with a primer derived from the original human ZC1 clone 125-33-5 (5′-ATGAAGAGGAAGAAATCAAACTG(SEQ ID NO:51)) using sscDNA from human SNB19 glioblastoma as a template.
  • PCR clone R65-12-2 was identified and was found to contain a 3611 bp insert with a 3534 bp ORF encoding the C-terminal portion of human ZC1 (1178 amino acids).
  • the full-length human ZC1 cDNA (SEQ ID NO. 9) is 3798 bp long. Clone 3R25-24-2 spans positions 1-527, and clone R65-12-2 spans positions 188-3798. The overlap between these two clones exhibits 100% sequence identity.
  • the human ZC1 contains a 3717 bp ORF (17-3723) flanked by a 6 bp 5′ UTR and a 75 bp (3724-3798) 3′ UTR. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human ZC1.
  • the human ZC2 cDNA sequence is composed of four overlapping PCR clones, G75-31-17, R65-24-6, 2R28-8-1, and R99-6-10.
  • a human ZC2 clone, G75-31-17 was first identified from a PCR screen with degenerate oligos, ROS 1(5′-GCNTTYGGNGARGTNTAYGARGG(SEQ ID NO:34)) and CCK4b (5′-GCTGGATCCYTCNGGNSWCATCCA(SEQ ID NO:35)), applied to sscDNA generated from the human HLT383 primary non-small cell lung cancer tissue. Sequence analysis of the 492 bp insert identified a 492 ORF (164 amino acids) with the potential to encode a novel human STK related to the C. elegans ZC504.4 gene product.
  • PCR clone R99-6-10 was isolated as follows: A Smith-Waterman search of the EST database with C. elegans ZC504.4 gene (GB:Z50029) as a query identified two overlapping human EST fragments (AA115844 and R51245) whose ORFs were related to the C. elegans gene and terminate in an identical residue (Trp).
  • a primer was designed 3′ to the stop codon found in R51245 (5′-AGATGGACTGTACTGGGAGG(SEQ ID NO:52)) and used in a PCR reaction with a primer derived from AA115844 (5′-ACTTTGTGCAGCTCTGTGGG(SEQ ID NO:53)) using human fetal brain sscDNA as a template.
  • PCR clone R99-6-10 was identified and was found to contain a 1095 bp insert with a 930 bp ORF encoding the C-terminal portion of human ZC2 (310 amino acids).
  • PCR clone R65-24-6 was isolated from human HT29 colon cancer cell line sscDNA using primers 5′-AAGGTTATGGATGTCACAGGG(SEQ ID NO:54) and 5′-AGATGGACTGTACTGGGAGG(SEQ ID NO:52) derived from G75-31-17 and R51245, respectively.
  • the 3′ primer used in this PCR reaction misprimed between positions 1634-1653 of this gene leading to the formation of a truncated product.
  • R65-24-6 contains a 1593 bp insert and an ORF of 1593 bp (531 amino acids).
  • PCR clone 2R28-8-1 was isolated from human colon cancer cell line HT29 sscDNA using primers 5′-CTCACAAGGTTGCCAACAGG(SEQ ID NO:55) and 5′-AGTCCCCACCAGAAGGTTTAC(SEQ ID NO:56) derived from R65-24-6 and R99-6-10, respectively.
  • 2R28-8-1 contains a 1538 bp insert and an ORF of 1536 bp (512 amino acids).
  • the partial human ZC2 cDNA (SEQ ID NO. 10) is 4055 bp long. Clone G75-31-17 spans positions 1-492, clone R65-24-6 spans positions 58-1650, clone 2R28-8-1 spans positions 1466-3003 and clone R99-6-10 spans positions 2961-4055. The overlaping regions between these clones exhibit 100% sequence identity except for a single guanine (G75-31-17) to adenosine (R65-24-6) mismatch at position 280 resulting in a Glu to Lys change. Based on the presence of an acidic residue in this position in human ZC1 and ZC3 and C.
  • the human ZC2 gene contains a 3891 bp ORF (1-3891) flanked by 164 bp (3892-4055) 3′ UTR. No polyadenylation signal (AATAAA) or polyadenylated region is present in the 3′ UTR.
  • the human ZC3 cDNA sequence is composed of four overlapping PCR clones, G75-30-30, 3R33-5-3, 3R19-17-6, and R99-43-11.
  • a human ZC3 clone, G75-30-30 was first identified from a PCR screen with degenerate oligos, ROS1 and CCK4b, applied to sscDNA generated from a human HLT370 primary non-small cell lung cancer tissue. Sequence analysis of the 492 bp insert identified a 492 ORF (164 amino acids) with the potential to encode a novel human STK related to the C. elegans ZC504.4 gene product.
  • PCR clone R99-43-11 was isolated as follows: A Smith-Waterman search of the EST database with the C. elegans ZC504.4 gene (GB:Z50029) as a query identified a human EST (R54563) whose ORF is related to the C. elegans gene and terminates in an identical residue (Trp).
  • a primer was designed 3′ to the stop codon found in R54563 (5′-TCAGGGGTCAGAGGTCACG(SEQ ID NO:57)) and used in a PCR reaction with a primer derived from the 5′ end of R54563 (5′-CCCAAACCCTACCACAAATTC(SEQ ID NO:58)) using sscDNA from human fetal brain as a template.
  • PCR clone R99-43-11 was identified and was found to contain a 719 bp insert with a 564 bp ORF encoding the C-terminal portion of human ZC3 (188 amino acids).
  • PCR clone 3R19-17-6 was isolated from human A549 lung cancer cell line sscDNA using primers 5′-CCCCCGGGAAACGATGACCA and 5′-AGCCGCTGCCCCTCCTCTACTGT derived from G75-30-30 and R99-43-11, respectively.
  • the 3′ primer used in this PCR reaction misprimed leading to the formation of a truncated product.
  • 3R19-17-6 contains a 1172 bp insert and an ORF of 1170 bp (390 amino acids).
  • PCR clone 3R33-5-3 was isolated from human A549 lung cancer cell line sscDNA using primers 5′-ACCGCAACATCGCCACCTACTAC(SEQ ID NO:61) and 5′-CTCGACGTCGTGGACCACC(SEQ ID NO:62) derived from G75-30-30 and 3R19-17-6, respectively.
  • 3R33-5-3 contains a 2465 bp insert and an ORF of 2463 bp (821 amino acids).
  • the full-length human ZC3 cDNA (SEQ ID NO. 11) is 4133 bp long. Clone G75-30-30 spans positions 1-483, clone 3R33-5-3 spans positions 134-2598, clone 3R19-17-6 spans positions 2356-3512 and clone R99-43-11 spans positions 3415-4133. The overlaps between these clones exhibit 100% sequence identity.
  • the human ZC3 gene contains a 3978 bp ORF (1-3978) flanked by a 152 bp 3′ UTR (3979-4133). No polyadenylation signal (AATAAA) or polyadenylated region is present in the 3′ UTR.
  • the human ZC4 cDNA sequence represented by PCR fragment 3R25-27-1, was first identified in the human genomic cosmid 82J11 (GB:Z833850) containing exon sequences that displayed strong homology to the ZC504.4 C. elegans gene.
  • PCR clone 3R25-27-1 was isolated from human fetal liver sscDNA and primers 5′-CAATGTTAACCCACTCTATGTCTC(SEQ ID NO:63) and 5′-AGTTTGCCGATGTTTTTCTTTTC(SEQ ID NO:64) derived from a potential ORF (positions 25729-25852) from the 82J11 cosmid and from an EST (R98571) encoding the C-terminus of the human ZC4 gene, respectively.
  • the partial human ZC4 cDNA (SEQ ID NO.12) is 1459 bp long and consists of a 1047 bp ORF (2-1048) flanked by a 411 bp (1049-1459) 3′ UTR region. No polyadenylation signal (AATAAA) or polyadenylated region is present in the 3′ UTR.
  • the N-terminal coding sequence for ZC4_h was extended by building a contiguous DNA sequence of 233,137 bp containing Z83850 and four other sequences: cU84B10 and cU230B10 (from the Sanger Human Genome Sequencing Project) and Z97356 and Z69734 (available from the National Institute for Biotechnology Information. The position of each sequence in the contig is represented in the table below. Accession Length Start End cUS4B10 43273 0 43273 Z97356 21848 43171 65018 Z69734 37077 63073 100149 cU230B10 11841 88416 100256 Z83850 132981 100156 233137
  • the resulting human ZC4 coding sequence (SEQ ID NO:104) is 3,681 bp long (excluding the stop codon) and encodes for a STE20 kinase of 1227 amino acids.
  • the human KHS2 cDNA sequence is composed of four overlapping clones, 3R25-51-2, 3R16-34-2, 3R16-31-2, and T79916.
  • a human KHS2 clone AA250855
  • KHS1 GB:U77129
  • Sequence analysis of the 1112 bp insert identified a 618 bp ORF (206 amino acids) with the potential to encode a novel STK related to the human KHS1 gene product.
  • AA250855 as a query
  • a second EST (AA446022) was found whose sequence was shown to contain the initiator methionine for human KHS2 based on a comparison with KHS1.
  • PCR clone 3R25-51-2 was isolated from human testicular cancer sscDNA using primers 5′-CCGCCATGAACCCCGGCTT(SEQ ID NO:65) and 5′-CGATTGCCAAAGACCGTGTCA(SEQ ID NO:66) derived from AA446022 and AA250855, respectively.
  • 3R25-51-2 contains an 850 bp insert and an ORF of 849 bp (283 amino acids).
  • EST clone, T79916, was identified using a Smith-Waterman search of the EST database with the C-terminus of KHS1 (GB:U77129) as a query. Sequence analysis of the 2107 bp insert identified this EST to contain an ORF of 345 bp (115 amino acids disrupted by a single stop codon) encoding the C-terminus of human KHS2, followed by 1762 bp 3′ UTR.
  • PCR clone 3R16-34-2 was isolated from human testis sscDNA using primers 5′-AGAAGTTGCAGCTGTTGAGAGGA(SEQ ID NO:67) and 5′-TATGGCCCGTGTAAGGATTTC(SEQ ID NO:68) derived from AA250885 and T79916, respectively.
  • 3R16-34-2 contains an 1516 bp insert and an ORF of 1128 bp (376 amino acids).
  • PCR clone 3R16-31-2 was isolated from normal human colon sscDNA using primers 5′-GTGCCAGAAGTGTTGTGTTGTAA(SEQ ID NO:69) and 5′-TATGGCCCGTGTAAGGATTTC(SEQ ID NO:68) derived from EST T79916.
  • 3R16-31-2 contains a 728 bp insert and an ORF of 669 bp (223 amino acids). This clone lacked the stop codon present within EST T79916 (postion 2662 in the KHS2 sequence).
  • the full-length human KHS2 cDNA (SEQ ID NO.17) is 4023 bp long. Clone 3R25-51-2 spans positions 1-855, clone AA250885 spans positions 336-923, clone 3R16-34-2 spans positions 545-2061, and clone T79916 spans positions 1917-4023. The overlaping regions between these clones exhibit 100% sequence identity, except for 4 nucleotide differences, two of which are silent, a third corrects the internal stop codon at position 2662, and the fourth at position 247 (T to C change) results in a Pro to Leu change.
  • the human KHS2 cDNA contains a 2682 bp ORF (6-2687) flanked by a 5 bp (1-5) 5′UTR and a 1336 bp (2688-4023) 3′ UTR.
  • a potential polyadenylation signal (AATAAA) is found at positions 4008-4013. No polyadenylated region is present in the 3′ UTR.
  • the sequence flanking the first ATG is in a poor context for translational initiation, however, a 134 bp 5′UTR sequence from EST AA446022 did not reveal any additional ATG's and displayed two in-frame stop codons 5′ to the putative start ATG for human KHS2.
  • the human SULU1 cDNA sequence is composed of three overlapping clones, N40091, 2R90-1-1 and R90907.
  • a human SULU1 clone, N40091 was first identified using a Smith-Waterman search of the EST database with the C. elegans SULU gene (GB: U32275) as a query. Sequence analysis of the 1321 bp insert identified a 906 bp ORF (302 amino acids) with the potential to encode a novel human STK related to the C. elegans SULU gene product.
  • EST clone R90907 was first identified using a Smith-Waterman search of the EST database with the 3′ end of the C. elegans SULU gene (GB: U32275) as a query. Sequence analysis of the 1647 bp insert identified a 578 bp ORF (192 amino acids) with the potential to encode the C-terminus of the human SULU1 gene product.
  • PCR clone 2R90-1-1 was isolated from human HT29 colon cancer cell sscDNA using primers 5′-TATTGAATTGGCGGAACGGAAG(SEQ ID NO:70) and 5′-TTGTTTTGTGCTCATTCTTTGGAG(SEQ ID NO:71) derived from N40091 and R90907, respectively.
  • 2R90-1-1 contains a 1625 bp insert and an ORF of 1623 bp (541 amino acids).
  • the full-length human SULU1 cDNA (SEQ ID NO.19) is 4177 bp long Clone N40091 spans positions 1-1321, clone 2R90-1-1 spans positions 1048-2671, and clone R90907 spans positions 2531-4177. The overlaping regions between these clones exhibit 100% sequence identity.
  • the human SULU1 cDNA contains a 2694 bp ORF (415-3108) flanked by a 414 bp (1-414) 5′UTR and a 1069 bp (3109-4177) 3′ UTR followed by a 19 nucleotide polydenylated region.
  • a potential polyadenylation signal (AATAAA) is found at positions 4164-4169. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human SULU1.
  • the murine SULU3 cDNA sequence is represented by PCR fragment 2R92-1-6.
  • a murine SULU3 clone, G83-4-5 was first identified from a PCR screen with degenerate oligos, CCK4c and CCK4b, applied to sscDNA generated from murine day-12 embryos. Sequence analysis of the 473 bp insert identified a 471 ORF (157 amino acids) with the potential to encode a novel human STK related to the C. elegans SULU gene (GB: U32275) product. The antisense strand of G83-4-5 is identical at the nucleic acid level to the 5′UTR of the murine etsl protooncogenic transcription factor (GB:X53953). This homology is likely the result of a cloning artifact attached to the 5′-end of the database entry for murine ets1.
  • PCR clone 3R19-17-6 was isolated from human A549 cell sscDNA using primers 5′-CCCCCGGGAAACGATGACCA(SEQ ID NP:59) and 5′-AGCCGCTGCCCCTCCTCTACTGT(SEQ ID NO:60) derived from G75-30-30 and R99-43-11, respectively.
  • the 3′ primer used in this PCR reaction misprimed leading to the formation of a truncated product.
  • 3R19-17-6 contains a 1172 bp insert and an ORF of 1170 bp (390 amino acids).
  • PCR clone 2R92-1-6 was isolated from murine d8 embryo sscDNA using primers 5′-ACCGCAACATCGCCACCTACTAC(SEQ ID NO:61) and 5′-GATTGCTTTGTGCTCATTCTTTGG(SEQ ID NO:72) derived from the 5′ UTR of the etsl gene and the human EST AA234623, respectively.
  • the latter (shown herein) encodes the C-terminus of human SULU3.
  • 2R92-1-6 contains a 2249 bp insert and an ORF of 2244 bp (748 amino acids).
  • the partial murine SULU3 cDNA (SEQ ID NO.21) is 2249 bp long and consists of a 2244 bp ORF (6-2249) flanked by a 5 bp (1-5) 5′UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for murine SULU3.
  • the human SULU3 cDNA sequence is composed of two overlapping clones, 2R90-22-1 and AA234623.
  • PCR clone 2R90-22-1 was isolated from human SKMel128 melanoma cell line sscDNA using primers 5′-TATTGAATTGGCGGAACGGAAG(SEQ ID NO:70) and 5′-TTGTTCTAAGAGTGCCCTCCG(SEQ ID NO:73) derived from the murine SULU3 2R92-1-6 clone and from AA234623, respectively.
  • 2R92-1-6 contains a 1897 bp insert and an ORF of 1896 bp (632 amino acids).
  • the partial human SULU3 cDNA (SEQ ID NO.20) is 3824 bp long. Clone 2R90-22-1 spans positions 1-1897 and clone AA234623 spans positions 1173. The overlaping region between these clones exhibits 100% sequence identity.
  • the human SULU3 cDNA contains a 2358 bp ORF (2-2359) flanked by a 1465 bp (2360-3824) 3′ UTR followed by a 19 nucleotide polydenylated region.
  • a potential polyadenylation signal (AATAAA) is found at positions 2602-2607. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • the human GEK2 cDNA sequence is composed of three overlapping clones, AA459448, 3R25-48-1 and GEK2_h#3.
  • a human GEK2 clone, AA459448 was first identified using a Smith-Waterman search of the EST database with the human SLK gene (GB: AB002804) as a query. Sequence analysis of the 1286 bp insert identified a 1227 bp ORF (409 amino acids) with the potential to encode the N-terminus of a novel human STK related to the human SLK gene product. An additional Smith-Waterman search using the C-terminus of the SLK gene as a query yielded three additional EST's, AA323687, AA380492 and AA168869, that encode the C-terminal region of human GEK2.
  • PCR clone 2R98-41-17 was isolated from human testis sscDNA using primers 5′-AAGACCATGCCGTGCGCCG(SEQ ID NO:74) and 5′-ATTCCTTCAGGTTCTGGTTATGG(SEQ ID NO:75) derived from AA323687 and from AA380492, respectively. 2R98-41-17 contains a 851 bp insert and an ORF of 849 bp (283 amino acids).
  • PCR clone GEK2_h#3 was isolated from human sscDNA made from the H23 tumor cell line using primers 5′-GCAGCAAGTGGAGAAGATGG(SEQ ID NO: 109) and 5′-GGAAGCATCCCCAGAGCTGTAG(SEQ ID NO: 110) derived from the sequence of clone 3R25-48-1 and from the 3′ end of murine LOK (GB:D89728), respectively.
  • GEK2_h#3 contains a 1042bp insert and an ORF of 1041 bp (347 amino acids).
  • the full-length human GEK2 cDNA (SEQ ID NO:106) is 2962 bp long. Clone AA459448 spans positions 1-1286, clone 3R25-48-1 spans positions 1100-2449 and clone GEK2_h#3 spans positions 1920-2962. The overlapping regions between these clones exhibit 100% sequence identity.
  • the human GEK2 cDNA contains a 2904 bp ORF (59-2962) flanked by a 58 bp (1-58) 5′UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human GEK2.
  • the human PAK4 cDNA sequence is represented by clone SNB2#1.
  • a human PAK4 clone, R88460 was first identified using a Smith-Waterman search of the EST database with the human PAK gene (GB: U24152) as a query. Sequence analysis of the 2332 bp insert identified a 930 bp ORF (310 amino acids) with the potential to encode the C-terminus of a novel human STK related to the human PAK gene product.
  • cDNA clone SNB2#1 was isolated from human glioblastoma cell line SNB75 cDNA library using a probe derived from R88460.
  • SNB2#1 contains a 3604 bp insert and an ORF of 2043 bp (681 amino acids).
  • the full-length human PAK4 cDNA (SEQ ID NO.27) is 3604 bp long and consists of a 2043 bp ORF (143-2185) flanked by a 142 bp (1-142) 5′UTR and a 1419 3′ UTR followed by a 22 nucleotide polydenylated region.
  • a potential polyadenylation signal (AATTAAA) is found at positions 3582-3588.
  • the sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human PAK4.
  • the 3′ UTR of the PAK4 gene contains a GT dinucleotide repeat prone to undergo expansion based on the number of repeats found in clones SNB#1 and R88460, 32 and 23, respectively.
  • Several neurologic disorders have been correlated with the expansion of di- or tri-nucleotide repeats similar to those found in the PAK4 sequence, suggesting PAK 4 may also be a disease target and that this repeat in its 3′ UTR may serve as a diagnostic marker.
  • the full-length human PAK5 cDNA sequence is composed of two overlapping clones, H450#1-1 and SNB8#5.
  • a human PAK5 clone, RI 8825 was first identified using a Smith-Waterman search of the EST database with the human PAK4 gene as a query. Sequence analysis of the 1248 bp insert identified a 420 bp ORF (140 amino acids) with the potential to encode the C-terminus of a novel human STK related to the human PAK4 gene product.
  • cDNA clone SNB8#5 was isolated from human SNB75 cDNA library using a probe derived from R18825.
  • SNB2#1 contains a 2028 bp insert and an ORF of 1194 bp (398 amino acids).
  • the partial human PAK5 cDNA (SEQ ID NO.28) is 2028 bp long and consists of a 1194 bp ORF (2-1195) flanked by an 833 bp (1196-2028) 3′ UTR followed by a 22 nucleotide polydenylated region.
  • a potential polyadenylation signal (AATTAAA) is found at positions 2004-2010. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • Clone H460#1-1 was isolated from a human lung H460 cDNA library using a probe derived from the partial SNB2#1 cDNA clone described above. Sequence analysis of the 2526 bp insert identified a 1773 bp ORF (592 amino acids) with the potential to encode a full-length PAK5.
  • the human PAK5 cDNA (SEQ ID NO:102) is 2,806 bp long and consists of a 1,773 bp ORF flanked by a 201 bp 5′ UTR and a 833 bp 3′ UTR.
  • the sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiating methionine, and is believed to be the translational start site for PAK5.
  • PAK5 shares 99% sequence identity over 2795 bp to a recent database entry, AF005046. These sequences are presumed to be from the same gene, with minor polymorphic variations.
  • Northern blots were prepared by running 10 ⁇ g total RNA isolated from 60 human tumor cell lines (HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, SNB-19, SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, K-562, MOLT-4, HL-60, RPMI 8226, SR, DU-145, PC-3, HT-29, HCC-2998, HCT-116, SW620, Colo 205, HTC15, KM-12, UO-31, SN12C, A498, CaKil, RXF-393, ACHN, 786-0, TK-10, LOX IMVI, Malme-3M, SK-M
  • Filters were hybridized with random primed [ ⁇ 32 P]dCTP-labeled probes synthesized from the inserts of several of the STE20-related kinase genes. Hybridization was performed at 42° C. overnight in 6X SSC, 0.1% SDS, 1 ⁇ Denhardt's solution, 100 ⁇ g/mL denatured herring sperm DNA with 1-2 x 10 6 cpm/mL of 32 P-labeled DNA probes. The filters were washed in 0.1 ⁇ SSC/0.1% SDS, 65° C., and exposed on a Molecular Dynamics phosphorimager.
  • Plasmid DNA array blots were prepared by loading 0.5 ⁇ g denatured plasmid for each STE20-related kinase on a nylon membrane.
  • the [ ⁇ 32 P]dCTP labeled single stranded DNA probes were synthesized from the total RNA isolated from several human immune tissue sources or tumor cells (thymus, dendrocytes, mast cells, monocytes, B cells (primary, Jurkat, RPMI8226, SR), T cells (CD8/CD4+, TH 1, TH2, CEM, MOLT4), K562 (megakaryocytes). Hybridization was performed at 42° C.
  • ZC1, ZC2, and ZC3 RNA expression was analyzed by quantitative PCR from multiple human normal tissues, cultured primary epithelial and endothelial cells, and tumor cell lines. The results are summarized in Tables 1 and 2, with relative expression values ranging from 0 (undetectable) to 23 (very strong). An “x” refers to sample not tested. ZC1, ZC2, and ZC3 were all expressed at very low levels in most normal human tissues, however ZC1 and ZC2 were more abundant in cultured epithelial cells and ZC3 in normal kidney and breast tissue.
  • STLK2 is widely expressed; the highest expression levels were found in placenta, spleen and PBL.
  • STLK4 is also widely expressed in normal tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood lymphocytes. STLK4 was also detected in Jurkat T cells.
  • ZC1 is highly overexpressed in the following human cancer cell lines: HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62 (lung); OVCAR-3, OVCAR-4, OVCAR-5 (ovary); SNB-19, U251, SF-268, SF-295, SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3 (prostate); HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12 (colon); UO-31, CAKi-1, RXF-393, 786-0, TK-10 (renal); LOXIMVI, Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); and MCF-7, MCF-7/ADR, H
  • ZC2 is expressed in brain and testis. It is highly overexpressed in the following human cancer cell lines: TK-10 (renal); SK-MEL-28, UACC-62 (melanoma); T47D (breast).
  • SULU1 is overexpressed in the following human cancer cell lines: HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62 (lung); OVCAR-3, OVCAR-4, OVCAR-5, SK-OV-3 (ovary); SNB-19, U251, SF-268, SF-295, SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3 (prostate); HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12 (colon); UO-31, CAKi-1, RXF-393, 786-0, TK-10 (renal); LOX, IMVI, Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); MCF-7, M
  • SULU3 showed a broad pattern of expression in the normal tissue panel of RNAs.
  • GEK2 was expressed in spleen, thymus and testis. Expression was high in the cell lines RBL-2H3 and H441.
  • PAK4 was expressed in the normal tissues: brain, testis and prostate, and in the human cancer cell lines: HNCI-H23 (lung); OVCAR-3 (ovary); SNB-19, U251 (CNS); RPMI-8226 (leukemia); DU-145 (prostate); COLO-205, HCT-15 (colon).
  • PAK5 showed weak expression levels in the normal tissues: brain, testes, bladder, colon, adrenal medulla, spleen, fetal liver, breast, cerebral cortex, cerebellum, thymus, salivary gland, lung, stomach, duodenum, uterus, prostate, skeletal muscle and placenta.
  • PAK5 was overexpressed in the human cancer cell lines: HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62 (lung); OVCAR-3, OVCAR-4, OVCAR-5, SK-OV-3 (ovary); SNB-19, U251, SF-268, SF-295, SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3 (prostate); HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12 (colon); UO-31, CAKi-1, RXF-393, 786-0, TK-10 (renal); LOXIMVI, Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); MCF-7, MCF-7/ADR, H
  • K350,351A (Lys at aa positions 350 and 351 changed to Ala).
  • Proteins in SDS PAGE are transferred to immobilon membrane.
  • the washing buffer is PBST (standard phosphate-buffered saline pH 7.4+0.1% triton x 100).
  • Blocking and antibody incubation buffer is PBST +5% milk.
  • Antibody dilutions varied from 1:1000 to 1:2000.
  • STE20-related protein kinase GST fusion protein immunogens and their specificity in recognizing endogenous protein by Western blots or immunoprecipitations.
  • Protein domain Aa positions West.
  • IP ZC1 Coiled-coil/pro/B/C 350-867 Y Y ZC1 B 615-732 Y Y ZC2 Coiled-coil/pro/B 348-762 ND ND ZC2 B 658-762 Y Y PAK4 Nterm 252-426 ND ND PAK4 Kinase/Cterm 350-681 ND Y PAK5 A/Nterm 53-330 ND ND PAK5 A/Nterm 53-309 ND ND
  • the 50 kD STLK2 protein was expressed highly in several hematopoietic cell lines including Jurkat, pGL10, Ramos, A20, WEHI-231, K562, HEL and freshly isolated thyrnocytes from C57/BL6 mice. High levels of STLK2 expression were also detected in several tumor cell lines including Calu6, Colo205, LS 180, MDAM231 and A549.
  • the 160 kD ZC1 protein was detected in Jurkat T cells, Colo205, HCT116, RIE-1, 293T, MDAMB231, and SK-MEL28.
  • the pcDNA expression plasmids (10 ⁇ g DNA/100 mm plate) containing the STE20-related kinase constructs are introduced into 293 cells with lipofectamine (Gibco BRL). After 72 hours, the cells are harvested in 0.5 mL solubilization buffer (20 mM HEPES, pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/mL aprotinin).
  • solubilization buffer (20 mM HEPES, pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/mL
  • ZC1 Assay buffer 20 mM Tris pH 7.4, 200 mM NaCl, 0.5 mM DTT, 3 mM MgCl2, 0.3 mM MnCl2, 100 ⁇ M 32 P ⁇ ATP.
  • SULU1 Assay buffer This buffer is identical to that for ZC1, except for 5 mM MgCl2. Under these conditions, other STE20 family members (PAK4, ZC1) were inhibited for autophosphorylation and required reducing the [Mn] to ⁇ 0.3 mM for an efficient autophosphorylation reaction.
  • Substrates MBP, phosvitin, or ⁇ -casein at 0.28 mg/mL.
  • PAK4, PAK5 Assay Buffer 20 mM Hepes pH 7.2, 130 mM KCl, 10 mM MgCl2, 1 mM NaF, 20 mM B-glycerolphosphate, 0.5 mM DTT, 50 ⁇ M ATP, 0.5 ⁇ Ci 32 P ⁇ ATP.
  • STLK2 Assay buffer Similar to that described above, except for the inclusion of 5 mM MgCl 2 , 5 mM MnCl 2 and 5 ⁇ Ci 32 P ⁇ ATP.
  • NIH3T3 fibroblasts displaying normal morphology (flat, non-refractile cellular morphology), as well as low rates of spontaneous transformation, were used in transformation assays.
  • NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL) and kept in an humidified incubator at 37° C. and 5% CO 2 .
  • Foci were scored after 3 weeks by fixing 10 min in 10% methanol, 10% acetic acid for 10 min, followed by staining with 0.4% (w/v) crystal violet in 10% methanol for 10 min, and washing with deionized water and drying at room temperature.
  • ZC1 is constitutively active as a full-length kinase when expressed either in vitro (TNT rabbit reticulocyte system) or in NIH 3T3, 293T, or H1299 tissue culture cells.
  • the endogenously expressed kinase is also active when immunoprecipitated from carcinoma cell lines.
  • RE/AP is a composite in the IL-2 gene promoter containing both a NF ⁇ B-like site and an AP-1 site.
  • Optimal activation of both RE/AP-luciferase and NF ⁇ B-luciferase reporter genes in Jurkat T cells requires signals generated from stimulation of both T cell receptor and the costimulator receptor CD28.
  • Cotransfection of wild-type ZC1 with either the RE/AP-luciferase or the NF ⁇ B-luciferase reporter results in the activation of RE/AP or NF ⁇ B when costimulated with the anti-T cell receptor monoclonal antibody or the pharmacological reagents PMA and ionomycin that bypass proximal T cell receptor. No activation was seen when costimulated with an anti-CD28 monoclonal antibody.
  • PAK5 Design of Specific Peptide Substrates
  • PAK5 Activation Loop Peptides as Kinase Substrates SEQ Peptide # Kinase Sequence Aa ID Kinase substrate 1 PAK5 (C)RRKSLVGTPYWMA 471-485 120 PAK5 yes PE 2 PAK5 (C)RRKSLVG T PYWMA 471-485 120 PAK5 yes PE 3 PAK5 (C)RRK S LVGTPYWMA 471-485 120 PAK5 no PE 4 KHS1 KRKSFIGTPYWMAPE 171-185 122 PAK5 yes 5 STLK2 KRNTFVGTPFWMA 175-189 123 PAK5 poor PE 6 SULU1 PANSFVGTPYWMAPE 174-188 124 PAK5 poor 7 ZC1 RRNTFIGTPYWMAPE 184-198 125 PAK5 poor 8 ZC1 RRNTFIG T PYWMAPE 184-198 126 PAK5 poor 9 STLK4 RNKVRKTFVGTPCWM 66-83 127 PAK5
  • PAK5 Equally well as MBB 2
  • PAK5 S is the site of phosphorylation 4 KHS1 Similar to peptide 1 5 STLK2 6 SULU1 7 ZC1 8 ZC1 Better than 7 9 STLK4 10
  • PAK5 Same Km as phosph. by PAK5
  • PAK4 and PAK5 Interaction with Cdc42
  • PAK 4 interacts with CDC42 small G-protein but not Rac, RhoA, or Ras as determined by co-transfection of recombinant genes and detection by kinase assays.
  • PAK5 also interacts with Cdc42. Coding sequences of activated alleles of small G proteins (ras, Cdc42, Rac, Rho) tagged with a Myc epitope were transiently expressed in 293T cells, various alleles of 35S-labeled PAK5 tagged with HA epitope were expressed in vitro with the reticulocyte (TNT) system.
  • TNT reticulocyte
  • STE20 protein kinases STLK3, STLK4, ZC1, ZC2, ZC3, KHS2, SULU1, PAK4, and PAK5 were mapped using the GeneBridge 4 Radiation Hybrid Panel, RH02.05 (Research Genetics).
  • the GeneBridge 4 Panel consists of 91 hybrid panel samples, in addition to one human positive control (HFL), and one hamster negative control (A23).
  • HFL human positive control
  • A23 hamster negative control
  • the standard reaction conditions used to test and conduct PCR reactions using the GeneBridge 4 Panel are available from Research Genetics.
  • Oligonucleotide sequences (all 5′ to 3′) used for PCR mapping were: STLK3: CTCCCATTTCCTAGCAAAATCA, (SEQ ID NO:128) AGAGGCAGTATTGTCAGATGTA (SEQ ID NO:129) STLK4: CCACACATGCGTATCTCTGTTG, (SEQ ID NO:130) TTGCTAGAATTCACATCAGGTACA (SEQ ID NO:131) ZC1: ATCCCTGGATCACACTGCTTCT, (SEQ ID NO:132) CAAGGTGTTCTTTGCCTCTGTT (SEQ ID NO:133) ZC2: AGATGGACTGTACTGGGAGGG, (SEQ ID NO:134) AGAAGAGCACTTGGCACTTATC (SEQ ID NO:135) ZC3: CATCATGAACTGGTGACGGG, (SEQ ID NO:136) CCAGTGAAATCAAACCAGTAAAA (SEQ ID NO:137) SULU1: CAAAACCTGGCCGTCTCTCTCTC
  • Non-small cell lung cancer 2q31-q32, 1 of 50
  • Non-small cell lung cancer 12q24.1-24.3, 2 of 50.
  • non-small cell lung cancer 17p, 1_of — 50
  • PAK5_h 19q13.2-q13.3 (Sugen)
  • Nylon membranes were purchased from Boehringer Mannheim. Denaturing solution contains 0.4 M NaOH and 0.6 M NaCl. Neutralization solution contains 0.5 M Tris-HCL, pH 7.5 and 1.5 M NaCl. Hybridization solution contains 50% formamide, 6 ⁇ SSPE, 2.5 ⁇ Denhardt's solution, 0.2 mg/mL denatured salmon DNA, 0.1 mg/mL yeast tRNA, and 0.2% sodium dodecyl sulfate. Restriction enzymes were purchased from Boehringer Mannheim. Radiolabeled probes were prepared using the Prime-it 11 kit by Stratagene. The beta actin DNA fragment used for a probe template was purchased from Clontech.
  • Genomic DNA was isolated from 20 different tumor cell lines: MCF-7, MDA-MB-231, Calu-6, A549, HCT-15, HT-29, Colo 205, LS-180, DLD-1, HCT-116, PC3, CAPAN-2, MIA-PaCa-2, PANC-1, AsPc-1, BxPC-3, OVCAR-3, SKOV3, SW 626 and PA-1, and from two normal cell lines: human mammary epithelial cells and human umbilical vein endothelial cells.
  • a 600 base pair fragment (EcoR I-Sac I) of the PAK5 gene was used as a template for a radiolabeled DNA probe which was hybridized to the blots at 42° C. for 48 hours in hybridization solution using standard methods (supra).
  • the blots were exposed to a phosphorimager screen for 4 days, then scanned and analyzed using a Molecular Dynamics Storm 840 phosphorimager.
  • the relative mass and gene copy number values of the PAK5 DNA fragments were calculated from the band density values obtained.
  • the blots were re-hybridized with a radiolabeled probe copied from a fragment of human beta actin DNA and developed as above to confirm the sample mass loading equivalency.
  • the PAK5 gene was determined to exhibit 3-fold amplification compared to the normal DNA copy number in PANC-1 (pancreatic epithelioid carcinoma) and OVCAR-3 (ovarian adenocarcinoma) human cell lines, and approximately 2 times the normal copy number in the BxPC-3 (primary pancreatic adenocarcinoma) human cell line.
  • Phage display provides a method for isolating molecular interactions based on affinity for a desired bait. cDNA fragments cloned as fusions to phage coat proteins are displayed on the surface of the phage. Phage(s) interacting with a bait are enriched by affinity purification and the insert DNA from individual clones is analyzed.
  • Protein domains to be used as baits were generated as C-terminal fusions to GST and expressed in E. coli .
  • Peptides were chemically synthesized and biotinylated at the N-terminus using a long chain spacer biotin reagent.
  • bound phage was eluted in 100 ⁇ L of 1% SDS and plated on agarose plates to obtain single plaques.
  • the phage display data suggest potential interactions of SULU3 with SLK and SULU1 with GEK2 through their coiled-coil domains. Therefore two members of the SULU subfamily of STE20 kinases interact with two members of a separate STE20 family, the prototype being SLK.
  • the invention is meant to also cover the final formulation formed by the combination of these excipients.
  • the invention includes formulations in which one to all of the added excipients undergo a reaction during formulation and are no longer present in the final formulation, or are present in modified forms.

Abstract

The present invention relates to the nucleic acid molecules encoding an STE20-related family of novel protein kinases, ZC1, ZC2, ZC3, ZC4, STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, KHS2, SULU1, SULU3, GEK2, PAK4 and PAK5, segments and domains thereof, as well as various methods useful for the diagnosis and treatment of various kinase-related diseases and conditions. Mammalian nucleic acid molecules encoding these kinases are particularly disclosed, and more specifically human sources of these nucleic acids are disclosed.

Description

    RELATED APPLICATIONS
  • This application is a divisional of U.S. application Ser. No. 09/688,188 filed Oct. 16, 2000, which is a divisional of U.S. application Ser. No. 09/291,417, filed Apr. 13, 1999, which in turn claims priority to U.S. Provsional Application Ser. No. 60/081,784, filed Apr. 14, 1998. This application claims only subject matter disclosed in the parent application and therefore presents no new matter. [0001]
  • The instant application contains a “lengthy” Sequence Listing which has been submitted via triplicate CD-R in lieu of a printed paper copy, and is hereby incorporated by reference in its entirety. Said CD-R are labeled “CRF”, '[0002] opy 1 ” and “Copy 2”, respectively, and each contains only one identical 329 Kb file (38602329.APP).
    • FIELD OF THE INVENTION
  • The present invention relates to novel kinase polypeptides, nucleotide sequences encoding the novel kinase polypeptides, as well as various products and methods useful for the diagnosis and treatment of various kinase-related diseases and conditions. [0003]
    • BACKGROUND OF THE INVENTION
  • The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be or to describe prior art to the invention. [0004]
  • Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure and function. [0005]
  • The best characterized protein kinases in eukaryotes phosphorylate proteins on the hydroxyl moiety of serine, threonine and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylating serines and threonines, and those specific for phosphorylating tyrosines. Some kinases, referred to as “dual specificity” kinases, are able to phosphorylate on tyrosine as well as serine/threonine residues. [0006]
  • Protein kinases can also be characterized by their location within the cell. Some kinases are transmembrane receptor-type proteins capable of directly altering their catalytic activity in response to the external environment such as the binding of a ligand. Others are non-receptor-type proteins lacking any transmembrane domain. They can be found in a variety of cellular compartments from the inner surface of the cell membrane to the nucleus. [0007]
  • Many kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state. Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway. [0008]
  • Protein kinases are one of the largest families of eukaryotic proteins with several hundred known members. These proteins share a 250-300 amino acid domain that can be subdivided into 12 distinct subdomains that comprise the common catalytic core structure. These conserved protein motifs have recently been exploited using PCR-based cloning strategies leading to a significant expansion of the known kinases. [0009]
  • Multiple alignment of the sequences in the catalytic domain of protein kinases and subsequent parsimony analysis permits the segregation of related kinases into distinct branches or subfamilies including: tyrosine kinases, cyclic-nucleotide-dependent kinases, calcium/calmodulin kinases, cyclin-dependent kinases and MAP-kinases, serine-threonine kinase receptors, and several other less defined subfamilies. [0010]
    • SUMMARY OF THE INVENTION
  • Through the use of a targeted PCR cloning strategy and of a “motif extraction” bioinformatics script, mammalian members of the STE20-kinase family have been identified as part of the present invention. Multiple alignment and parsimony analysis of the catalytic domain of all of these STE20-family members reveals that these proteins cluster into 9 distinct subgroups. Classification in this manner has proven highly accurate not only in predicting motifs present in the remaining non-catalytic portion of each protein, but also in their regulation, substrates, and signaling pathways. The present invention includes the partial or complete sequence of new members of the STE20-family, their classification, predicted or deduced protein structure, and a strategy for elucidating their biologic and therapeutic relevance. [0011]
  • Thus, a first aspect of the invention features an isolated, enriched, or purified nucleic acid molecule encoding a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. [0012]
  • By “isolated” in reference to nucleic acid is meant a polymer of nucleotides conjugated to each other, including DNA and RNA, that is isolated from a natural source or that is synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes. [0013]
  • By the use of the term “enriched” in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term “significant” is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as [0014] pUC 19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term “purified” in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10[0015] 6-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • By a “kinase polypeptide” is meant 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; 250 (preferably 255, more preferably 260, most preferably 270) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; 27 (preferably 30, more preferably 40, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO: 18; 16 (preferably 20, more preferably 25, most preferably 35) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO: 103 or the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO: 101, or the corresponding full-length amino acid sequence; 78 (preferably 80, more preferably 85, most preferably 90) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:107 or functional derivatives thereof as described herein. For sequences for which the full-length sequence is not given, the remaining sequences can be determined using methods well-known to those in the art and are intended to be included in the invention. In certain aspects, polypeptides of 100, 200, 300 or more amino acids are preferred. The kinase polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained, not to include fragments containing only amino acids 1-22 of SEQ ID NO: 13 or only amino acids 1-33 of SEQ ID NO:107. [0016]
  • The amino acid sequence will be substantially similar to the sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or fragments thereof, not to include fragments consisting only of the amino acid sequences 1-22 of SEQ ID NO:13 or 1-33 of SEQ ID NO:107. A sequence that is substantially similar to the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107. [0017]
  • By “identity” is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. “Gaps” are spaces in an alignment that are the result of additons or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Blast (Altschul, et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul, et al. (1990) J. mol. biol. 215:403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol. 147:195-197). [0018]
  • In preferred embodiments, the invention features isolated, enriched, or purified nucleic acid molecules encoding a kinase polypeptide comprising a nucleotide sequence that: (a) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO: 105, or SEQ ID NO: 107; (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecule of (a) and encodes a naturally occurring kinase polypeptide; (d) encodes a kinase polypeptide having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 107, except that it lacks one or more, but not all, of the following segments of amino acid residues: 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489 or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ ID NO:7, 1-22, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO: 18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281, 284-430, 431-640, 641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO: 107; (e) is the complement of the nucleotide sequence of (d); (f) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31; SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 from amino acid residues 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489, or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ ID NO:7, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281, 282-430, 431-640, 641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO: 107; (g) is the complement of the nucleotide sequence of (f); (h) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, except that it lacks one or more of the domains selected from the group consisting of a N-terminal domain, a catalytic domain, a C-terminal domain, a coiled-coil structure region, a proline-rich region, a spacer region, an insert, and a C-terminal tail; or (i) is the complement of the nucleotide sequence of (h). [0019]
  • The term “complement” refers to two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. A nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence. [0020]
  • The term “domain” refers to a region of a polypeptide which contains a particular function. For instance, N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal. Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains. [0021]
  • The term “N-terminal domain” refers to the extracatalytic region located between the initiator methionine and the catalytic domain of the protein kinase. The N-terminal domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the N-terminal boundary of the catalytic domain. Depending on its length, the N-terminal domain may or may not play a regulatory role in kinase function. An example of a protein kinase whose N-terminal domain has been shown to play a regulatory role is PAK65, which contains a CRIB motif used for Cdc42 and rac binding (Burbelo, P. D. et al. (1995) J. Biol. Chem. 270, 29071-290740). [0022]
  • The N-terminal domain spans amino acid residues 1-21 of the sequence set forth in SEQ ID NO:5, amino acid residues 1-31 of the sequence set forth in SEQ ID NO:6, amino acid residues 1-22 of the sequence set forth in SEQ ID NO: 13, amino acid residues 1-13 of the sequence set forth in SEQ ID NO: 18, amino acid residues 1-21 of the sequence set forth in SEQ ID NO:22, amino acid residues 1-25 of the sequence set forth in SEQ ID NO:24, amino acid residues 1-51 of the sequence set forth in SEQ ID NO:29, amino acid residues 1-25 of the sequence set forth in SEQ ID NO:31, amino acid residues 1-57 of the sequence set forth in SEQ ID NO:99, amino acid residues 1-52 of the sequence set forth in SEQ ID NO: 103, amino acid residues 1-24 of the sequence set forth in SEQ ID NO:105, or amino acid residues 1-33 of the sequence set forth in SEQ ID NO:107. [0023]
  • The term “catalytic domain” refers to a region of the protein kinase that is typically 25-300 amino acids long and is responsible for carrying out the phosphate transfer reaction from a high-energy phosphate donor molecule such as ATP or GTP to itself (autophosphorylation) or to other proteins (exogenous phosphorylation). The catalytic domain of protein kinases is made up of 12 subdomains that contain highly conserved amino acid residues, and are responsible for proper polypeptide folding and for catalysis. The catalytic domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database. [0024]
  • The catalytic domain spans amino acid residues 22-274 of the sequence set forth in SEQ ID NO:5, residues 32-308 of the sequence set forth in SEQ ID NO:6, residues 1-178 of the sequence set forth in SEQ ID NO:7, residues 23-289 of the sequence set forth in SEQ ID NO:13, residues 1-255 of the sequence set forth in SEQ ID NO:14, residues 1-255 of the sequence set forth in SEQ ID NO: 15, residues 14-273 of the sequence set forth in SEQ ID NO:18, residues 22-277 of the sequence set forth in SEQ ID NO:22, residues 1-66 of the sequence set forth in SEQ ID NO:23, residues 26-273 of the sequence set forth in SEQ ID NO:24, residues 394-658 of the sequence set forth in SEQ ID NO:29, residues 26-281 of the sequence set forth in SEQ ID NO:31, residues 1-278 of the sequence set forth in SEQ ID NO:97, residues 58-369 of the sequence set forth in SEQ ID NO:99, residues 1-103 of the sequence set forth in SEQ ID NO:101, residues 308-572 of the sequence set forth in SEQ ID NO:103, residues 25-289 of the sequence set forth in SEQ ID NO:105, or residues 34-294 of the sequence set forth in SEQ ID NO: 107. [0025]
  • The term “catalytic activity”, as used herein, defines the rate at which a kinase catalytic domain phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a phosphorylated product as a function of time. Catalytic activity can be measured by methods of the invention by holding time constant and determining the concentration of a phosphorylated substrate after a fixed period of time. Phosphorylation of a substrate occurs at the active-site of a protein kinase. The active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated. [0026]
  • The term “substrate” as used herein refers to a molecule phosphorylated by a kinase of the invention. Kinases phosphorylate substrates on serine/threonine or tyrosine amino acids. The molecule may be another protein or a polypeptide. [0027]
  • The term “C-terminal domain” refers to the region located between the catalytic domain or the last (located closest to the C-terminus) functional domain and the carboxy-terminal amino acid residue of the protein kinase. By “functional” domain is meant any region of the polypeptide that may play a regulatory or catalytic role as predicted from amino acid sequence homology to other proteins or by the presence of amino acid sequences that may give rise to specific structural conformations (i.e. coiled-coils). The C-terminal domain can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C-terminal boundary of the catalytic domain or of any functional C-terminal extracatalytic domain. Depending on its length and amino acid composition, the C-terminal domain may or may not play a regulatory role in kinase function. An example of a protein kinase whose C-terminal domain may play a regulatory role is PAK3 which contains a heterotrimeric G[0028] b subunit-binding site near its C-terminus (Leeuw, T. et al (1998) Nature, 391, 191-195).
  • The C-terminal domain spans amino acid residues 275-416 of the sequence set forth in SEQ ID NO:5, residues 309-489 of the sequence set forth in SEQ ID NO:6, residues 179-414 of the sequence set forth in SEQ ID NO:7, residues 897-1239 of the sequence set forth in SEQ ID NO:13, residues 955-1297 of the sequence set forth in SEQ ID NO:14, residues 984-1326 of the sequence set forth in SEQ ID NO:15, residues 535-894 of the sequence set forth in SEQ ID NO: 18, residues 752-898 of the sequence set forth in SEQ ID NO:22, residues 279-330 of the sequence set forth in SEQ ID NO:97, residues 370-418 of the sequence set forth in SEQ ID NO:99, or residues 873-1227 of the sequence set forth in SEQ ID NO:105. [0029]
  • The term “signal transduction pathway” refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response. The polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, [0030] SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine binding (PTB and PH) domain containing proteins), proline-rich binding proteins (SH3 domain containing proteins), nucleotide exchange factors, and transcription factors.
  • The term “coiled-coil structure region” as used herein, refers to a polypeptide sequence that has a high probability of adopting a coiled-coil structure as predicted by computer algorithms such as COILS (Lupas, A. (1996) Meth. Enzymology 266:513-525). Coiled-coils are formed by two or three amphipathic □-helices in parallel. Coiled-coils can bind to coiled-coil domains of other polypeptides resulting in homo- or heterodimers (Lupas, A. (1991) Science 252:1162-1164). Coiled-coil-dependent oligomerization has been shown to be necessary for protein function including catalytic activity of serine/threonine kinases (Roe, J. et al. (1997) J. Biol. Chem. 272:5838-5845). [0031]
  • The coiled-coil structure region spans amino acid residues 290-526 of the sequence set forth in SEQ ID NO: 13, residues 256-442 of the sequence set forth in SEQ ID NO:14, residues 256-476 of the sequence set forth in SEQ ID NO:15, residues 428-637 of the sequence set forth in SEQ ID NO:22, residues 216-425 or 540-786 of the sequence set forth in SEQ ID NO:23, residues 423-632 of the sequence set forth in SEQ ID NO:24, residues 431-640 or 755-901 of the sequence set forth in SEQ ID NO:31, residues 291-398 or 629-668 of the sequence set forth in SEQ ID NO: 105, or residues 473-724 or 725-968 of the sequence set forth in SEQ ID NO:107. [0032]
  • The term “proline-rich region” as used herein, refers to a region of a protein kinase whose proline content over a given amino acid length is higher than the average content of this amino acid found in proteins (i.e., >10%). Proline-rich regions are easily discernable by visual inspection of amino acid sequences and quantitated by standard computer sequence analysis programs such as the DNAStar program EditSeq. Proline-rich regions have been demonstrated to participate in regulatory protein -protein interactions. Among these interactions, those that are most relevant to this invention involve the “PxxP” (SEQ ID NO: 148) proline rich motif found in certain protein kinases (i.e., human PAK1) and the SH3 domain of the adaptor molecule Nck (Galisteo, M. L. et al. (1996) J. Biol. Chem. 271:20997-21000). Other regulatory interactions involving “PxxP” (SEQ ID NO: 148) proline-rich motifs include the WW domain (Sudol, M. (1996) Prog. Biochys. Mol. Bio. 65:113-132). [0033]
  • The proline-rich region spans amino acid residues 527-640 of the sequence set forth in SEQ ID NO:13, residues 443-626 of the sequence set forth in SEQ ID NO:14, residues 477-680 of the sequence set forth in SEQ ID NO:15, residues 347-534 of the sequence set forth in SEQ ID NO: 18, residues 398-628 of the sequence set forth in SEQ ID NO:105, or residues 338-472 of the sequence set forth in SEQ ID NO:107. [0034]
  • The term “spacer region” as used herein, refers to a region of the protein kinase located between predicted functional domains. The spacer region has no detectable homology to any amino acid sequence in the database, and can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C- and N-terminal boundaries of the flanking functional domains. Spacer regions may or may not play a fundamental role in protein kinase function. Precedence for the regulatory role of spacer regions in kinase function is provided by the role of the src kinase spacer in inter-domain interactions (Xu, W. et al. (1997) Nature 385:595-602). [0035]
  • The spacer region spans amino acid residues 641-896 of the sequence set forth in SEQ ID NO:13, residues 627-954 of the sequence set forth in SEQ ID NO:14, residues 681-983 of the sequence set forth in SEQ ID NO: 15, residues 274-346 of the sequence set forth in SEQ ID NO:18, residues 278-427 or 638-751 of the sequence set forth in SEQ ID NO:22, residues 67-215 or 426-539 of the sequence set forth in SEQ ID NO:23, residues 274-422 or 633-748 of the sequence set forth in SEQ ID NO:24, residues 225-393 of the sequence set forth in SEQ ID NO:29, residues 282-430 or 641-754 of the sequence set forth in SEQ ID NO:31, residues 174-307 of the sequence set forth in SEQ ID NO:103, residues 669-872 of the sequence set forth in SEQ ID NO:105, or residues 295-337 of the sequence set forth in SEQ ID NO:107. [0036]
  • The term “insert” as used herein refers to a portion of a protein kinase that is absent from a close homolog. Inserts may or may not by the product alternative splicing of exons. Inserts can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Inserts may play a functional role by presenting a new interface for protein-protein interactions, or by interfering with such interactions. Inserts span amino acid residues 52-224 of the sequence set forth in SEQ ID NO:29 or residues 53-173 of the sequence set forth in SEQ ID NO:103. [0037]
  • The term “C-terminal tail” as used herein, refers to a C-terminal domain of a protein kinase, that by homology extends or protrudes past the C-terminal amino acid of its closest homolog. C-terminal tails can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Depending on its length, a C-terminal tail may or may not play a regulatory role in kinase function. [0038]
  • The C-terminal tail spans amino acid residues 490-516 of the sequence set forth in SEQ ID NO:6, residues 787-887 of the sequence set forth in SEQ ID NO:23, residues 659-681 of the sequence set forth in SEQ ID NO:29, residues 994-1093 of the sequence set forth in SEQ ID NO:31, or residues 573-591 of the sequence set forth in SEQ ID NO:103. [0039]
  • Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. These conditions are well-known to those skilled in the art. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides, more preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 50 contiguous nucleotides, most preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 100 contiguous nucleotides. In some instances, the conditions may prevent hybridization of nucleic acids having more than 5 mismatches in the full-length sequence. [0040]
  • By stringent hybridization assay conditions is meant hybridization assay conditions at least as stringent as the following: hybridization in 50% formamide, 5× SSC, 50 mM NaH[0041] 2PO4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5× Denhart solution at 42° C. overnight; washing with 2× SSC, 0.1% SDS at 45 DC; and washing with 0.2× SSC, 0.1% SDS at 45° C. Under some of the most stringent hybridization assay conditions, the second wash can be done with 0.1× SSC at a temperature up to 70° C. (Berger et al. (1987) Guide to Molecular Cloning Techniques pg 421, hereby incorporated by reference herein including any figures, tables, or drawings.). However, other applications may require the use of conditions falling between these sets of conditions. Methods of determining the conditions required to achieve desired hybridizations are well-known to those with ordinary skill in the art, and are based on several factors, including but not limited to, the sequences to be hybridized and the samples to be tested.
  • In other preferred embodiments, the invention features isolated, enriched, or purified nucleic acid molecules encoding kinase polypeptides, further comprising a vector or promoter effective to initiate transcription in a host cell. The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO:106, or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a kinase polypeptide and a transcriptional termination region functional in a cell. Specific vectors and host cell combinations are discussed herein. [0042]
  • The term “vector” relates to a single or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome. A circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art. A nucleic acid molecule encoding a kinase can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together. [0043]
  • The term “transfecting” defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies. [0044]
  • The term “promoter” as used herein, refers to nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to persons skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5′-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. [0045]
  • In preferred embodiments, the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100 SEQ ID NO: 102, SEQ ID NO: 104, or SEQ ID NO: 106, or the corresponding full-length sequence, encodes the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO: 107, or the corresponding full-length amino acid sequence, a functional derivative thereof, or at least 40, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or of the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or of the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or of the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, or the corresponding full-length amino acid sequence; 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO: 101, or the corresponding full-length amino acid sequence; or at least 80, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO: 107, or functional derivatives thereof. The kinase polypeptides, selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, comprise, consist essentially of, or consist of at least at least 40, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 35, 40, 45, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:103; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99; 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101; or at least 80, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or the corresponding full-length sequences or derivatives thereof. The nucleic acid may be isolated from a natural source by cDNA cloning or by subtractive hybridization. The natural source may be mammalian, preferably human, blood, semen, or tissue, and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer. [0046]
  • The term “mammal” refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes, and most preferably to humans. [0047]
  • In yet other preferred embodiments, the nucleic acid is a conserved or unique region, for example those useful for: the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, obtaining antibodies to polypeptide regions, and designing antisense oligonucleotides. [0048]
  • By “conserved nucleic acid regions”, are meant regions present on two or more nucleic acids encoding a kinase polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding kinase polypeptides are provided in Abe, et al. (J. Biol. Chem. 19:13361-13368, 1992), hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables. Preferably, conserved regions differ by no more than 5 out of 20 nucleotides, even more preferably 2 out of 20 nucleotides or most preferably 1 out of 20 nucleotides. [0049]
  • By “unique nucleic acid region” is meant a sequence present in a nucleic acid coding for a kinase polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably encode 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; 250 (preferably 255, more preferably 260, most preferably 270) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; 27 (preferably 30, more preferably 40, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO: 18; 16 (preferably 20, more preferably 25, most preferably 35) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101, or the corresponding full-length amino acid sequence; or 78 (preferably 80, more preferably 85, most preferably 90) or more contiguous amino acids set forth in the amino acid sequence SEQ ID NO:107, or functional derivatives thereof. In particular, a unique nucleic acid region is preferably of mammalian origin. [0050]
  • A second aspect of the invention features a nucleic acid probe for the detection of nucleic acid encoding a kinase polypeptide in a sample, wherein said polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. Preferably, the nucleic acid probe encodes a kinase polypeptide that is a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO: 107, or the corresponding full-length amino acid sequences, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO: 13 or amino acids 1-33 of SEQ ID NO:107. The nucleic acid probe contains a nucleotide base sequence that will hybridize to a sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO: 102, SEQ ID NO: 104, or SEQ ID NO: 106, or the corresponding full-length sequence, or a functional derivative thereof. [0051]
  • In preferred embodiments, the nucleic acid probe hybridizes to nucleic acid encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or functional derivatives thereof. [0052]
  • Methods for using the probes include detecting the presence or amount of kinase RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to kinase RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a kinase polypeptide may be used in the identification of the sequence of the nucleic acid detected (Nelson et al., in Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Kricka, ed., p. 275, 1992, hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe. [0053]
  • In a third aspect, the invention describes a recombinant cell or tissue comprising a nucleic acid molecule encoding a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. In such cells, the nucleic acid may be under the control of the genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By “exogenous” it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the kinase polypeptides. [0054]
  • The polypeptide is preferably a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107. By “fragment,” is meant an amino acid sequence present in a kinase polypeptide. Preferably, such a sequence comprises at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or of the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, OR SEQ ID NO:105, or of the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO: 103, or of the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101; at least 78, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO:107, or the corresponding full-length amino acid sequence; or a functional derivative thereof. [0055]
  • In a fourth aspect, the invention features an isolated, enriched, or purified kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. [0056]
  • By “isolated” in reference to a polypeptide is meant a polymer of amino acids (2 or more amino acids) conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it. [0057]
  • By the use of the term “enriched” in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2-fold, more preferably at least 5- to 10-fold or even more. The term also does not imply that there is no amino acid sequence from other sources. The other source of amino acid sequences may, for example, comprise amino acid sequence encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to increase the proportion of the desired amino acid sequence. [0058]
  • It is also advantageous for some purposes that an amino acid sequence be in purified form. The term “purified” in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment. Compared to the natural level this level should be at least 2-5 fold greater (e.g., in terms of mg/mL). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure. [0059]
  • In preferred embodiments, the kinase polypeptide is a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequences, not to include fragments consisting only of amino acids 1-22 of SEQ ID NO:13 or amino acids 1-33 of SEQ ID NO:107. Preferably, the kinase polypeptide contains at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the corresponding full-length amino acid sequence; at least 250, 255, 275, 300, or 400 contiguous amino acids of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:105, or the corresponding full-length amino acid sequence; at least 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:18; at least 16, 25, 35, 50, 100, 200, or 300 contiguous amino acids of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:103, or the corresponding full-length amino acid sequence; 6 (preferably 10, more preferably 15, most preferably 25) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:97 or SEQ ID NO:99, 22 (preferably 30, more preferably 35, most preferably 45) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:101, or the corresponding full-length amino acid sequence; or at least 78, 85, 90, 100, 200, or 300 contiguous amino acids of SEQ ID NO: 107, or a functional derivative thereof. [0060]
  • In preferred embodiments, the kinase polypeptide comprises an amino acid sequence having (a) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107; (b) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, except that it lacks one or more, but not all, of the following segments of amino acid residues: 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489 or 490-516 of SEQ ID NO:6,1-178 or 179-414 of SEQ ID NO:7, 1-22, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-281, 282-430, 431-640, 641-754, 755-901, or 902-1001 of SEQ ID NO:31, 1-10, 11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-668, 669-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; (c) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107 from amino acid residues 1-21, 22-274, or 275-416 of SEQ ID NO:5, 1-31, 32-308, 309-489, or 490-516 of SEQ ID NO:6, 1-178 or 179-414 of SEQ ID NO:7, 23-289, 290-526, 527-640, 641-896, or 897-1239 of SEQ ID NO:13, 1-255, 256-442, 443-626, 627-954, or 955-1297 of SEQ ID NO:14, 1-255, 256-476, 477-680, 681-983, or 984-1326 of SEQ ID NO:15, 1-13, 14-273, 274-346, 347-534, or 535-894 of SEQ ID NO:18, 1-21, 22-277, 278-427, 428-637, 638-751, or 752-898 of SEQ ID NO:22, 1-66, 67-215, 216-425, 426-539, 540-786, or 787-887 of SEQ ID NO:23, 1-25, 26-273, 274-422, 423-632, or 633-748 of SEQ ID NO:24, 1-51, 52-224, 225-393, 394-658, or 659-681 of SEQ ID NO:29, 1-25, 26-273, 274-422, 423-632, 633-746, 747-993, or 994-1093 of SEQ ID NO:31, 1-10,11-321, or 322-373 of SEQ ID NO:97, 1-57, 58-369, or 370-418 of SEQ ID NO:99, 1-52, 53-173, 174-307, 308-572, or 573-591 of SEQ ID NO:103, 1-24, 25-289, 290-397, 398-628, 629-668, 669-872, or 873-1227 of SEQ ID NO:105, or 1-33, 34-294, 295-337, 338-472, 473-724, or 725-968 of SEQ ID NO:107; or (d) the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, except that it lacks one or more, but not all, of the domains selected from the group consisting of a C-terminal domain, a catalytic domain, an N-terminal domain, a spacer region, a proline-rich region, a coiled-coil structure region, an insert, and a C-terminal tail. [0061]
  • The polypeptide can be isolated from a natural source by methods well-known in the art. The natural source may be mammalian, preferably human, blood, semen, or tissue, and the polypeptide may be synthesized using an automated polypeptide synthesizer. The isolated, enriched, or purified kinase polypeptide is preferably: a STLK2, STLK3, STLK4; STLK5, STLK6, or STLK7 polypeptide; a ZC1, ZC2, ZC3, or ZC4 polypeptide; a KHS2 polypeptide; a SULU1 or SULU3 polypeptide; a GEK2 polypeptide; or a PAK4 or PAK5 polypeptide. [0062]
  • In some embodiments the invention includes a recombinant kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. By “recombinant kinase polypeptide” is meant a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature. [0063]
  • In a fifth aspect, the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain or fragment where the polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. By “specific binding affinity” is meant that the antibody binds to the target kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions. Antibodies or antibody fragments are polypeptides that contain regions that can bind other polypeptides. The term “specific binding affinity” describes an antibody that binds to a kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions. [0064]
  • The term “polyclonal” refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species. [0065]
  • “Monoclonal antibodies” are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art (Kohler et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110, both of which are hereby incorporated by reference herein in their entirety including any figures, tables, or drawings). [0066]
  • The term “antibody fragment” refers to a portion of an antibody, often the hyper variable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hyper variable region is a portion of an antibody that physically binds to the polypeptide target. [0067]
  • Antibodies or antibody fragments having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by probing the sample with the antibody under conditions suitable for kinase-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide. Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for the kinase as well as a conjugate of a binding partner of the antibodies or the antibodies themselves. [0068]
  • An antibody or antibody fragment with specific binding affinity to a kinase polypeptide of the invention can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above. [0069]
  • Antibodies having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor. [0070]
  • In a sixth aspect, the invention features a hybridoma which produces an antibody having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain, where the polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. By “hybridoma” is meant an immortalized cell line that is capable of secreting an antibody, for example an antibody to a kinase of the invention. In preferred embodiments, the antibody to the kinase comprises a sequence of amino acids that is able to specifically bind a kinase polypeptide of the invention. [0071]
  • In a seventh aspect, the invention features a kinase polypeptide binding agent able to bind to a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK6, STLK7, STLK5, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. The binding agent is preferably a purified antibody that recognizes an epitope present on a kinase polypeptide of the invention. Other binding agents include molecules that bind to kinase polypeptides and analogous molecules that bind to a kinase polypeptide. Such binding agents may be identified by using assays that measure kinase binding partner activity, such as those that measure PDGFR activity. [0072]
  • The invention also features a method for screening for human cells containing a kinase polypeptide of the invention or an equivalent sequence. The method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying the kinases of the invention (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.). [0073]
  • In an eighth aspect, the invention features methods for identifying a substance that modulates kinase activity comprising the steps of: (a) contacting a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5 with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide. [0074]
  • The term “modulates” refers to the ability of a compound to alter the function of a kinase of the invention. A modulator preferably activates or inhibits the activity of a kinase of the invention depending on the concentration of the compound exposed to the kinase. [0075]
  • The term “activates” refers to increasing the cellular activity of the kinase. The term inhibit refers to decreasing the cellular activity of the kinase. Kinase activity is preferably the interaction with a natural binding partner. [0076]
  • The term “modulates” also refers to altering the function of kinases of the invention by increasing or decreasing the probability that a complex forms between the kinase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the kinase and the natural binding partner depending on the concentration of the compound exposed to the kinase, and most preferably decreases the probability that a complex forms between the kinase and the natural binding partner. [0077]
  • The term “complex” refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another. For instance, a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand. [0078]
  • The term “natural binding partner” refers to polypeptides, lipids, small molecules, or nucleic acids that bind to kinases in cells. A change in the interaction between a kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of kinase/natural binding partner complex. [0079]
  • The term “contacting” as used herein refers to mixing a solution comprising the test compound with a liquid medium bathing the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the test compound or compounds into the cells of the methods. The solution comprising the test compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipet-based device or syringe-based device. [0080]
  • In a ninth aspect, the invention features methods for identifying a substance that modulates kinase activity in a cell comprising the steps of: (a) expressing a kinase polypeptide in a cell, wherein said polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5; (b) adding a test substance to said cell; and (c) monitoring a change in cell phenotype or the interaction between said polypeptide and a natural binding partner. [0081]
  • The term “expressing” as used herein refers to the production of kinases of the invention from a nucleic acid vector containing kinase genes within a cell. The nucleic acid vector is transfected into cells using well known techniques in the art as described herein. [0082]
  • In a tenth aspect, the invention provides methods for treating a disease by administering to a patient in need of such treatment a substance that modulates the activity of a kinase selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5. Preferably, the disease is selected from the group consisting of immune-related diseases and disorders, organ transplantation, myocardial infarction, cardiovascular disease, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer. Most preferably, the immune-related diseases and disorders include, but are not limited to, rheumatoid arthritis, artherosclerosis, and autoimmune disorders. [0083]
  • In preferred embodiments, the invention provides methods for treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, ZC4, KHS2, PAK4, and PAK5. Preferably, the disease or disorder is selected from the group consisting of rheumatoid arthritis, artherosclerosis, autoimmune disorders, and organ transplantation. The invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of STLK1, STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7. Preferably the disease or disorder is selected from the group consisting of immune-related diseases and disorders, myocardial infarction, cardiomyopathies, stroke, renal failure, and oxidative stress-related neurodegenerative disorders. Most preferably, the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation. [0084]
  • The invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of ZC1, ZC2, ZC3, and ZC4. Preferably the disease is selected from the group consisting of immune-related diseases and disorders, cardiovascular disease, and cancer. Most preferably, the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation. [0085]
  • Substances useful for treatment of kinase-related disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (Examples of such assays are provided in the references in section VI, below; and in Example 7, herein). Examples of substances that can be screened for favorable activity are provided and referenced in section VI, below. The substances that modulate the activity of the kinases preferably include, but are not limited to, antisense oligonucleotides and inhibitors of protein kinases, as determined by methods and screens referenced in section VI and Example 7, below. [0086]
  • The term “preventing” refers to decreasing the probability that an organism contracts or develops an abnormal condition. [0087]
  • The term “treating” refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism. [0088]
  • The term “therapeutic effect” refers to the inhibition or activation factors causing or contributing to the abnormal condition. A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells. Compounds demonstrating efficacy against abnormal conditions can be identified as described herein. [0089]
  • The term “abnormal condition” refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival. [0090]
  • Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation. [0091]
  • Abnormal differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates. [0092]
  • Abnormal cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated. A number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death. [0093]
  • The term “aberration”, in conjunction with the function of a kinase in a signal transduction process, refers to a kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner. [0094]
  • The term “administering” relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications. For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques. [0095]
  • The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism. The effect of administering a compound on organism function can then be monitored. The organism is preferably a mouse, rat, rabbit, guinea pig, or goat, more preferably a monkey or ape, and most preferably a human. [0096]
  • In an eleventh aspect, the invention features methods for detection of a kinase polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a kinase polypeptide selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease. [0097]
  • In preferred embodiments of the invention, the disease or disorder is selected from the group consisting of rheumatoid arthritis, artherosclerosis, autoimmune disorders, organ transplantation, myocardial infarction, cardiomyopathies, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer. In other preferred embodiments, the kinase polypeptide is selected from the group consisting of PAK4 and PAK5, or the polypeptide is selected from the group consisting of ZC1, ZC2, ZC3, and ZC4, and the disease is cancer. [0098]
  • The kinase “target region” is the nucleotide base sequence set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO: 106, or the corresponding full-length sequences, a functional derivative thereof, or a fragment thereof to which the nucleic acid probe will specifically hybridize. Specific hybridization indicates that in the presence of other nucleic acids the probe only hybridizes detectably with the kinase of the invention's target region. Putative target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database. [0099]
  • In preferred embodiments the nucleic acid probe hybridizes to a kinase target region encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or the corresponding full-length amino acid sequence, or a functional derivative thereof. Hybridization conditions should be such that hybridization occurs only with the kinase genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra. [0100]
  • The diseases for which detection of kinase genes in a sample could be diagnostic include diseases in which kinase nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells. By “amplification” is meant increased numbers of kinase DNA or RNA in a cell compared with normal cells. In normal cells, kinases are typically found as single copy genes. In selected diseases, the chromosomal location of the kinase genes may be amplified, resulting in multiple copies of the gene, or amplification. Gene amplification can lead to amplification of kinase RNA, or kinase RNA can be amplified in the absence of kinase DNA amplification. [0101]
  • “Amplification” as it refers to RNA can be the detectable presence of kinase RNA in cells, since in some normal cells there is no basal expression of kinase RNA. In other normal cells, a basal level of expression of kinase exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, kinase RNA, compared to the basal level. [0102]
  • The diseases that could be diagnosed by detection of kinase nucleic acid in a sample preferably include cancers. The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized. [0103]
  • In a final aspect, the invention features a method for detection of a kinase polypeptide in a sample as a diagnostic tool for a disease or disorder, wherein the method comprises: (a) comparing a nucleic acid target region encoding the kinase polypeptide in a sample, where the kinase polypeptide is selected from the group consisting of STLK2, STLK3, STLK4, STLK5, STLK6, STLK7, ZC1, ZC2, ZC3, ZC4, KHS2, SULU1, SULU3, GEK2, PAK4, and PAK5, or one or more fragments thereof, with a control nucleic acid target region encoding the kinase polypeptide, or one or more fragments thereof; and (b) detecting differences in sequence or amount between the target region and the control target region, as an indication of the disease or disorder. Preferably, the disease or disorder is selected from the group consisting of immune-related diseases and disorders, organ transplantation, myocardial infarction, cardiovascular disease, stroke, renal failure, oxidative stress-related neurodegenerative disorders, and cancer. Immune-related diseases and disorders include, but are not limited to, those discussed previously. [0104]
  • The term “comparing” as used herein refers to identifying discrepancies between the nucleic acid target region isolated from a sample, and the control nucleic acid target region. The discrepancies can be in the nucleotide sequences, e.g. insertions, deletions, or point mutations, or in the amount of a given nucleotide sequence. Methods to determine these discrepancies in sequences are well-known to one of ordinary skill in the art. The “control” nucleic acid target region refers to the sequence or amount of the sequence found in normal cells, e.g. cells that are not diseased as discussed previously. [0105]
  • The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. For example, in some instances the nucleotide sequence of the ZC4 kinase polypeptide may not be part of a preferred embodiment. [0106]
  • The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.[0107]
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIGS. 1A, 1B and [0108] 1C show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 84-85, 5-7, respectively, in order of appearance) of the STE20-STE20 family kinases.
  • FIGS. 2A and 2B show a multiple sequence alignment of the amino acid sequences ([0109] SEQ ID NOS 84, 86-87 & 8, respectively, in order of appearance) of the STE20-STLK5 family kinases.
  • FIGS. 3A, 3B, [0110] 3C, 3D, 3E, 3F and 3G show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 88-89, 13-16, respectively, in order of appearance) of STE20-ZC family kinases.
  • FIGS. 4A, 4B and [0111] 4C show a pairwise sequence (SEQ ID NOS 91 & 18, respectively, in order of appearance) alignment of STE20-KHS family kinases.
  • FIGS. 5A, 5B, [0112] 5C and 5D show a multiple sequence alignment of the amino acid sequences ( SEQ ID NOS 90, 22, 24 & 151 respectively, in order of appearance) of STE20-SULU family kinases.
  • FIGS. 6A, 6B and [0113] 6C show a pairwise sequence (SEQ ID NOS 92 & 26, respectively, in order of appearance) alignment of STE20-GEK family kinases.
  • FIGS. 7A, 7B and [0114] 7C show a multiple sequence alignment of the amino acid sequences (SEQ ID NOS 93-95, 29-30 respectively, in order of appearance) of STE20-PAK family kinases.
  • FIGS. 8A, 8B, [0115] 8C, 8D, 8E, 8F and 8G show the amino acid sequences of human STLK2(SEQ ID NO:5), human STLK3(SEQ ID NO:6), human STLK4(SEQ ID NO:7), human STLK5(SEQ ID NO:8), human ZC1(SEQ ID NO:13), human ZC2(SEQ ID NO:14), human ZC3(SEQ ID NO:15), human ZC4(SEQ ID NO:16), human KHS2(SEQ ID NO:18), human SULU1(SEQ ID NO:22), human SULU3(SEQ ID NO:23), murine SULU3(SEQ ID NO:24), human GEK2(SEQ ID NO:26), human PAK4(SEQ ID NO:29), and human PAK5(SEQ ID NO30).
  • FIGS. 9A, 9B, [0116] 9C, 9D, 9E, 9F, 9G, 9H, 91, 9J, 9K, 9L, 9M, 9N, 90, 9P, 9Q, 9R, 9S, 9T, 9U and 9V show the nucleic acid sequences of human STLK2(SEQ ID NO: 1), human STLK3(SEQ ID NO:2), human STLK4(SEQ ID NO:3), human STLK5(SEQ ID NO:4), human ZC1(SEQ ID NO:9), human ZC2(SEQ ID NO:10), human ZC3(SEQ ID NO:11), human ZC4(SEQ ID NO:12), human KHS2(SEQ ID NO:17), human SULU1(SEQ ID NO:19), human SULU3(SEQ ID NO:20), murine SULU3(SEQ ID NO:21), human GEK2(SEQ ID NO:25), human PAK4(SEQ ID NO:27), and human PAK5(SEQ ID NO:28).
  • FIGS. 10A, 10B and [0117] 10C show the full-length amino acid sequences of human STLK5 (SEQ ID NO: 97), human PAK5 (SEQ ID NO:103), and human ZC4 (SEQ ID NO: 105), as well as the partial amino acid sequences of human full-length STLK6 (SEQ ID NO: 99) and human STLK7 (SEQ ID NO: 101) and human GEK2 (SEQ ID NO: 107).
  • FIGS. 11A, 11B, [0118] 11C, 11D, 11E, 11F, 11G and 11H show the full-length nucleic acid sequences of human STLK5 (SEQ ID NO:96), human PAK5 (SEQ ID NO:102), and human ZC4 (SEQ ID NO:104), as well as the partial nucleic acid sequences of human STLK6 (SEQ ID NO: 98) and human STLK7 (SEQ ID NO: 100) and human GEK2 (SEQ ID NO: 106).
  • FIGS. 12A and 12B show a multiple sequence alignment among human SPAK (SEQ ID NO: 153), human STLK6 (SEQ ID NO: 99), human STLK7 (SEQ ID NO: 101) and full-length human STLK5 (SEQ ID NO: 152). [0119]
  • FIGS. 13A, 13B and [0120] 13C show a multiple sequence alignment among human PAK1 (SEQ ID NO: 93), human PAK4 (SEQ ID NO: 29) and human PAK5 (SEQ ID NO: 103).
  • FIGS. 14A, 14B and [0121] 14C show a pair-wise sequence alignment between human ZC1 (SEQ ID NO: 15) and human ZC4 (SEQ ID NO: 105).
  • FIGS. 15A, 15B and [0122] 15C show a pair-wise sequence alignment between LOK1 (SEQ ID NO: 154) and full-length GEK2 (SEQ ID NO: 155).
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention relates in part to kinase polypeptides, nucleic acids encoding such polypeptides, cells containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. The present invention is based upon the isolation and characterization of new kinase polypeptides. The polypeptides and nucleic acids may be produced using well-known and standard synthesis techniques when given the sequences presented herein. [0123]
  • The recent elucidation of the DNA sequence of [0124] Saccharomyces cerevesiae has provided the first complete example of the genetic information contained in a simple eukaryotic organism. Analysis of this yeast genome revealed that it contains at least 113 protein kinases. These kinases were further subdivided into several structurally related groups. One of these newly defined groups was termed the STE20-family to represent its founding member STE20, which is a protein kinase involved in the yeast pheromone response pathway that initiates a protein kinase cascade in response to a G-protein mediated signal. S. cerevesiae has two additional members of this family, CLA4, and YOL113W (HRA655).
  • Several mammalian homologues have recently been identified that belong to the STE20-family, including SOK-1 (human STE20), GC-kinase, KHS, HPK1, NIK, SLK, GEK, PAK1, PAK65, MST1, and CDC7. Furthermore, the [0125] Drosophila and the C. elegans genome efforts have identified additional protein kinases which belong to the STE20-family, yet have structurally unique extracatalytic domains, including ZC504.4 and SULU kinases from C. elegans, and NINAC of Drosophila.
  • STE20-related protein kinases have been implicated as regulating a variety of cellular responses, including response to growth factors or cytokines, oxidative-, UV-, or irradiation-related stress pathways, inflammatory signals (i.e., TNF□), apoptotic stimuli (i.e., Fas), T and B cell costimulation, the control of cytoskeletal architecture, and cellular transformation. Typically, the STE20-related kinases serve as upstream regulators of MAPK cascades. Examples include: HPK1, a protein-serine/threonine kinase (STK) that possesses a STE20-like kinase domain that activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK; PAK1, an STK with an upstream CDC42-binding domain that interacts with Rac and plays a role in cellular transformation through the Ras-MAPK pathway; and murine NIK, which interacts with upstream receptor tyrosine kinases and connects with downstream STE11-family kinases. [0126]
  • The STE20-kinases possess a variety of non-catalytic domains that are believed to interact with upstream regulators. Examples include proline-rich domains for interaction with SH3-containing proteins, or specific domains for interaction with Rac, Rho, and Rab small G-proteins. These interactions may provide a mechanism for cross-talk between distinct biochemical pathways in response to external stimuli such as the activation of a variety of cell surface receptors, including tyrosine kinases, cytokine receptors, TNF receptor, Fas, T cell receptors, CD28, or CD40. [0127]
  • I. The Nucleic Acids of the Invention [0128]
  • Included within the scope of this invention are the functional equivalents of the herein-described isolated nucleic acid molecules. The degeneracy of the genetic code permits substitution of certain codons by other codons that specify the same amino acid and hence would give rise to the same protein. The nucleic acid sequence can vary substantially since, with the exception of methionine and tryptophan, the known amino acids can be coded for by more than one codon. Thus, portions or all of the kinase genes of the invention could be synthesized to give a nucleic acid sequence significantly different from that shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO: 104, and SEQ ID NO: 106. The encoded amino acid sequence thereof would, however, be preserved. [0129]
  • In addition, the nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5′-end and/or the 3′-end of the nucleic acid formula shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, or SEQ ID NO:106, or a derivative thereof. Any nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, which is encoded by the nucleotide sequence. For example, the present invention is intended to include any nucleic acid sequence resulting from the addition of ATG as an initiation codon at the 5′-end of the inventive nucleic acid sequence or its derivative, or from the addition of TTA, TAG or TGA as a termination codon at the 3′-end of the inventive nucleotide sequence or its derivative. Moreover, the nucleic acid molecule of the present invention may, as necessary, have restriction endonuclease recognition sites added to its 5′-end and/or 3′-end. [0130]
  • Such functional alterations of a given nucleic acid sequence afford an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto. All variations of the nucleotide sequence of the kinase genes of the invention and fragments thereof permitted by the genetic code are, therefore, included in this invention. [0131]
  • Further, it is possible to delete codons or to substitute one or more codons with codons other than degenerate codons to produce a structurally modified polypeptide, but one which has substantially the same utility or activity as the polypeptide produced by the unmodified nucleic acid molecule. As recognized in the art, the two polypeptides are functionally equivalent, as are the two nucleic acid molecules that give rise to their production, even though the differences between the nucleic acid molecules are not related to the degeneracy of the genetic code. [0132]
  • Mammalian STLK2 [0133]
  • The full-length human STLK2 cDNA (SEQ ID NO: 1) is 3268 bp long and consists of a 1248 bp open reading frame (ORF) flanked by a 181 [0134] bp 5′ untranslated region (UTR; 1-181) and a 1784 bp 3′ UTR (1433-3216) that is followed by a 52 nucleotide polyadenylated region. A polyadenylation signal (AATAAA) is found at positions (3193-3198). The sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiating methionine, and is believed to be the translational start site for STLK2. Furthermore, human STLK2 and the related SOK-1 and MST3 proteins conserve the amino acid sequence immediately following this presumed initiating methionine.
  • Several EST fragments span the complete STLK2 sequence with AA191319 at the 5′ end and W16504 at the 3′ end. [0135]
  • Mammalian STLK3 [0136]
  • The partial human STLK3 cDNA (SEQ ID NO:2) is 3030 bp long and consists of a 1548 bp ORF flanked by a 1476 [0137] bp 3′ UTR (1550-3025) and a 5 nucleotide polyadenylated region. A potential polyadenylation signal (AATAAA) begins at position 3004. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • Multiple EST fragments span the complete STLK3 sequence with AA278967 at the 5′ end and AA628477 and others at the 3′ end. [0138]
  • Mammalian STLK4 [0139]
  • The partial human STLK4 cDNA (SEQ ID NO:3) is 3857 bp long and consists of a 1242 bp ORF flanked by a 2596 [0140] bp 3′ UTR (1244-3839) and an 18 nucleotide polyadenylated region. A potential polyadenylation signal (AATAAA) is found at positions 2181-3822. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. A near full-length murine STLK4 cDNA is represented in the 1773 bp EST AA117438. It extends an additional 21 nucleotides 5′ of the human STLK4 consensus, but since its coding region is open throughout the 5′ extent of the sequence, this is also apparently a partial cDNA clone lacking the N-terminal start methionine.
  • Several EST fragments span the complete STLK3 sequence with AA297759 at the 5′ end and AA100484 and others at the 3′ end. [0141]
  • Mammalian STLK5 [0142]
  • The full-length human STLK5 cDNA (SEQ ID NO:96) is 2110 bp long and consists of a 1119 bp ORF flanked by a 229 [0143] bp 5′ UTR and a 762 bp 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for STLK5. Several EST fragments span the complete STLK5 sequence with AA297059 and F07734 at the 5′ end, and R46686 and F03423 and others at the 3′ end.
  • Mammalian STLK6 [0144]
  • The full-length human STLK6 cDNA (SEQ ID NO:98) is 2,001 bp long and consists of a 1,254 bp ORF flanked by a 75 [0145] bp 5′ UTR and a 673 bp 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for STLK6.
  • Mammalian STLK7 [0146]
  • The partial human STLK7 cDNA (SEQ ID NO: 100) is 311 bp long and consists of a 309 bp ORF. Since the coding region is open throughout both the 5′ and 3′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine and C-terminal stop codon. [0147]
  • Mammalian ZC1 [0148]
  • The full-length human ZC1 cDNA (SEQ ID NO:9) is 3798 bp long and consists of a 3717 bp ORF (7-3723) flanked by a 6 [0149] bp 5′ UTR and a 75 bp (3724-3798) 3′ UTR. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human ZC1.
  • Multiple EST fragments (W81656) match the 3′ end of the human ZC1 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0150]
  • Mammalian ZC2 [0151]
  • The partial human ZC2 cDNA (SEQ ID NO:10) is 4055 bp long and consists of a 3891 bp ORF (1-3891) and a 164 bp (3892-4055) 3′ UTR. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR. [0152]
  • Multiple EST fragments (R51245) match the 3′ end of the human ZC2 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0153]
  • Mammalian ZC3 [0154]
  • The partial human ZC3 cDNA (SEQ ID NO:11) is 4133 bp long and consists of a 3978 bp ORF (1-3978) and a 152 bp (3979-4133) 3′ UTR region. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR. [0155]
  • Multiple EST fragments (R54563) match the 3′ end of the human ZC3 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0156]
  • Mammalian ZC4 [0157]
  • The full-length human ZC4 cDNA (SEQ ID NO:104) is 3,684 bp long and was originally assembled from X chromosome genomic DNA sequence. [0158]
  • Multiple EST fragments (R98571) match the 3′ end of the human ZC4 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. ZC4 gene is also contained within the human genomic clone Z83850. [0159]
  • Mammalian KHS2 [0160]
  • The full-length human KHS2 cDNA (SEQ ID NO:17) is 4023 bp long and consists of a 2682 bp ORF (6-2687) flanked by a 5 bp (1-5) 5′UTR and a 1336 bp (2688-4023) 3′ UTR. A potential polyadenylation signal (AATAAA) is found at positions 4008-4013. No polyadenylated region is present in the 3′UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human KHS2. [0161]
  • Multiple EST fragments match the 5′end (AA446022) as well as the 3′ end (R37625) of the human KHS2 gene. [0162]
  • Mammalian SULU1 [0163]
  • The full-length human SULU1 cDNA (SEQ ID NO:19) is 4177 bp long and consists of a 2694 bp ORF (415-3108) flanked by a 414 bp (1-414) 5′UTR and a 1069 bp (3109-4177) 3′ UTR followed by a 19 nucleotide polydenylated region. A potential polyadenylation signal (AATAAA) is found at positions 4164-4169. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human SULU1. [0164]
  • Multiple EST fragments match the 5′end (N27153) as well as the 3′ end (R90908) of the human SULU1 gene. [0165]
  • Mammalian (Murine) SULU3 [0166]
  • The partial murine SULU3 cDNA (SEQ ID NO:21) is 2249 bp long and consists of a 2244 bp ORF (6-2249) flanked by a 5 bp (1-5) 5′UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for murine SULU3. The 3′ end of the murine SULU3 cDNA shares 90% DNA sequence identity over 1620 nucleotides with human SULU3, suggesting that these two genes are functional orthologues. [0167]
  • One EST fragment (AA446022) matches the 3′ end of the partial murine SULU3 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0168]
  • Mammalian (Human) SULU3 [0169]
  • The partial human SULU3 cDNA (SEQ ID NO:20) is 3824 bp long and consists of a 2358 bp ORF (2-2359) flanked by a 1465 bp (2360-3824) 3′ UTR followed by a 19 nucleotide polydenylated region. A potential polyadenylation signal (AATAAA) is found at positions 2602-2607. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. The 5′ end of the human SULU3 cDNA shares 90% DNA sequence identity over 1620 nucleotides with murine SULU3, suggesting that these two genes are functional orthologues. [0170]
  • Multiple EST fragments (R02283) match the 3′end of the human SULU3 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0171]
  • Mammalian GEK2 [0172]
  • The full-length human GEK2 cDNA (SEQ ID NO:106) is 2962 bp long and consists of a 2737 bp ORF (59-2795) flanked by a 58 bp (1-58) 5′UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human GEK2. [0173]
  • Multiple EST fragments (AA465671) match the 5′end, but at the time of filing, the inventors believe that only one (AA380492) matches the 3′ end of the human GEK2 gene. [0174]
  • Mammalian PAK4 [0175]
  • The full-length human PAK4 cDNA (SEQ ID NO:27) is 3604 bp long and consists of a 2043 bp ORF (143-2185) flanked by a 142 bp (1-142) 5′UTR and a 1419 3′UTR followed by a 22 nucleotide polydenylated region. A potential polyadenylation signal (AATTAAA) is found at positions 3582-3588. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human PAK4. [0176]
  • Multiple EST fragments (AA535791) match the 3′end of the human PAK4 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0177]
  • Mammalian PAK5 [0178]
  • The full-length human PAK5 cDNA (SEQ ID NO:102) is 2806 bp long and consists of a 1773 bp ORF flanked by a 201 [0179] bp 5′ UTR and a 833 bp 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for PAK5.
  • Multiple EST fragments (AA442867) match the 3′end of the human PAK5 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0180]
  • II. Nucleic Acid Probes, Methods, and Kits for Detection of STE20-Related Kinases. [0181]
  • A nucleic acid probe of the present invention may be used to probe an appropriate chromosomal or cDNA library by usual hybridization methods to obtain other nucleic acid molecules of the present invention. A chromosomal DNA or cDNA library may be prepared from appropriate cells according to recognized methods in the art (cf. “Molecular Cloning: A Laboratory Manual”, second edition, Cold Spring Harbor Laboratory, Sambrook, Fritsch, & Maniatis, eds., 1989). [0182]
  • In the alternative, chemical synthesis can be carried out in order to obtain nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions of the amino acid sequence of the polypeptide of interest. The synthesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, “A Guide to Methods and Applications”, Academic Press, Michael, et al., eds., 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment of the present invention. [0183]
  • One skilled in the art can readily design such probes based on the sequence disclosed herein using methods of computer alignment and sequence analysis known in the art (“Molecular Cloning: A Laboratory Manual”, 1989, supra). The hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes may be visualized using known methods. [0184]
  • The nucleic acid probes of the present invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art. The nucleic acid probe may be immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art. [0185]
  • The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized. [0186]
  • One method of detecting the presence of nucleic acids of the invention in a sample comprises (a) contacting said sample with the above-described nucleic acid probe under conditions such that hybridization occurs, and (b) detecting the presence of said probe bound to said nucleic acid molecule. One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue. [0187]
  • A kit for detecting the presence of nucleic acids of the invention in a sample comprises at least one container means having disposed therein the above-described nucleic acid probe. The kit may further comprise other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe. Examples of detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin). [0188]
  • In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like. One skilled in the art will readily recognize that the nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art. [0189]
  • III. DNA Constructs Comprising a STE20-Related Nucleic Acid Molecule and Cells Containing These Constructs. [0190]
  • The present invention also relates to a recombinant DNA molecule comprising, 5′ to 3′, a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules. In addition, the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecule. The present invention also relates to a nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in said cell. The above-described molecules may be isolated and/or purified DNA molecules. [0191]
  • The present invention also relates to a cell or organism that contains an above-described nucleic acid molecule and thereby is capable of expressing a polypeptide. The polypeptide may be purified from cells which have been altered to express the polypeptide. A cell is said to be “altered to express a desired polypeptide” when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels. One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells. [0192]
  • A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked”[0193] 0 to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5′-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • If desired, the [0194] non-coding region 3′ to the sequence encoding a kinase of the invention may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3′-region naturally contiguous to the DNA sequence encoding a kinase of the invention, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3′ region functional in the host cell may be substituted.
  • Two DNA sequences (such as a promoter region sequence and a sequence encoding a kinase of the invention) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a gene sequence encoding a kinase of the invention, or (3) interfere with the ability of the gene sequence of a kinase of the invention to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. Thus, to express a gene encoding a kinase of the invention, transcriptional and translational signals recognized by an appropriate host are necessary. [0195]
  • The present invention encompasses the expression of a gene encoding a kinase of the invention (or a functional derivative thereof) in either prokaryotic or eukaryotic cells. Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system for kinases of the invention. Prokaryotes most frequently are represented by various strains of [0196] E. Coli. However, other microbial strains may also be used, including other bacterial strains.
  • In prokaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used. Examples of suitable plasmid vectors may include pBR322, pUC118, pUC 119 and the like; suitable phage or bacteriophage vectors may include γgt10, γgt11 and the like; and suitable virus vectors may include pMAM-neo, pKRC and the like. Preferably, the selected vector of the present invention has the capacity to replicate in the selected host cell. [0197]
  • Recognized prokaryotic hosts include bacteria such as [0198] E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • To express a kinase of the invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the kinase of the invention to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene sequence of pBR322, and the cat promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ(P[0199] L and PR), the trp, recA, λacZ, λacI, and gal promoters of E. coli, the α-amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and the c-28-specific promoters of B. subtilis (Gilman et al., Gene Sequence 32:11-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478, 1986). Prokaryotic promoters are reviewed by Glick (Ind. Microbiot. 1:277-282, 1987), Cenatiempo (Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
  • Proper expression in a prokaryotic cell also requires the presence of a ribosome-binding site upstream of the gene sequence-encoding sequence. Such ribosome-binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981). The selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene. As used herein, “cell”, “cell line”, and “cell culture” may be used interchangeably and all such designations include progeny. Thus, the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell. [0200]
  • Host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the kinase polypeptide of interest. Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as [0201] IMR 332, which may provide better capacities for correct post-translational processing.
  • In addition, plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences. Another preferred host is an insect cell, for example the [0202] Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can be engineered to express large amounts of kinases of the invention in insect cells (Jasny, Science 238:1653, 1987; Miller et al., In: Genetic Engineering, Vol. 8, Plenum, Setlow et al., eds., pp. 277-297, 1986).
  • Any of a series of yeast expression systems can be utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes that are produced in large quantities when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. Yeast provides substantial advantages in that it can also carry out post-translational modifications. A number of recombinant DNA strategies exist utilizing strong promoter sequences and high copy number plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian genes and secretes peptides bearing leader sequences (i.e., pre-peptides). Several possible vector systems are available for the expression of kinases of the invention in a mammalian host. [0203]
  • A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and the like, may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation. [0204]
  • Expression of kinases of the invention in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984). [0205]
  • Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes a kinase of the invention (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the kinase of the invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the kinase of the invention coding sequence). [0206]
  • A nucleic acid molecule encoding a kinase of the invention and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome. [0207]
  • A vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama (Mol. Cell. Biol. 3:280-, 1983). [0208]
  • The introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species. [0209]
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in [0210] E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, □VX; “Molecular Cloning: A Laboratory Manual”, 1989, supra). Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, NY, pp. 307-329, 1982). Suitable Streptomyces plasmids include plJ101 (Kendall et al., J. Bacteriol. 169:4177-4183, 1987), and streptomyces bacteriophages such as □C31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary, pp. 45-54, 1986). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978).
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982; Broach, In: “The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance”, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon et al., J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980). [0211]
  • Once the vector or nucleic acid molecule containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene(s) results in the production of a kinase of the invention, or fragments thereof. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like). A variety of incubation conditions can be used to form the peptide of the present invention. The most preferred conditions are those which mimic physiological conditions. [0212]
  • IV. The Proteins of the Invention [0213]
  • A variety of methodologies known in the art can be utilized to obtain the polypeptides of the present invention. The polypeptides may be purified from tissues or cells that naturally produce the polypeptides. Alternatively, the above-described isolated nucleic acid fragments could be used to express the kinases of the invention in any organism. The samples of the present invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The samples will vary based on the assay format, the detection method, and the nature of the tissues, cells or extracts used as the sample. [0214]
  • Any eukaryotic organism can be used as a source for the polypeptides of the invention, as long as the source organism naturally contains such polypeptides. As used herein, “source organism” refers to the original organism from which the amino acid sequence of the subunit is derived, regardless of the organism the subunit is expressed in and ultimately isolated from. [0215]
  • One skilled in the art can readily follow known methods for isolating proteins in order to obtain the polypeptides free of natural contaminants. These include, but are not limited to: size-exclusion chromatography, HPLC, ion-exchange chromatography, and immuno-affinity chromatography. [0216]
  • Mammalian STLK2 [0217]
  • Analysis of the deduced amino acid sequence predicts STLK2 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. STLK2 contains a 21 amino acid N-terminal domain, a 253 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, followed by a 142 amino acid C-terminal domain. [0218]
  • STLK2 is most closely related to human STE20-subfamily kinases, MST3 (GB:AF024636) and SOK-1 (GB:X99325) and a [0219] C. elegans kinase yk34b11.5 (GB:U53153) sharing 72.7%, 68.7%, and 69.3% amino acid identity, respectively.
  • The 21 amino acid N-terminal domain of human STLK2 is 71.4% identical to the N-terminus of MST3 (GB:AF024636). Human STLK2 lacks a glycine residue at [0220] position 2, and is therefore unlikely to undergo myristylation. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • The 253 amino acid catalytic domain of human STLK2 is most related to human SOK-1 (X99325), MST3 (GB:AF024636), [0221] C. elegans yk32b11.5 (GB:U53153), and STLK3 (SEQ ID NO:6) sharing 88.9%, 87.4%, 78.3%, and 49% amino identity respectively, placing it in the STLK-subfamily of STE20-related kinases. The STLK2 kinase domain displayed lesser homology to other STE20-related kinases including: 55.9% to human MST2 (GB:U26424), 49.2% to human GCK (GB:U07349), 49.2% to human KHS1 (GB:U77129), and 44.2% to human HPK1 (GB:U66464). The activation loop of human STLK2 catalytic domain is identical to that of human SOK-1 and MST3 including the presence of four potential threonine phosphorylation sites that could serve an autoregulatory role on kinase activity.
  • The 142 amino acid C-terminal domain of human STLK2 is most related to human SOK-1 (X99325), MST3 (GB:AF024636), and [0222] C. elegans yk32b11.5 (GB:U53153), sharing 39.9%, 39.9%, and 33.3% amino acid identity, respectively. This C-terminal domain shares some significant amino acid similarity to the C-terminal domains of the related human STLK3 (SEQ ID NO:6) and STLK4 (SEQ ID NO:7).
  • The C-terminus of the related human SOK-1 (GB:X99325) kinase has been shown to be inhibitory to the catalytic activity of this kinase (Pombo, C. M., Bonventre, J. V., Molnar, A., Kyriakis, J. and Force, T. EMBO J. 15, 4537-4546 (1996)). Based on the sequence identity between the C-termini of human SOK-1 (GB:X99325) and human STLK2 (39.2%), the C-terminus of human STLK2 may also function as an inhibitory domain for its kinase. [0223]
  • Mammalian STLK3 [0224]
  • The 3030 bp human STLK3 nucleotide sequence of the partial cDNA clone encodes a polypeptide of 516 amino acids (SEQ ID NO:6) with a predicted molecular mass of 56,784 daltons. Analysis of the deduced amino acid sequence predicts STLK3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-termius is not known. STLK3 contains a 31 amino acid N-terminal domain, a 277 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, followed by a 181 amino acid C-terminal domain containing a 25 amino acid insert and a 27 amino acid tail relative to the sequence of human STLK2. [0225]
  • STLK3 is most closely related to human STE20-subfamily kinases, STLK4 (SEQ ID. NO:7), MST3 (GB:AF024636), SOK-1 (GB:X99325) and STLK2 (SEQ ID NO:5) sharing 71.1%, 37.6%, 38.1%, and 38.4% amino acid identity respectively. [0226]
  • The 31 amino acid N-terminal domain of human STLK3 lacked any significant amino acid sequence homologies using a Smith-Waterman search of the nonredundant protein database, other than sequence similarity to proline-alanine repeats. [0227]
  • The 277 amino acid catalytic domain of human STLK3 is most related to human STLK4 (SEQ ID NO:7), SOK-1 (GB:X99325), MST3 (GB:AF024636), and STLK2 (SEQ ID NO:5) sharing 88.2%, 49.2%, 49%, and 49% amino acid identity, respectively. It also shares strong homology to other STKs from lower organisms including 51.7% to [0228] A. thaliana (GB: AC002343), 43.1% to A. thaliana (GB: Z97336), 42.1% to A. thaliana (GB: U96613), and 43.3% to C. elegans (GB: U53153). The activation loop of the human STLK3 catalytic domain conserves three potential threonine phosphorylation sites with other members of the STLK-subfamily of STE20-related kinases (human STE20, MST3, STLK2, STLK4) that could serve an autoregulatory role on kinase activity.
  • The 181 amino acid C-terminal domain of human STLK3 shares 55.5% amino acid identity to human STLK4 (SEQ ID NO:7), and is 100% identical to a partial human cDNA DCHT (GB:AF017635). The C-terminal domain of human STLK3 contains a 26 amino acid insert relative to human STE20. A similar (87.5% amino acid identity) 26 amino acid insert is also present in human STLK4. [0229]
  • The 27 amino acid C-terminal tail of human STLK3 shares 77.8% amino acid identity to human STLK4, but is absent from other STLK-family members. This high degree of homology between the C-tail of two STLK-family members suggests they may be involved in an as yet unidentified protein-protein interaction. [0230]
  • The weak sequence homology between the C-termini of human STLK3 and STE20, suggests it may also function as an inhibitory domain for its kinase. [0231]
  • Mammalian STLK4 [0232]
  • The 3857 bp human STLK4 nucleotide sequence of the partial cDNA clone encodes a polypeptide of 414 amino acids (SEQ ID NO:7) with a predicted molecular mass of 45,451 daltons. Analysis of the deduced amino acid sequence predicts STLK4 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-terminus is not known. The partial STLK4 protein sequence contains a 178 amino acid catalytic domain corresponding to the C-terminal motifs VI-XI of a serine/threonine kinase, followed by a 236 amino acid C-terminal domain containing two inserts of 25 and 41 amino acids each, relative to the sequence of human STLK2. [0233]
  • STLK4 is most closely related to human STE20-subfamily kinases, STLK3 (SEQ ID. NO 6), MST3 (GB:AF024636), STLK2 (SEQ ID NO:5), and SOK-1 (GB:X99325) sharing 71.0%, 46.8%, 43.9%, and 37.7% amino acid identity, respectively. [0234]
  • The 178 amino acid catalytic domain of human STLK4 is most related to human STLK3 (SEQ ID NO. 7), SOK-1 (GB:X99325), MST3 (GB:AF024636), STLK2 (SEQ ID NO:5), and MST1 (GB:U18297), sharing 88.2%, 54.2%, 54.0%, 53.7 and 45.7% amino acid identity, respectively. It also shares strong homology to other STKs from lower organisms including 56.9% to [0235] A. thaliana (GB: AC002343), 52.5% to C. elegans (GB: U53153), 46.2% to A. thaliana (GB: Z97336) and 45.7% to A. thaliana (GB: U96613). The activation loop of the human STLK4 catalytic domain conserves three potential threonine phosphorylation sites with other members of the STLK-subfamily of STE20-related kinases (human STE20, MST3, STLK2 and STLK3) that could serve an autoregulatory role on kinase activity.
  • The 236 amino acid C-terminal domain of human STLK4 shares 58.1% amino acid identity to both human STLK3 (SEQ ID NO:6) and to a partial human cDNA, DCHT (GB:AF017635). The C-terminal domain of human STLK4 contains a 25 amino acid insert relative to human SOK-1 and shares 87.5% amino acid identity to an insert present in human STLK3. [0236]
  • The weak sequence homology between the C-termini of human STLK4 and STE20, suggests it may also function as an inhibitory domain for its kinase. [0237]
  • Mammalian STLK5 [0238]
  • The full-length 2110 bp human STLK5 cDNA encodes a polypeptide of 373 amino acids (SEQ ID NO:97) with a predicted molecular mass of 41,700 daltons. Analysis of the deduced amino acid sequence predicts STLK5 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and transmembrane domain. STLK5 contains a 10 amino acid N-terminal domain, a 311 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, and a 52 amino acid C-terminal domain. [0239]
  • STLK5 is most closely related to the human STE20-subfamily kinases STLK6 (SEQ ID No. 99) and SPAK (AFO99989), sharing 51% and 33% amino acid identity, respectively, over its full extent. It also shares significant homology to database entries from [0240] Arabidopsis thaliana (GB:AC002343) and C. elegans (GB:AL023843, GB:AL023843).
  • The 10 amino acid N-terminal domain of human STLK5 does not reveal any significant homologies to the protein database. [0241]
  • The 311 amino acid catalytic domain of human STLK5 shares 51% and 34% identity to STLK6 and SPAK, respectively. The catalytic domain of STLK5 contains a 45 amino acid insert between kinase subdomains X and XI relative to human STE20. Multiple human EST fragments as well as a murine EST (GB:AA575647) contain this insert providing evidence that this region is an integral part of STLK5. [0242]
  • The 52 amino acid C-terminal tail of human STLK5 shares 41.3% amino acid identity to human SOK-1 (GB:X99325). The weak sequence homology between the C-termini of human STLK5 and STE20, suggests it may also function as an inhibitory domain for its kinase. [0243]
  • Mammalian STLK6 [0244]
  • The 2,001 bp human STLK6 nucleotide sequence of the complete cDNA encodes a polypeptide of 418 amino acids (SEQ ID NO:99) with a predicted molecular mass of 47,025 daltons. Analysis of the deduced amino acid sequence predicts STLK6 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and transmembrane domain. STLK6 contains a 57 amino acid N-terminal domain, a 312 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, followed by a 49 amino acid C-terminal domain. [0245]
  • STLK6 is most closely related to human STE20-subfamily kinases STLK5 (SEQ ID NO:97), STLK7 (SEQ ID NO:101), and SPAK (AFO99989), sharing 50%, 35%, and 30% amino acid identity over its full extent. It also shares significant homology to database entries from [0246] Arabidopsis thaliana (GB:AC002343) and C. elegans (GB:U53153).
  • The 57 amino acid N-terminal domain of human STLK6 does not reveal any significant homologies in the protein database. [0247]
  • The 312 amino acid catalytic domain of human STLK6 shares 51 and 30% identity to human STLK5 and SPAK, respectively. [0248]
  • The 49 amino acid C-terminal tail of human STLK6 shares low amino acid sequence identity (29%) with STLK5 and SPAK. [0249]
  • Mammalian STLK7 [0250]
  • The 311 bp human STLK7 nucleotide sequence of the partial cDNA encodes a polypeptide of 103 amino acids (SEQ ID NO: 101). Analysis of the deduced amino acid sequence predicts STLK7 to be an internal fragment of an intracellular STE20-family kinase. This sequence lacks the N- and C-terminal portions of STLK7 and contains only the N-[0251] terminal 103 amino acids of the predicted catalytic domain.
  • Human STLK7 is most closely related to human STE20-subfamily kinases SPAK (AFO99989), STLK5 (SEQ ID NO:97), and STLK6 (SEQ ID NO:99), sharing 86%, 38%, and 35% amino acid identity within this region of the kinase domain. It also shares significant homology to database entries from [0252] Arabidopsis thaliana (GB:AC002343) and Drosophila melanogaster (GB:AF006640).
  • Mammalian ZC1 [0253]
  • The 3798 bp human ZC1 nucleotide sequence encodes a polypeptide of 1239 amino acids (SEQ ID NO: 13) with a predicted molecular mass of 142,140 daltons. Analysis of the deduced amino acid sequence predicts ZC1 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. The full-length ZC1 protein contains a 22 amino acid N-terminus, a 267 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 237 amino acid region predicted to form a coiled-coil structure, a 114 amino acid proline-rich region, a 256 amino acid spacer region, followed by a 343 amino acid C-terminal domain containing a potential Rab/Rho-binding region. [0254]
  • ZC1 is most closely related to the human STE20-subfamily kinases ZC2 (SEQ ID NO:14), ZC3 (SEQ ID NO:15), and ZC4 (SEQ ID NO:16), sharing 61.7%, 60.9%, and 43.8% amino acid identity, respectively. ZC1 also shares 45.5% amino acid identity to a [0255] C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029). ZC1 exhibits 90.0% amino acid homology to murine NIK (GB:U88984), suggesting it may be the human orthologue of this STK.
  • The 22 amino acid N-terminal domain of human ZC1 is 58.8% identical to the [0256] C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029), and 100% identical to murine NIK (GB: U88984). Human ZC1 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • The 267 amino acid catalytic domain of human ZC1 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15), ZC2 (SEQ ID NO: 14), KHS2 (SEQ ID NO:18), SOK-1 (GB:X99325), GCK (GB:U07349), and GEK2 (SEQ ID NO:107), and to the [0257] C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029) sharing 90.6%, 90.2%, 50.6%, 47.4%, 45.4%, 42.5% and 82.6% amino acid identity, respectively. The ZC1 kinase domain shares 98.1% identity to murine NIK (GB:U88984). ZC1 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • Immediately C-terminal to the kinase domain of human ZC1 is a 237 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (Lupas, A. Meth. Enzymol. 266, 513-525 (1996)). This region of ZC1 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15), ZC2 (SEQ ID NO: 14), and GEK2 (SEQ ID NO: 107), as well as to human PITSLRE (GB:U04824) sharing 65.5%, 65.4%, 25.3%, and 29.0% amino acid identity, respectively. The ZC1 coiled-coil domain also shares 90.6% amino acid homology to murine NIK. The [0258] C. elegans homologue ZC504.4 shares 32.2% sequence identity over this region.
  • Within the predicted coiled-coil domain of human ZC1, and the related ZC3, is a region predicted to form a leucine zipper (Leu-X6-Leu-X6-Leu-X6-Leu-X20-Leu-X6-Leu) (SEQ ID NO: 149). The fact that this leucine repeat exists within a predicted coiled-coil structure suggests that the leucine zipper may have a high probability of serving as a dimerization interface (Hirst, J. D. et al Protein Engineering 9657-662 (1996)) mediating a potential inter- or intra-molecular dimerization of human ZC1. [0259]
  • The 114 amino acid proline-rich region of human ZC1 is most related to human STE20-subfamily kinases, ZC2 (SEQ ID NO: 14) and ZC3 (SEQ ID NO: 15), sharing 35.8%, and 24.9%, respectively. The ZC1 proline-rich domain shares 36.4% amino acid homology to murine NIK (GB:U88984). Three potential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (I, II and III) are found within the proline-rich region of human ZC1. Motif I is conserved in human ZC1 and [0260] C. elegans ZC504.4 (GB:Z50029). Motif II is conserved in ZC1, ZC2, ZC3, ZC4 and C. elegans ZC504.4. Motif III is conserved in ZC1, ZC2, ZC3 and ZC4. Motifs II and III of murine NIK have been shown to bind the SH3 motif of the adaptor molecule Nck (Su, Y-C. et al, EMBO J. 16, 1279-1290 (1997)). From this evidence, human ZC1 may have the potential to bind to Nck or other SH3 or WW domain-containing proteins and participate in growth factor-induced signaling pathways.
  • The 256 amino acid spacer region of human ZC1 is most related to human STE20-subfamily kinases, ZC2 (SEQ ID NO: 14) and ZC3 (SEQ ID NO: 15), as well as to human PITSLRE (GB:U04824), sharing 59.9%, 33.1%, 29.6%, and 26.4% amino acid identity, respectively. It also shares 59.9% amino acid homology to murine NIK. The [0261] C. elegans homologue ZC504.4 has only limited sequence similarity in this spacer region.
  • The 343 amino acid C-terminal of human ZC1 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15), ZC2 (SEQ ID NO: 14), and ZC4 (SEQ ID NO:16), sharing 89.2%, 88.9%, and 42.3%, amino acid identity, respectively. The ZC1 C-terminal domain also shares 98.8% amino acid identity to murine NIK. The [0262] C. elegans homologue ZC504.4 also shares 68.7% amino acid identity with the C-tail of human ZC1. A lower, yet significant, homology is also evident to human KHS2 (SEQ ID NO: 18), GCK (GB:U07349), and murine citron (GB:U07349) with 26.6%, 23.1% and 36.2% amino acid identity, respectively. GCK is a STE20-family kinase whose C-terminal domain has been shown to bind the small G-protein Rab8 (Ren, M. et al., Proc. Natl. Acad. Sci. 93, 5151-5155 (1996)). Citron is a non-kinase Rho-binding protein (Madaule, P. et al., FEBS Lett. 377, 243-238 (1995)).
  • The sequence similarity of the C-terminal region of ZC1 to proteins that have potential Rab- or Rho-binding domains suggests that ZC1 may signal through a small G-protein-dependant pathway. [0263]
  • Mammalian ZC2 [0264]
  • The 4055 bp human ZC2 nucleotide sequence of the partial cDNA encodes a polypeptide of 1297 amino acids (SEQ ID NO:14) with a predicted molecular mass of 147,785 daltons. Analysis of the deduced amino acid sequence predicts ZC2 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-terminus is not known. The N-terminally truncated ZC2 protein contains a 255 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 187 amino acid region predicted to form a coiled-coil structure, a 184 amino acid proline-rich region, a 328 amino acid spacer region, followed by a 343 amino acid C-terminal domain containing a potential Rab/Rho-binding region. [0265]
  • ZC2 is most closely related to the human STE20-subfamily kinases ZC3 (SEQ ID NO:15), ZC1 (SEQ ID NO:13), and ZC4 (SEQ ID NO:16), sharing 88.3%, 61.7%, and 41.9% amino acid identity, respectively, and shares 41.7% amino acid identity to a [0266] C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029).
  • The 255 amino acid catalytic domain of human ZC2 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO: 13), ZC3 (SEQ ID NO: 15), SOK-1 (GB:X99325), KHS2 (SEQ ID NO:18), MST1 (GB:U18297), and GCK (GB:U07349), and to the [0267] C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029) sharing 90.2%, 89.8%, 49.0%, 48.6%, 47.9%, 45.0 and 76.7% amino acid identity, respectively. ZC2 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • Immediately C-terminal to the kinase domain of human ZC2 is a 187 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (supra). This region of ZC2 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and GEK2 (SEQ ID NO:107), as well as to human PITSLRE (GB:U04824), sharing 65.8%, 61.5%, 29.7% and 29.6% amino acid identity, respectively. The [0268] C. elegans homologue ZC504.4 shares 30.8% sequence identity over this region. Human ZC2 lacks the potential leucine zipper found in ZC1 as a consequence of a 29 amino acid deletion relative to ZC1 and ZC3.
  • The 184 amino acid proline-rich region of human ZC2 is most related to human STE20-subfamily kinases, ZC3 (SEQ ID NO: 15) and ZC1 (SEQ ID NO: 13), sharing 35.9% and 28.6%, amino acid identity, respectively. Significant homology is also evident to the murine WW domain-binding protein WBP7 (GB:U92455), and to the human SH3 domain-binding protein 3BP-1 (GB:X87671), with 27.7% and 25.3% amino acid identity, respectively. [0269]
  • ZC2 contains two of the potential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (II and III) found within the proline-rich region of human ZC1. Motif II is conserved in ZC1, ZC3, ZC4 and [0270] C. elegans ZC504.4, and Motif III is conserved in ZC1, ZC3 and ZC4. Motifs II and III of murine NIK have been shown to bind the SH3 motif of the adaptor molecule Nck. From this evidence, human ZC1 may have the potential to bind to Nck or other SH3 or WW domain-containing proteins, and to participate in growth factor-induced signaling pathways.
  • The 328 amino acid spacer region of human ZC2 is most related to human STE20-subfamily kinases ZC1 (SEQ ID NO:13) and ZC3 (SEQ ID NO:15), and to murine NIK (GB:U88984), sharing 31.6%, 26.9% and 25.9% amino acid identity, respectively. The [0271] C. elegans homologue ZC504.4 has only limited sequence similarity in this spacer region.
  • The 343 amino acid C-terminal of human ZC2 is most related to human STE20-subfamily kinases ZC1 (SEQ ID NO: 13), ZC3 (SEQ ID NO: 15) and ZC4 (SEQ ID NO:16), and to murine NIK (GB:U88984), sharing 88.9%, 88.3%, 41.9%, and 88.0%, amino acid identity, respectively. The [0272] C. elegans homologue, ZC504.4, also shares 67.2% amino acid identity with the C-tail of human ZC2. A lower, yet significant, homology is also evident to human GCK (GB:U07349), murine citron (GB:U07349), and the S. cerevisiae ROM2 protein (GB:U19103), a Rho1 GDP/GTP exchange factor, with 22.3%, 22.2% and 21.9% amino acid identity, respectively.
  • The sequence similarity of the C-terminal region of ZC2 to proteins that have potential Rab- or Rho-binding domains suggests that ZC2, like ZC1, may also signal through a small G-protein-dependant pathway. [0273]
  • Mammalian ZC3 [0274]
  • The 4133 bp human ZC3 nucleotide sequence of the partial cDNA encodes a polypeptide of 1326 amino acids (SEQ ID NO: 15) with a predicted molecular mass of 149,906 daltons. Analysis of the deduced amino acid sequence predicts ZC3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain, however the cDNA clone lacks an initiating ATG, so the full extent of it N-termius is not known. The N-terminally truncated ZC3 protein contains a 255 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase: a 221 amino acid region predicted to form a coiled-coil structure, a 204 amino acid proline-rich region, and a 303 amino acid spacer region followed by a 343 amino acid C-terminal domain containing a potential Rab/Rho-binding region. [0275]
  • ZC3 is most closely related to the human STE20-subfamily kinases ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14), and ZC4 (SEQ ID NO:16), sharing 62.0%, 61.0%, and 42.5% amino acid identity, respectively and shares 46.7% amino acid identity to a [0276] C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029).
  • The 255 amino acid catalytic domain of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO: 13), ZC2 (SEQ ID NO: 14), SOK-1 (GB:X99325), KHS2 (SEQ ID NO:18), GCK (GB:U07349), SULU1 (SEQ ID NO:22), and GEK2 (SEQ ID NO: 107), and to the [0277] C. elegans kinase encoded by the cosmid ZC504.4 (GB:Z50029) sharing 90.6%, 89.3%, 49.0%, 48.3%, 45.0%, 43.1%, 42.3% and 76.7% amino acid identity, respectively. ZC1 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • Immediately C-terminal to the kinase domain of human ZC3 is a 221 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (supra). This region of ZC3 is most homologous to human STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14), and GEK2 (SEQ ID NO:107), sharing 66.9%, 61.5%, and 27.5% identity, as well as to rat PLC-beta (GB:A45493) and human PITSLRE (GB:H54024) sharing 29.6% and 25.9% amino acid identity, respectively. The [0278] C. elegans homologue ZC504.4 shares 26.8% sequence identity over this region.
  • Within the predicted coiled-coil domain of human ZC3, and the related ZC1, is a region predicted to form a leucine zipper (Leu-X6-Leu-X6-Leu-X6-Leu-X20-Leu-X6-Leu) (SEQ ID NO: 149). The fact that this leucine repeat exists within a predicted coiled-coil structure suggests that the leucine zipper may have a high probability of serving as a dimerization interface (Hirst, J. D. et al Protein Engineering 9657-662 (1996)) mediating a potential inter- or intra-molecular dimerization of human ZC3. [0279]
  • The 204 amino acid proline-rich region of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO: 13) and ZC2 (SEQ ID NO: 14), sharing 66.9% and 61.5% amino acid identity, respectively. [0280]
  • ZC3 contains two of the potential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (II and III) found within the proline-rich region of human ZC1. Motif II is conserved in ZC1, ZC2, ZC4 and [0281] C. elegans ZC504.4; Motif III is conserved in ZC1, ZC2 and ZC4. Motifs II and III of murine NIK have been shown to bind the SH3 motif of the adaptor molecule Nck. From this evidence, human ZC3 may have the potential to bind to Nck or other SH3 or WW domain-containing proteins and participate in growth factor-induced signaling pathways.
  • The 303 amino acid acid spacer region of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO: 13) and ZC2 (SEQ ID NO: 14) sharing 30.1%, and 27.1% amino acid identity, respectively. The [0282] C. elegans homologue ZC504.4 lacks nearly the entire spacer region of ZC3.
  • The 343 amino acid C-terminal of human ZC3 is most related to human STE20-subfamily kinases, ZC1 (SEQ ID NO:13), ZC2 (SEQ ID NO:14) and ZC4 (SEQ ID NO: 16), sharing 89.2%, 88.9%, and 42.5%, amino acid identity, respectively. The [0283] C. elegans homologue ZC504.4 also shares 67.2% amino acid identity with the C-tail of human ZC3. A lower, yet significant, homology is also evident to human GCK (GB:U07349), as well as to the non-kinases murine citron (GB:U07349) and the S. cerevisiae ROM2 protein (GB:U 19103), a Rho1 GDP/GTP exchange factor, with 21.6%, 32.4% and 22.9% amino acid identity, respectively.
  • The sequence similarity of the C-terminal region of ZC3 to proteins that have potential Rab- or Rho-binding domains suggests that ZC3, like ZC1 and ZC2, may signal through a small G-protein-dependant pathway. [0284]
  • Mammalian ZC4 [0285]
  • The 3,684 bp human ZC4 nucleotide sequence of the complete cDNA encodes a polypeptide of 1,227 amino acids (SEQ ID NO:105) with a predicted molecular mass of 138,205 Daltons. Analysis of the deduced amino acid sequence predicts ZC4 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and a transmembrane domain. The full-length ZC4 protein contains a 25 amino acid N-terminus, a 265 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 108 amino acid region predicted to form a coiled-coil structure, a 231 amino acid proline-rich region, a 40 amino acid region predicted to form a coiled-coil structure spacer region, a 204 amino acid spacer region (domain B), followed by a 355 amino acid C-terminal domain containing a potential Rab/Rho-binding region (domain C). [0286]
  • ZC4 is most closely related to human ZC1 (SEQ ID NO:13, also known as human HGK, human KIAA0687, murine NIK, human AC005035, human NIK, and [0287] C. elegans MIG-15), ZC2 (SEQ ID NO:14, similar to partial sequence human KIAA0551), and ZC3 (SEQ ID NO:15). An assembled genomic fragment in the database (Z83850) is identical to ZC4, except for inappropriate identification of the exon boundaries. (Abo et al. (1998) EMBO J. 17: 6527-6540.)
  • The 25 amino acid N-terminal domain of human ZC4 shares weak homology to human ZC1 in its C-terminal extent, but otherwise does not reveal any significant homologies to the protein database. [0288]
  • The 265 amino acid catalytic domain of human ZC4 is most related to human ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO:14), sharing 63%, 64% and 62% amino acid identity, respectively. [0289]
  • Immediately C-terminal to the kinase domain of human ZC4 is a 108 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (supra). This region is most related to human ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO: 14), sharing 29%, 25% and 20% amino acid identity, respectively. [0290]
  • The 231 amino acid proline-rich region of human ZC4 does not reveal any significant homologies to the protein database. This region of ZC4 contains two “PxxP” (SEQ ID NO: 148) motifs that could potentially bind to proteins containing SH3 or WW domains and allow ZC4 to participate in growth factor activated signaling pathways. In addition, within the pro-rich domain of human ZC4 is a region predicted to form a leucine zipper (Leu-X6-Leu-X6-Leu-X6-Leu-X20-Leu-X6-Leu) (SEQ ID NO: 149) which may serve as a dimerization interface. The ZC STE20 subfamily kinases (ZC1, ZC2 and ZC3) have similarly located “PxxP” (SEQ ID NO: 148) motifs and potential Leu zippers. [0291]
  • Immediately C-terminal to the proline-rich region of human ZC4 is a 40 amino acid region also predicted to form a coiled-coil structure based on the Lupas algorithm. This region of human ZC4 does not reveal any significant homologies to the protein database. [0292]
  • The 204 amino acid acidic- and serine-rich domain “B” of ZC4 does not reveal any significant homologies to the protein database. [0293]
  • The 355 amino acid C-terminal of human ZC4 is most related to human ZC1 (SEQ ID NO:13), ZC3 (SEQ ID NO:15), and ZC2 (SEQ ID NO:14), sharing 43%, 42% and 42% amino acid identity, respectively. [0294]
  • The sequence similarity of the C-terminal region of ZC4 to proteins that have potential Rab- or Rho-binding domains suggests that ZC4, like other ZC-subfamily STE20 kinases, may signal through a small G-protein-dependant pathway. Mammalian KHS2 [0295]
  • The 4023 bp human KHS2 nucleotide sequence encodes a polypeptide of 894 amino acids (SEQ ID NO: 18) with a predicted molecular mass of 101,327 daltons. Analysis of the deduced amino acid sequence predicts KHS2 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. The full-length KHS2 protein contains a 13 amino acid N-terminus, a 260 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 73 amino acid spacer region, a 188 proline-rich region, followed by a 360 amino acid C-terminal domain containing a potential Rab/Rho-binding site. [0296]
  • KHS2 is most closely related to the human STE20-subfamily kinases KHS1 (GB:U177129), GCK (GB:U07349), and HPK1 (GB:U07349), sharing 65.5%, 51.9%, and 44.9% amino acid identity, respectively and shares 38.5% amino acid identity to a [0297] C. elegans STK (GB:U55363).
  • The 13 amino acid N-terminal domain of human KHS2 does not reveal any significant homologies that might suggest a potential function for this domain when examined by a Smith-Waterman alignment to the nonredundant protein database. Human KHS2 lacks a glycine residue at [0298] position 2, and is therefore unlikely to undergo myristylation.
  • The 260 amino acid catalytic domain of human KHS2 is most related to human STE20-subfamily kinases KHS1 (GB:U177129), GCK (GB:U07349), HPK1 (GB:U66464), SOK-1 (GB:X99325), MST1 (GB:U18297), ZC1 (SEQ ID NO:13), and to the [0299] C. elegans kinase (GB:U55363), sharing 85.4%, 75.1%, 67.7%, 51.4%, 48.1%, 49.8% and 72.0% amino acid identity, respectively. KHS2 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, SULU1, SULU3, PAK4 and PAK5.
  • The 73 amino acid acid spacer region of human KHS2 is most related to human STE20-subfamily kinases, KHS1 (GB:U177129), HPK1 (GB:U66464) and GCK (GB:U07349), sharing 60.3%, 43.5% and 44.0%, amino acid identity, respectively. [0300]
  • The 188 amino acid proline-rich region of human KHS2 is most related to human STE20-subfamily kinases, HPK1 (GB:U66464), GCK (GB:U07349) and KHS1 (GB:U177129), sharing 33.3%, 31.9% and 31.4%, amino acid identity, respectively. [0301]
  • Two potential “PxxP” (SEQ ID NO: 148) SH3 domain-binding motifs (I and II) are found within the proline-rich region of human KHS2. Motif I is conserved with human KHS1 and HPK1; motif II is conserved with GCK and KHS2. A 192 amino acid region of human HPK1 containing motif II has been shown to bind to the C-terminal SH3 motif of the adaptor molecule Grb2 (Anafi, M et al, J. Biol. Chem. J. 272, 27804-27811 (1997)). Human KHS2 may bind SH3 or WW domain-containing proteins through this proline-rich region. [0302]
  • The 360 amino acid C-terminal of human KHS2 is most related to KHS1 (GB:U177129), GCK (GB:U07349) and HPK1 (GB:U66464), and to the [0303] C. elegans kinase (GB:U55363), sharing 74.9%, 54.8%, 42.9%, and 31.0%, amino acid identity, respectively. GCK is a STE20-family kinase whose C-terminal domain has been shown to bind the small G-protein Rab8 (Ren, M. et al., Proc. Natl. Acad. Sci. 93, 5151-5155 (1996)).
  • Mammalian SULU1 [0304]
  • The 4196 bp human SULU1 nucleotide sequence encodes a polypeptide of 898 amino acids (SEQ ID NO:22) with a predicted molecular mass of 105,402 daltons. Analysis of the deduced amino acid sequence predicts SULU1 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. The full-length SULU1 protein contains a 21 amino acid N-terminus, a 256 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 150 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure, a 114 amino acid spacer region and a 147 amino acid C-terminal domain predicted to form a coiled-coil structure. [0305]
  • SULU1 is most closely related to the STE20-subfamily kinases murine SULU3 (SEQ ID NO:24), human SULU3 (SEQ ID NO:23), and to the [0306] C. elegans kinase SULU (GB:U11280), sharing 68.9%, 72.2% and 38.2% amino acid identity, respectively.
  • The 21 amino acid N-terminal domain of human SULU1 is most related to murine SULU3 (SEQ ID NO:24) and to the [0307] C. elegans kinase SULU (GB:U11280), sharing 86.3% and 62.3% amino acid identity. Human SULU1 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristoylation. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • The 256 amino acid catalytic domain of human SULU1 is most related to murine SULU3 (SEQ ID NO:24), and to human SOK-1 (GB:X99325), STLK2 (SEQ ID NO:5), MST1 (GB:U18297), PAK1 (GB:U24152), ZC2 (SEQ ID NO:14), and KHS2 (SEQ ID NO:18) sharing 86.3%, 48.1%, 46.9%, 45.2%, 43.3%, 43.1% and 42.0% amino acid identity, respectively. The [0308] C. elegans SULU STK (GB:U11280) shares 62.3% sequence identity over this region. SULU1 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU3, PAK4 and PAK5.
  • The 150 amino acid spacer region of human SULU1 is most related to human SULU3 (SEQ ID NO:23) and to the [0309] C. elegans kinase (GB:U11280), sharing 53.5% and 10.4% amino acid identity, respectively.
  • Immediately C-terminal to the spacer region of human SULU1 is a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of SULU1 is most related to SULU3 (SEQ ID NO:23), the [0310] C. elegans SULU kinase (GB:U11280), GEK 2 (SEQ ID NO:107) and ZC1 (SEQ ID NO:13), sharing 68.6%,26.8%,23.2%, and 22.8% amino acid identity, respectively.
  • The 114 amino acid spacer region human SULU1 is most related to human SULU3 (SEQ ID NO:24) with 73.7% amino acid sequence identity. A lower, yet significant, homology is also evident to murine PITSLRE (GB:U04824) and DLK (GB:A55318), human ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107), as well as to the [0311] C. elegans SULU STK (GB:U11280), sharing 39.7%, 35.4%, 29.5%, 23.6% and 37.6% amino acid identity, respectively.
  • Immediately C-terminal to the second spacer region of human SULU1 is a 147 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of SULU1 is most related to human SULU3 (SEQ ID NO:24), ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107), as well as to the [0312] C. elegans SULU STK (GB:U11280), sharing 73.3%, 28.4%, 26.1% and 39.5%, amino acid identity, respectively.
  • Mammalian (human) SULU3 [0313]
  • The 3824 bp partial cDNA human SULU3 nucleotide sequence encodes a polypeptide of 786 amino acids (SEQ ID NO:23) with a predicted molecular mass of 92,037 daltons. Analysis of the deduced amino acid sequence predicts SULU3 to be an intracellular serine/threonine kinase lacking a transmembrane domain. The N-terminally truncated human SULU3 protein contains a 66 amino acid partial catalytic domain followed by a 149 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure, a second spacer region of 114 amino acids, a 247 amino acid C-terminal region predicted to form a second coiled-coil structure and a 100 amino acid C-terminal tail. [0314]
  • Human SULU3 is most closely related murine SULU3 (SEQ ID NO:24), human SULU1 (SEQ ID NO:22), and to the [0315] C. elegans SULU kinase (GB:U 11280), sharing 66.3%, 68.9% and 32.9% amino acid identity, respectively. The high sequence homology between murine and human SULU3 suggests that these two proteins are orthologs of each other.
  • The 66 amino acid partial catalytic domain of human SULU3 is most related to murine SULU3 (SEQ ID NO:24), and to the human STE20 subfamily kinases ZC1 (SEQ ID NO:13), STE20 (GB:X99325), KHS1(GB:U177129) and GEK 2 (SEQ ID NO: 107), as well as to the [0316] C. elegans SULU kinase (GB:U11280), sharing 83.3%, 47.0%, 45.5%, 43.5%,41.8% and 55.6% amino acid identity, respectively.
  • The 149 amino acid spacer region of human SULU3 is most related to murine SULU3 (SEQ ID NO:24), human STE20 (GB:X99325), MST1 (GB:U18297), and to the [0317] C. elegans SULU kinase (GB:U11280) sharing 98.7%, 21.9% and 21.8% amino acid identity, respectively.
  • Immediately C-terminal to the first spacer region of human SULU3 is a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of SULU3 is most related to murine SULU3 (SEQ ID NO:24), and to human SULU1 (SEQ ID NO:22), ZC1 (SEQ ID NO:13) and GEK 2 (SEQ ID NO:107), as well as to the [0318] C. elegans SULU kinase (GB:U11280), sharing 99.5%, 68.6%, 27.4% and 22.5% amino acid identity, respectively.
  • The 114 amino acid second spacer region of human SULU3 is most related to murine SULU3 (SEQ ID NO:24), and to human SULU1 (SEQ ID NO:22) GEK 2 (SEQ ID NO:107), and ZC1 (SEQ ID NO:13), as well as to the [0319] C. elegans SULU kinase (GB:U11280), sharing 99.1%, 73.7%, 24.6%,24.1% and 41.2% amino acid identity, respectively.
  • Immediately C-terminal to the second spacer region of human SULU3 is a 247 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm (supra). This region of SULU3 is most related to human SULU1 (SEQ ID NO:22) and ZC1 (SEQ ID NO:13) as well as to rat PKN-(GB:D26180) murine pl60 ROCK1 (GB:U58512), and the [0320] C. elegans SULU kinase (GB:U11280), sharing 73.7%, 26.7%, 24.0% and 21.0% amino acid identity, respectively.
  • The 100 amino acid C-tail of human SULU3 is most related to a human prion protein (GB:L38993), with 45.0% amino acid identity. [0321]
  • Mammalian (murine) SULU3 [0322]
  • The 2249 bp murine, partial cDNA SULU3 nucleotide sequence encodes a polypeptide of 748 amino acids (SEQ ID NO:24) with a predicted molecular mass of 87,520 daltons. Analysis of the deduced amino acid sequence predicts SULU3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. The partial murine SULU3 protein contains a 25 amino acid N-terminus, a 248 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 149 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure, and a 116 amino acid spacer region. [0323]
  • Murine SULU3 is most closely related to human SULU3 (SEQ ID NO:23) and SULU1 (SEQ ID NO:22), as well as to the [0324] C. elegans SULU kinase (GB:U112 80), sharing 97.0%, 72.3% and 38.4% amino acid identity, respectively. The high sequence homology between murine and human SULU3 suggests that these two proteins are orthologs.
  • The 25 amino acid N-terminal domain of murine SULU3 is most related to human SULU1 (SEQ ID NO:22) and to the [0325] C. elegans SULU kinase (GB:U11280), sharing 70.0% and 44.4% amino acid identity, respectively.
  • Murine SULU3 lacks a glycine residue at [0326] position 2, and is therefore unlikely to undergo myristoylation. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • The 248 amino acid catalytic domain of murine SULU3 is most related to human SULU1 (SEQ ID NO:22), STE20 (GB:X99325), ZC1 (SEQ ID NO:13), and KHS1 (GB:U77129), as well as to the [0327] C. elegans SULU kinase (GB:U11280), sharing 86.7%, 46.6%, 43.3%, 59.4% amino acid identity, respectively. Murine SULU3 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • The 149 amino acid spacer of murine SULU3 is most related to human SULU3 (SEQ ID NO:23), SULU1 (SEQ ID NO:22), and STE20 (GB:X99325), as well as to the [0328] C. elegans SULU (GB:U11280) and the S. cerevisiae STE20 (GB:L04655) kinases, sharing 98.7%, 53.4%, 21.9%, 59.4% and 21.9% amino acid identity, respectively.
  • Immediately C-terminal to the spacer region of murine SULU3 is a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of murine SULU3 is most related to human SULU3 (SEQ ID NO:23), ZC1 (SEQ ID NO:13), and GEK 2 (SEQ ID NO:107), as well as to the [0329] C. elegans SULU kinase (GB:U11280), sharing 99.5%, 27.4%, 22.5% and 29.2% amino acid identity, respectively.
  • The 116 amino acid C-terminal spacer region of murine SULU3 is most related to human SULU3 (SEQ ID NO:23), GEK 2 (SEQ ID NO:107), and ZC1 (SEQ ID NO: 13), well as to the [0330] C. elegans SULU kinase (GB:U11280), sharing 98.3%, 24.6%, 24.1% and 40.5% amino acid identity, respectively.
  • Mammalian (Murine/Human) SULU3 [0331]
  • The 2249 bp murine SULU3 and the 3824 bp human SULU3 cDNAs contain a 1620 nucleotide overlap (541 amino acids) with 90% and 98% DNA and amino acid sequence identity, respectively. Owing to the high degree of sequence identity in this extended overlap, we propose that these are functional orthologues of a single gene. The combined murine/human 4492 bp SULU3 sequence encodes a polypeptide of 1001 amino acids (SEQ ID NO:31) with a predicted molecular mass of 116,069 daltons. Analysis of the deduced amino acid sequence predicts SULU3 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. SULU3 contains a 25 amino acid N-terminus, a 248 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 149 amino acid spacer region, a 210 amino acid region predicted to form a coiled-coil structure and a second spacer region of 114 amino acids, a 247 amino acid C-terminal region predicted to form a second coiled-coil structure and a 100 amino acid C-terminal tail. The murine SULU3 clone lacks the region from the second C-terminal coiled-coil to the C-terminus, whereas the human clone lacks the N-terminal domain, and all but 66 amino acids of the 248 amino acid kinase domain. [0332]
  • SULU3 is most closely related to SULU1 (SEQ ID NO:22) and the [0333] C. elegans SULU kinase (GB:U11280) sharing 72.3% and 38.4% amino acid identity, respectively.
  • The 25 amino acid N-terminal domain of SULU3 is most related to human SULU1 (SEQ ID NO:22) and to the [0334] C. elegans SULU kinase (GB:U11280), sharing 70.0% and 44.4% amino acid identity, respectively. SULU3 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • The 248 amino acid catalytic domain of SULU3 is most related to human SULU1 (SEQ ID NO:22), SOK-1 (GB:X99325), ZC1 (SEQ ID NO:13), KHS1 (GB:U77129) and the [0335] C. elegans SULU kinase (GB:U11280), sharing 86.7%, 46.6%, 43.3%, 42.0% and 59.4% amino acid identity, respectively. SULU3 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, PAK4 and PAK5.
  • The 149 amino acid spacer of SULU3 is most related to SULU1 (SEQ ID NO:22) and SOK-1 (GB:X99325), and to the [0336] C. elegans SULU (GB:U11280), and S. cerevisiae STE20 (GB:L04655) kinases, sharing 53.4%, 21.9%, 59.4% and 21.9% amino acid identity, respectively.
  • Immediately C-terminal to the spacer region of SULU3 is a 210 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region is most related to ZC1 (SEQ ID NO:13), GEK 2 (SEQ ID NO:107), and the [0337] C. elegans SULU kinase (GB:U11280), sharing 27.4% 22.5% and 29.2% amino acid identity, respectively.
  • The 114 amino acid spacer region of SULU3 is most related to human SULU1 (SEQ ID NO:22), GEK 2 (SEQ ID NO:107), ZC1 (SEQ ID NO:13), and to the [0338] C. elegans SULU kinase (GB:U11280), sharing 73.7%, 24.6%, 24.1% and 41.2% amino acid identity, respectively.
  • Immediately C-terminal to the second spacer region of SULU3 is a 247 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of SULU3 is most related to human SULU1 (SEQ ID NO:22) and ZC1 (SEQ ID NO:13), as well as to rat PKN (GB:D26180), murine pl60 ROCK1 (GB:U58512) and the [0339] C. elegans SULU kinase (GB:U11280), sharing 73.7%, 26.7%, 24.0%, 21.0% and 37.6% amino acid identity, respectively.
  • The 100 amino acid C-tail of SULU3 is most related to a human prion protein (GB:L38993) with 45.0% amino acid identity. [0340]
  • Mammalian GEK2 [0341]
  • The 2926 bp human GEK2 nucleotide sequence of the complete cDNA encodes a polypeptide of 968 amino acids (SEQ ID NO: 107) with a predicted molecular mass of 112,120 daltons. Analysis of the deduced amino acid sequence predicts GEK2 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. The complete GEK2 protein contains a 33 amino acid N-terminus, a 261 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, a 43 amino acid spacer region, a 135 amino acid proline-rich region, a 252 amino acid region predicted to form a coiled-coil structure followed by a 244 amino acid region also predicted to form a coiled-coil structure. [0342]
  • GEK2 is most closely related to rat AT1-46 (GB:U33472) (a partial cDNA that extends from the middle of the first potential coiled-coil domain of GEK2 to the C-terminus), murine LOK (GB:D89728), [0343] Xenopus laevis polo-like kinase 1 (GB:AF100165), and human SLK (GB:AB002804), sharing 91.3%, 88.5%, 65.0%, and 44.7% amino acid identity, respectively. The high sequence homology between human GEK2, murine LOK and rat AT 1-46 suggests that human GEK2 is a highly related protein to the rodent forms, or alternatively, its orthologue. Recently, a full-length version of GEK2 was reported (STK10 or human LOK AB015718). The 968 amino acid sequence is 99% identical to GEK2 (SEQ ID NO:107).
  • The 33 amino acid N-terminal domain of human GEK2 is most related to murine LOK (GB:D89728) and to human SLK (GB:AB002804), sharing 100% and 54.5% amino acid identity, respectively. [0344]
  • Human GEK2 lacks a glycine residue at [0345] position 2, and is therefore unlikely to undergo myristylation. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • The 261 amino acid catalytic domain of human GEK2 is most related to murine LOK (GB:D89728), rat AT1-46 (GB:D89728) and human SLK (GB:AB002804) as well as to a [0346] C. elegans kinase (GB:Z81460), sharing 97.7%, 90.8%, 54.5% and 55.9% amino acid identity, respectively. GEK2 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3, PAK4 and PAK5.
  • The 43 amino acid spacer region of human GEK2 is most related to murine LOK (GB:D89728) and to human SLK, sharing 83.7% and 77.6% amino acid identity, respectively. [0347]
  • The 135 amino acid proline-rich region of human GEK2 is most related to murine LOK (GB:D89728) with 66.2% amino acid identity, respectively. Within the proline-rich region of human GEK2 is a potential “PxxP” (SEQ ID NO: 148) SH3-binding domain conserved with murine LOK. [0348]
  • Immediately C-terminal to the proline-rich region of human GEK2 is a 252 amino acid region predicted to form a coiled-coil structure based on the Lupas algorithm. This region of human GEK2 is most related to rat AT1-46 (GB:D89728), murine LOK (GB:D89728) and human SLK (GB:AB002804), and ZC2 (SEQ ID NO:14), sharing 90.8%, 86.9%, 42.2%, and 29.7% amino acid identity, respectively. [0349]
  • Immediately C-terminal to the predicted coiled-coil structure of human GEK2 is a second potential coiled-coil structure of 244 amino acids predicted based on the Lupas algorithm. This region of human GEK2 is most related to rat AT1-46 (GB:D89728) and murine LOK (GB:D89728) as well as to human SLK (GB:AB002804) and ZC1 (SEQ ID NO:13), sharing 91.8%, 92.6%, 70.4% and 26.7% amino acid identity, respectively. The [0350] C. elegans kinase (GB:Z81460) shares 31.5% amino acid sequence identity over this region.
  • Mammalian PAK4 [0351]
  • The 3604 bp human PAK4 nucleotide sequence encodes a polypeptide of 681 amino acids (SEQ ID NO:29) with a predicted molecular mass of 74,875 daltons. Analysis of the deduced amino acid sequence predicts PAK4 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. The full-length PAK4 protein contains a 51 amino acid N-terminus predicted to contain a rac-binding motif, a 173 amino acid insert relative to the known mammalian PAK proteins, a 169 amino acid spacer region, a 265 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase and a 23 amino acid C-terminal tail. [0352]
  • PAK4 is most closely related to human PAK5 (SEQ ID NO:30), PAK1 (GB: U24152), and PAK65 (GB:U25975), as well as to a [0353] C. elegans kinase (GB: Z74029), sharing 76.8%, 49.5%, 49.8%, and 34.6% amino acid identity, respectively.
  • The 51 amino acid N-terminal domain of human PAK4 is most related to human PAK1 (GB:U24152), and PAK65 (GB:U25975), as well as to a [0354] C. elegans kinase (GB: Z74029), sharing 50.0%, 50.0% and 49.0% amino acid identity, respectively.
  • The 10 amino acid region at positions 13-23 of human PAK4 fits the consensus for a Cdc42/Rac-binding motif (SXPX4-6HXXH) (SEQ ID NO: 150) (Burbelo, P. D., Dreschel, D. and Hall, A. J. Bio. Chem. 270, 29071-29074 (1995)). [0355]
  • The 173 amino acid insert of human PAK4, relative to the known mammalian PAK proteins, is most related to a [0356] C. elegans kinase (GB: Z74029) with 39.0% amino acid identity. A Smith-Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this region.
  • The 169 amino acid spacer of human PAK4 does not reveal any significant homologies that might suggest a potential function for this region. [0357]
  • The equivalent spacer region in PAK1 binds to the guanine nucleotide exchange factor PIX (Manser, E. et al (1998) Molecular Cell, 1, 183-192). Since PAK4 differs substantially from PAK1 over this region, the spacer domain of PAK4 may differ in its guanine nucleotide exchange factor binding specificity, relative to PAK1. [0358]
  • The 265 amino acid catalytic domain of human PAK4 is most related to human PAK5 (SEQ ID NO:30), PAK1 (GB:U24152), GCK (GB:U07349), SOK-1 (GB:X99325), and SLK (GB:AB002804), as well as to the [0359] C. elegans (GB: Z74029), and S. cerevisiae STE20-related kinases (GB:L04655), sharing 95.9%, 51.7%, 41.3%, 39.8%, 37.4%, 60.2% and 47.9% amino acid identity, respectively. PAK4 contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3 and PAK5.
  • The 23 amino acid C-tail of human PAK4 contains a sequence that is homologous to a G-protein beta subunit binding site (Leeuw, T. et al. Nature, 391, 191-195 (1998)). PAK4 has, therefore, the potential to be activated by both Cdc42-as well as G-protein-dependant pathways. [0360]
  • Mammalian PAK5 [0361]
  • The 2,806 bp human PAK5 nucleotide sequence of the complete cDNA encodes a polypeptide of 591 amino acids (SEQ ID NO:103) with a predicted molecular mass of 64,071 Daltons. Analysis of the deduced amino acid sequence predicts PAK5 to be an intracellular STE20-subfamily kinase, lacking both a signal sequence and transmembrane domain. The full-length PAK5 protein contains a 52 amino acid N-terminus predicted to contain a p21 (small G-protein) binding domain (PDB or CRIB), a 121 amino acid insert relative to the known mammalian PAK proteins, a 134 amino spacer region, a 265 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase and a 19 amino acid C-terminal tail. [0362]
  • PAK5 is most closely related to Human PAK4 (SEQ ID NO:29), [0363] Drosophila melanogaster PAK (also known as “mushroom bodies tiny”) (AJ01578), C45B11.1b from C. elegans (Z74029), and human PAK3 (Q13177) sharing 48% (327/674 aa), 50% (330/651 aa), 43% (234/435 aa excluding gap), and 47% (190/405 aa excluding gap) amino acid identity, respectively. Recently, a full length version of PAK5 was reported (PAK4 AF005046) whose 591 amino acid sequence is identical to PAK5 (SEQ ID NO:103). (Abo, et al. (1998) EMBO J. 17:6527-6540).
  • The 52 amino acid N-terminal domain of human PAK5 is most related to human PAK4 (SEQ ID NO:29), [0364] Drosophila melanogaster PAK (AJ011578), C45B11.b from C. elegans (Z74029), and human PAK3 (Q13177), sharing 65%, 57%, 54%, and 53% amino acid identity, respectively.
  • The 10 amino acid region at positions 12-22 of human PAK5 (FIG. 18) fits the consensus for a small G-protein binding domain (PDB or CRIB) (SXPX4-6HXXH) (SEQ ID NO: 150) (Burbelo, P. D., Dreschel, D. and Hall, A. J. Bio. Chem. 270, 29071-29074 (1995), hereby incorporated by reference herein in its entirety including any figures, tables, or drawings.). [0365]
  • The 121 amino acid insert of [0366] human PAK5 shares 43% amino acid identity with a similar domain from PAK4 (SEQ ID NO:29), but that is absent from other known PAKs.
  • The equivalent spacer region in PAK1 binds to the guanine nucleotide exchange factor PIX (Manser, E. et al (1998) Molecular Cell, 1, 183-192 hereby incorporated by reference herein in its entirety including any drawings, figures, or tables.). Since PAK5 differs substantially from PAK1 over this region, the spacer domain of PAK5 may differ in its guanine nucleotide exchange factor binding specificity, relative to PAK1. [0367]
  • The 134 amino acid collagen-like region of human PAK5 shares 34% amino acid identity to pro-α I type collagen from several species and is not present in other known PAKs. [0368]
  • The 265 amino acid catalytic domain of human PAK5 is most related to human PAK4 (SEQ ID NO:29), [0369] Drosophila melanogaster PAK (AJ011578), C45B11.1b from C. elegans (Z74029), and human PAK3 (Q13177), sharing 78%, 80%, 61%, and 55% amino acid identity, respectively. PAK5 also contains the potential “TPY” regulatory phosphorylation site in its activation loop. This “TPY” motif is conserved in other STE20-related kinases, including ZC1, ZC2, ZC3, ZC4, GEK2, KHS2, SULU1, SULU3 and PAK4.
  • The 19 amino acid C-tail shares 80% amino acid identity to a PAK-like homologue identified from genomic DNA (AL031652). Furthermore, this C-terminal region of human PAK5 contains a sequence that is homologous to a G-protein beta subunit binding site (Leeuw, T. et al. Nature, 391, 191-195 (1998) hereby incorporated by reference herein in its entirety including any figures, tables, or drawings). PAK5 has, therefore, the potential to be activated by both, Cdc42 as well as G-protein-dependant pathways. [0370]
  • V. Antibodies, Hybridomas, Methods of Use and Kits for Detection of STE20-Related Kinases [0371]
  • The present invention relates to an antibody having binding affinity to a kinase of the invention. The polypeptide may have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107, or a functional derivative thereof, or at least 9 contiguous amino acids thereof (preferably, at least 20, 30, 35, or 40 or more contiguous amino acids thereof). [0372]
  • The present invention also relates to an antibody having specific binding affinity to a kinase of the invention. Such an antibody may be isolated by comparing its binding affinity to a kinase of the invention with its binding affinity to other polypeptides. Those which bind selectively to a kinase of the invention would be chosen for use in methods requiring a distinction between a kinase of the invention and other polypeptides. Such methods could include, but should not be limited to, the analysis of altered kinase expression in tissue containing other polypeptides. [0373]
  • The STE20-Related kinases of the present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction. [0374]
  • The kinases of the present invention can be used to produce antibodies or hybridomas. One skilled in the art will recognize that if an antibody is desired, such a peptide could be generated as described herein and used as an immunogen. The antibodies of the present invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting. [0375]
  • The present invention also relates to a hybridoma which produces the above-described monoclonal antibody, or binding fragment thereof. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody. [0376]
  • In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984; St. Groth et al., J. Immunol. Methods 35:1-21, 1980). Any animal (mouse, rabbit, and the like) which is known to produce antibodies can be immunized with the selected polypeptide. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on the animal which is immunized, the antigenicity of the polypeptide and the site of injection. [0377]
  • The polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity. Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or β-galactosidase) or through the inclusion of an adjuvant during immunization. [0378]
  • For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124, 1988). Hybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell, “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology”, supra, 1984). [0379]
  • For polyclonal antibodies, antibody-containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures. The above-described antibodies may be detectably labeled. Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well-known in the art, for example, see Stemberger et al., J. Histochem. Cytochem. 18:315, 1970; Bayer et al., Meth. Enzym. 62:308-, 1979; Engval et al., Immunol. 109:129-, 1972; Goding, J. Immunol._Meth. 13:215-, 1976. The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues which express a specific peptide. [0380]
  • The above-described antibodies may also be immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, [0381] Chapter 10, 1986; Jacoby et al., Meth. Enzym. 34, Academic Press, N.Y., 1974). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
  • Furthermore, one skilled in the art can readily adapt currently available procedures, as well as the techniques, methods and kits disclosed herein with regard to antibodies, to generate peptides capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides (Hurby et al., “Application of Synthetic Peptides: Antisense Peptides”, In Synthetic Peptides, A User's Guide, W. H. Freeman, NY, pp. 289-307, 1992; Kaspczak et al., Biochemistry 28:9230-9238, 1989). [0382]
  • Anti-peptide peptides can be generated by replacing the basic amino acid residues found in the peptide sequences of the kinases of the invention with acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine. [0383]
  • The present invention also encompasses a method of detecting a STE20-related kinase polypeptide in a sample, comprising: (a) contacting the sample with an above-described antibody, under conditions such that immunocomplexes form, and (b) detecting the presence of said antibody bound to the polypeptide. In detail, the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample. Altered levels of a kinase of the invention in a sample as compared to normal levels may indicate disease. [0384]
  • Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay. One skilled in the art will recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of the present invention. Examples of such assays can be found in Chard (“An Introduction to Radioimmunoassay and Related Techniques” Elsevier Science Publishers, Amsterdam, The Netherlands, 1986), Bullock et al. (“Techniques in Immunocytochemistry,” Academic Press, Orlando, FL Vol. 1, 1982; Vol. 2, 1983; Vol. 3, 1985), Tijssen (“Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1985). [0385]
  • The immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine. The test samples used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is testable with the system utilized. [0386]
  • A kit contains all the necessary reagents to carry out the previously described methods of detection. The kit may comprise: (i) a first container means containing an above-described antibody, and (ii) second container means containing a conjugate comprising a binding partner of the antibody and a label. In another preferred embodiment, the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies. [0387]
  • Examples of detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. The compartmentalized kit may be as described above for nucleic acid probe kits. One skilled in the art will readily recognize that the antibodies described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art. [0388]
  • VI. Isolation of Compounds Which Interact With STE20-Related Kinases [0389]
  • The present invention also relates to a method of detecting a compound capable of binding to a STE20-related kinase of the invention comprising incubating the compound with a kinase of the invention and detecting the presence of the compound bound to the kinase. The compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts. [0390]
  • The present invention also relates to a method of detecting an agonist or antagonist of kinase activity or kinase binding partner activity comprising incubating cells that produce a kinase of the invention in the presence of a compound and detecting changes in the level of kinase activity or kinase binding partner activity. The compounds thus identified would produce a change in activity indicative of the presence of the compound. The compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts. Once the compound is identified it can be isolated using techniques well known in the art. [0391]
  • The present invention also encompasses a method of agonizing (stimulating) or antagonizing kinase associated activity in a mammal comprising administering to said mammal an agonist or antagonist to a kinase of the invention in an amount sufficient to effect said agonism or antagonism. A method of treating diseases in a mammal with an agonist or antagonist of STE20-related kinase activity comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize STE20-related kinase associated functions is also encompassed in the present application. [0392]
  • In an effort to discover novel treatments for diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein kinases. Some small organic molecules form a class of compounds that modulate the function of protein kinases. Examples of molecules that have been reported to inhibit the function of protein kinases include, but are not limited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642, published Nov. 26, 1992 by Maguire et al.), vinylene-azaindole derivatives (PCT WO 94/14808, published Jul. 7, 1994 by Ballinari et al.), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992), styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 Al), seleoindoles and selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny et al.), tricyclic polyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT WO 91/15495, published Oct. 17, 1991 by Dow et al). [0393]
  • Compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous as therapeutics as they can become highly bioavailable after being administered orally to patients. However, many of these protein kinase inhibitors only weakly inhibit the function of protein kinases. In addition, many inhibit a variety of protein kinases and will cause multiple side-effects as therapeutics for diseases. [0394]
  • Some indolinone compounds, however, form classes of acid resistant and membrane permeable organic molecules. WO 96/22976 (published Aug. 1, 1996 by Ballinari et al.) describes hydrosoluble indolinone compounds that harbor tetralin, naphthalene, quinoline, and indole substituents fused to the oxindole ring. These bicyclic substituents are in turn substituted with polar moieties including hydroxylated alkyl, phosphate, and ether moieties. U.S. patent application Ser. No. 08/702,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al. and 08/485,323, filed Jun. 7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al. and International Patent Publication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., all of which are incorporated herein by reference in their entirety, including any drawings, describe indolinone chemical libraries of indolinone compounds harboring other bicyclic moieties as well as monocyclic moieties fused to the oxindole ring. Applications 08/702,232, filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease” by Tang et al., 08/485,323, filed Jun. 7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tang et al., and WO 96/22976, published Aug. 1, 1996 by Ballinari et al. teach methods of indolinone synthesis, methods of testing the biological activity of indolinone compounds in cells, and inhibition patterns of indolinone derivatives. [0395]
  • Other examples of substances capable of modulating kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines. The quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well known compounds such as those described in the literature. For example, representative publications describing quinazolines include Barker et al., EPO Publication No. 0 520 722 A1; Jones et al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No. 4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5,316,553; Kreighbaum and Comer, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO Publication No. 0 562 734 A1; Barker et al., [0396] Proc. of Am. Assoc. for Cancer Research 32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979); Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin et al., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research 43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44(2):173-178; Fry et al., Science 265:1093-1095 (1994); Jackman et al., Cancer Research 51:5579-5586 (1981); Jones et al. J. Med. Chem. 29(6):1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362 (1987); Lemus et al., J. Org. Chem. 54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975); Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Mini et al., Cancer Research 45:325-330 (1985); Phillips and Castle, J. Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al., Cancer Research 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol. and Immunother. 23:A65 (1986); Sikora et al., Cancer Letters 23:289-295 (1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all of which are incorporated herein by reference in their entirety, including any drawings.
  • Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No. 5,316,553, incorporated herein by reference in its entirety, including any drawings. [0397]
  • Quinolines are described in Dolle et al., [0398] J. Med. Chem. 37:2627-2629 (1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burke et al., J. Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganic Med. Chem. Letters 2:1771-1774 (1992), all of which are incorporated by reference in their entirety, including any drawings.
  • Tyrphostins are described in Allen et al., [0399] Clin. Exp. Immunol. 91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker et al., J. Cell Sci. 102:543-555 (1992); Bilder et al., Amer. Physiol. Soc. pp. 6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer. Assoc. Cancer Rsch. 33:558 (1992); Bryckaert et al., Experimental Cell Research 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60 (1993); Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al., J. Med. Chem. 32:2344-2352 (1989); Gazit et al., “J. Med. Chem. 36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994); Kaur et al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al., Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282 (1992); Lyall et al., J. Biol. Chem. 264:14503-14509 (1989); Peterson et al., The Prostate 22:335-345 (1993); Pillemer et al., Int. J. Cancer 50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683 (1993); Rendu et al., Biol. Pharmacology 44(5):881-888 (1992); Sauro and Thomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm. and Experimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J. Biol. Chem. 269(36):22470-22472 (1994); and Yoneda et al., Cancer Research 51:4430-4435 (1991); all of which are incorporated herein by reference in their entirety, including any drawings.
  • Other compounds that could be used as modulators include oxindolinones such as those described in U.S. patent application Ser. No. 08/702,232 filed Aug. 23, 1996, incorporated herein by reference in its entirety, including any drawings. [0400]
  • VII. Biological Significance, Applications and Clinical [0401]
  • Relevance of Novel STE20-Related Kinases [0402]
  • Human STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7 [0403]
  • STLK2, STLK4, STLK5, STLK6 and STLK7 belong to an expanding family of intracellular STKs that have varying degrees of sequence homology to SOK-1, a kinase implicated in oxidative stress agents (Pombo, CM et al, EMBO J. (17) 4537-4546, 1996). Our data shows that STLK2 is expressed highly in hematopoietic cells. Therefore, STLK2 may participate in the oxidative response pathway during inflammation. In addition, STLK2 could also be a possible component in the signaling pathways leading to T cell activation. High levels of STLK2 in several tumor cell lines could also imply that STLK2 might be involved in tumorigenesis. [0404]
  • STLK2 is most closely related to two human STE20-subfamily kinases: MST3 and SOK-1. MST3 is a 52,000 daltons cytoplasmic kinase that is ubiquitously expressed with its highest levels of expression found in heart, skeletal muscle and pancreas. The serine/threonine kinase activity of MST3 is activated by phosphorylation. Unlike SOK-1, MST3 prefers Mn[0405] ++ over Mg++ and can use both GTP and ATP as phosphate donors. MST3 may undergo dimerization. No agonists have yet been identified that activate MST3. The downstream signaling mechanism of this kinase is unknown (Schinkmann, K and Blenis, J. (1997) J. Biol. Chem. 272, 28695-28703).
  • SOK-1 is a 50,000 daltons cytoplasmic kinase expressed predominantly in testis, large intestine, brain and stomach and to a lesser extent in heart and lung. SOK-1 is also expressed in the germinal center B-cell line (RAMOS) and in a mature B cell line (HS Sultan). The serine/threonine kinase activity of SOK-1 is activated by phosphorylation. The C-terminus of SOK-1 has been shown to be inhibitory to the catalytic activity of this kinase. The only agonists known to activate SOK-1 are oxidant agents, like H[0406] 2O2 and menadione, a quinone that is a potent intracellular generator of reactive oxygen species (Pombo, C. M. et al. EMBO J. 15, 4537-4546). SOK-1 is also activated by chemical anoxia through the generation of reactive oxygen species and release of calcium into the cytoplasm from intracellular stores. SOK-1, therefore, may play an important role in ischemia, the cause of myocardial infarction, stroke and acute renal failure (Pombo, C. M. et al. J. Biol. Chem. 272, 29372-29379 (1997)). The activity of SOK-1 in the response to oxidant stress is inversely correlated with the activity of the stress-activated protein kinases (SAPKs): elevated SOK-1 activity correlates with absent SAPK activity and vice-versa. SOK-1 does not activate any of the four MAP kinase pathways, SAPKs, p38, ERK-1 or MEK-5/ERK-5 (Pombo, C. M. et al. EMBO J. 15, 4537-4546). The downstream signaling mechanism of this kinase remains unknown.
  • STLK2 is expressed in a wide variety of immune cell types and tissues including thymus, dendrocytes, mast cells, monocytes, B cells (primary, Jurkat, RPMI, SR), T cells (CD8/CD4+, TH1, TH2, CEM, MOLT4) and megakaryocytes (K562), whereas STLK3 is restricted to thymus and STLK4 is predominately expressed in thymus, T cells (CD4/CD8+, TH1, CEM) and B cells (Jurkat, RPMI). Consequently, these STKs might participate in the oxidative response pathway during inflammation, reperfusion injury (stroke, surgery, shock), TNFα-mediated signaling, insulin desensitization, atherogenesis, vascular injury, T or B cell costimulation, or alternatively, participate in other MAPK-related signal transduction processes. [0407]
  • STLK5 is more distantly related to this STE20-subfamily including SOK-1 and STLK2, STLK3 and STLK4. STLK5, may therefore mediate a signaling pathway that is distinct from the oxidative stress response pathway. [0408]
  • The high degree of sequence homology in the C-termini of SOK-1, STLK2, STLK3, STLK4, STLK5, and STLK6 raises the possibility that these novel STKs, like SOK-1, may be subject to autoinhibition through a conserved C-terminal motif. [0409]
  • Human ZC1, ZC2, ZC3 and ZC4 [0410]
  • ZC1 is a good candidate for any disease in which tyrosine kinase, cytokine, or heterotrimeric G-protein coupled receptors have been implicated. The mouse homologue binds to NCK, and is recruited to activated PDGF (Su et al., EMBO 16: 1279-1290, 1997). The [0411] Drosophila homolog has been shown to bind to TRAF2, implicating it in TNF-α signaling (Liu et al., (1999) Curr. Biol. 9:101-104, 1999)). While ZC1 does not contain the exact NCK- and TRAF2-binding domains, it is likely to bind to related proteins.
  • Of the ZC subfamily of STE20-related protein kinases, ZC1 has very broad over-expression in many tumor types, suggesting that it may be involved in cellular growth, transformation, or tumor progression. A truncated form of ZC1 containing only the C-terminal putative MEKK1-binding domain was found to reduce the number of foci generated by H-Ras-V12 in Rat Intestinal Epithelial cells (RIE-1). These data indicate that ZC1 may play a role in the ability for these cells to overcome contact inhibition and anchorage-dependent growth. [0412]
  • The ZC1 homolog, Misshapen (msn) in [0413] Drosophila melanogaster was cloned as a result of complementing a mutation in a developmental pathway required for dorsal closure, a process involving changes in cell shape and position in the embryo (Treisman et al. Gene 186 119-125, 1997). A D. melanogaster homolog of the JNK1/JNK2 kinases from mammals was shown to function downstream of msn in the dorsal-closure signaling pathway (Su et al. Genes Dev. 12:2371-2380, 1998).
  • While ZC1 could be involved in multiple aspects of tumorigenesis, by analogy with [0414] Drosophila, the role of misshapen in dorsal closure suggests a critical role in the regulation of the cytoskeleton for the processes of cell attachment, cell movement and perhaps migration.
  • The association of the ZC1 family members msn and NIK with TRAF2 may indicate a role for this kinase in cell survival and/or in apoptosis. The ZC1 family contains a highly conserved domain that in the mouse homolog, NIK, has been shown to bind to MEKK1 (Mitogen-activated/Extracellular-regulated Kinase Kinase 1) (Su et al., (1997) EMBO 16(6): 1279-90). MEKK1 is involved in cell survival and/or apoptosis in several systems (Schlesinger et al., Front. Biosci.3:D1181-6, 1998). Depending on the context, MEKK1 appears to be upstream of either the ERK1/MAPK or the JNK/SAPK pathway [Schlesinger et al., (1998 Front. Biosci. 3:D1181-6). Three homologues of ZC1: murine NIK (NCK-interacting kinase)(Su et al. EMBO 16:1279-90, 1997), [0415] Drosophila msn (Liu et al. Curr. Biol. 9:101-104, 1999) and human HGK (HPK/GCK-like kinase)(Yao et al., J. Biol. Chem. 274:2118-25, 1999) have all been shown to activate the JNK pathway when over-expressed in 293T cells.
  • ZC1 shares a high degree of homology with these other family members in both the kinase domain and the “MEKK”-binding domains, yet it differs in the intervening region, which contains several putative binding domains for upstream signaling adapter molecules (e.g. NCK, TRAF2). Unlike the other family members, ZC1 does not appear to activate the JNK pathway in 293T cells as seen by its ability to induce expression of either a JUN or ATF2-driven luciferase gene. Upon co-transfection into these cells with HA-tagged JNK, modest activation of JNK was detected. ZC1 also modestly activated co-transfected ERK1. Both the ERK and the JNK activation were very slight compared with the positive controls in the assay (activated forms of MEK1 and MEKK1, respectively). In both cases, activation required the full-length kinase. While the kinase domain alone is up to 5× more active in autophosphorylation and in phosphorylation of MBP, it does not lead to activation of these potential downstream kinases. Based on the strong sequence homology of ZC1 with the other family members, it is very likely that ZC1 will be important for either JNK or ERK activation once the proper context is found. [0416]
  • ZC1 profoundly inhibits ERK1 kinase expression in co-transfection assays. This effect is dependent on ZC1 kinase activity, occurring with the wild-type and the kinase domain alone, but not with the kinase-dead mutant even though all three forms of ZC1 are expressed at similar levels. This may suggest a role for this kinase in transcriptional or post-transcriptional regulation. [0417]
  • ZC1 may be an important component in the signaling pathways mediated by the co-stimulatory receptor CD28 in T cells and/or by the pro-inflammatory cytokine TNFα, since co-transfection of the wild-type ZC1 activated the RE/AP-luciferase and NFκB-luciferase reporter genes. While our data showed that ZC1 strongly activates NFκB in T-cells, no activation of NFκB driven luciferase was detectable in NIH 3T3 cells. A recent paper (J. Biol. Chem. 274:2118-25; 1999.) has shown that a human ZC1 splicing isoform, HGK, is involved in the TNFα-signaling pathways. [0418]
  • Given the importance of T cell activation in autoimmunity and transplantation, as well as the key role that TNFα plays in inflammatory diseases, it is possible that ZC1 could be a therapeutic target for immunological diseases which include but are not limited to: rheumatoid arthritus, chronic inflammatory bowel diseases (ie Crohn's disease), chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, and autoimmunity as well as organ transplantation and cardiovascular diseases. [0419]
  • ZC1 appears to be the human orthologue of murine NIK and possibly an orthologue of a [0420] C. elegans STE20-subfamily kinase encoded by the ZC504.4 cosmid.
  • Murine NIK is a 140,000 daltons kinase that is most highly expressed in brain and heart. NIK interacts with the SH3 domains of the adaptor molecule Nck through its proline-rich regions found in the C-terminal extra-catalytic region. The specific regions that mediate this interaction are two PxxP (SEQ ID NO: 148) motifs that are nearly uniformly conserved between NIK, ZC1,2,3 and the [0421] C. elegans STE20 ZC504.4 kinase. In addition, NIK binds MEKK1 through its 719 amino acid C-terminal (Su, Y-C. et al. (1997) EMBO J. 16, 1279-1290). MEKK1 is a membrane-associated kinase responsible for activating MKK4 (also known as SEKI), which in turn activates SAPK (Yan, M et al. (1994) Nature, 372, 798-800). NIK may function as a kinase that links growth factor activated pathways and the stress-response pathway mediated by SAPKs. According to this hypothesis, activation of growth factor receptors leads to receptor tyrosine phosphorylation, Nck binding to the phosphorylated tyrosines via its SH2 domain, NIK redistribution to a membrane compartment via binding to the SH3 domain of Nck, and juxtaposition to the membrane-associated MEKK1. The NIK-MEKK1 interaction would, in this fashion, turn on the SAPK pathway in response to growth factor stimulation (Su, Y-C. et al. (1997) EMBO J. 16, 1279-1290).
  • Given the high homology between ZC1, ZC2, ZC3, and ZC4 STKs and NIK, it is conceivable that these kinases may each function to connect growth factor- and stress-activated signaling pathways. The heterogeneity that the ZC kinases exhibit within their putative SH3-binding domain could provide signaling specificity in terms of the nature of the adaptor molecule that they bind. The high level of sequence conservation in the C-termini of the ZC1, ZC2 and ZC3 strongly suggests that these human kinases, like murine NIK, also may bind to MEKK1 and activate SAPKs. The ZC kinases also display strong homology at their C-termini to protein domains that bind small GTPase proteins such as Rab, Rho and Rac. For example, the C-termini of ZC1 is 36.2% identical to citron, a murine Rho-binding protein, and 23.1% identical to the rab-binding region of GC kinase. This suggests that, in addition to adaptor molecules, small GTPase proteins may also mediate membrane association and activation of the ZC kinases. The presence of a potential coiled-coil region located immediately C-terminal to the catalytic region strongly suggests that the ZC kinases may also be subject to regulation via homo or heterodimerization events. [0422]
  • The [0423] C. elegans STE20 ZC504.4 kinase is the product of the mig-15 gene. The product of this gene has been implicated in several developmental processes such as epidermal development, Q neuroblast migrations and muscle arm targeting in the developing worm (Zhu, X. and Hedgecock E. (1997) Worm Breeder's Gazette 14, 76). The high level of sequence conservation between the ZC kinases and the ZC504.4 C. elegans kinase will make C. elegans a valuable model organism to study, through epistatic analysis, the signaling properties of the human ZC kinases.
  • Human KHS2 [0424]
  • KHS1 (kinase homologous to SPS1/STE20) is a 100,000 dalton cytoplasmic STK that is expressed ubiquitously. KHS1 has been implicated in the mechanism of SAPK activation in response to inflammatory cytokines such as TNF□ as well as to ultraviolight light, which also uses the TNF signaling pathway. TNF□ binding to its receptors (TNFR1 and TNFR2) results in the sequential association with the receptor C-tail of multiple signaling molecules including TNFR1-associated death domain protein (TRADD), Fas-associated death domain protein (FADD or MORT1), TNFR-associated factor 2 (TRAF2), and the STK RIP (receptor interacting protein). The TRADD-TRAF2 interaction is mediated by a conserved region present at the C-terminus of TRAF2, the TRAF domain. Activation of the NFκB and SAPK pathways is mediated by the ring finger motif present at the N-terminus of TRAF2 (Curr. Opinion in Cell. Biol. (1997) 9:247-251). KHS1 is activated by TNFαstimulation in a TRAF2-dependant manner and inhibition of KHS1 blocks TNFα-induced SAPK activation but not NF□B activation. The mechanism by which TRAF2 activates KHS1 is not known. Cotransfection of TRAF2- and KHS1-expressing constructs in 293T cells failed to reveal a direct association between these two molecules. KHS1 activates the SAPK pathway by a direct association with the constitutively active kinase MEKK1. MEKK1 subsequently activates SEK1, which in turn activates SAPK. Neither the MAPK nor the p38 kinase pathways are activated by KHS1 (Shi, C-S and Kehrl. J. H. (1997) J. Biol. Chem. 272, 32102-32107). In addition to its catalytic domain, downstream signaling of KHS1 requires its conserved C-terminus (Diener, K. et al (1997) Proc. Natl. Acad. Sci. 94, 9687-9692). [0425]
  • GCK (germinal center kinase) is a constitutively active 97,000 dalton STK that is broadly expressed. GCK may participate in B-cell differentiation since its expression is localized to the germinal center within lymphoid follicles. GCK activates the SAPK pathway in response to TNFα via activation of SEK1. The upstream activators of GCK in response to cytokines as well as the immediate downstream target of this kinase are unknown. The C-terminus of GCK is sufficient to activate SEK1 (Pombo, C. M. et al (1995) Nature, 377, 750-754). [0426]
  • The murine orthologue of GCK, rab8ip (rab8-interacting protein), is a 97,000 dalton protein that fractionates with both the soluble cytoplasmic fraction as well as with a salt-sensitive fraction associated with the basolateral membrane of the trans-Golgi region in polarized MDCK epithelial cells. The C-terminus of rab8ip binds to rab8, a small GTP-binding protein required for vesicular transport from the Golgi apparatus (Ren, M. et al. (1996) Proc. Natl. Acad. Sci. 93, 5151-5155). In addition to inducing the transcriptional activation of cytokines like IL2 via SAPK, GCK may also promote the rab-dependent release of secretory proteins in response to TNFα(Buccione, R. et al (1995) Mol. Bio. [0427] Cell 6, 291).
  • HPK1 (hematopoietic protein kinase) is a constitutively active 90,000 dalton STK restricted to hematopoietic cells. HPK1 activates the SAPK pathway by directly binding to and activating MEKK1 (Hu, M. et al (1996) Genes and Dev. 10:2251-2264) as well as the ubiquitously expressed mixed-lineage kinase MLK-3 (Kiefer, F. et al (1996) EMBO J. 15:7013-7025). This function of HPK1 requires, in contrast to GCK, both its kinase domain as well as its C-terminus. The upstream activators of HPK1 remain unknown. HPK1 also plays a key role as a mediator of transforming growth factor-β-(TGFβ) signaling. HPK1 activates the TGFb-activated kinase (TAK), which in turn stimulates the SAPK pathway by phosphorylating SEK1 (Wang W. et al (1997) J. Biol. Chem. 272:22771-22775). [0428]
  • KHS2 is expressed in thymus, dendrocytes and monocytes. KHS2 could have a complementary function to that of KHS1 as a mediator of SAPK activation in the cellular response to inflammatory cytokines. KHS2 could have the potential to interact directly with TRAF2 since a STK with the predicted molecular weight of KHS2 (approximately 101,000 daltons) is found in the TNFR-TRAF2 complex upon TNF□ stimulation (VanArsdale, T. and Ware, C. F. (1994) J. Immunol. 153, 3043-3050). The presence of a putative binding domain for Rab or a Rab-like molecule at the C-terminus of KHS2 indicates that KHS2, in addition to having a potential role in the TRAF2-dependant TNFα cytokine response, could also mediate signaling events that utilize small GTPase proteins. Alternatively, the binding of a small GTPase protein to the C-terminus of KHS2 may be required for its potential TRAF2-dependant signaling to a downstream kinase such as MEKK1. [0429]
  • Human GEK2, SULU1 and SULU3 [0430]
  • A recent report (Y-W Qian et al., Science 282:1701-1704,1998) described xPlkk1 as the activator of Plx1 (the Xenopus Polo kinase). In Xenopus oocytes, the STK Plkk1 can phosphorylate and activate Plx1 STK (the mammalian Polo kinase or PLK). A dominant-negative (kinase-dead) form of xPlkk1 prevents Plx1 activation and delays germinal vesicle breakdown. Yet another unidentified kinase is probably responsible for xPlkk1 activation during mitosis. [0431]
  • The homology through the entire length of the xPlkk1 protein with GEK2 suggests that GEK2 might represent the human homologue for xPlkk1. Based on this, GEK2 might be upstream of PLK in mammalian cells. In addition, based on the phage display screen results using the SULU1 coiled-coil2 domain as bait, SULU1 might also interact in vivo with GEK2 and therefore regulate GEK2 (and/or SLK through the coiled-coil domain) activation leading to PLK activation and mitosis. [0432]
  • If such a cascade of events is required for mitosis in mammalian cells, interruption of this signaling cascade at any point might block mitosis and could be beneficial for cancer treatment. [0433]
  • A recently cloned STE20-subfamily kinase, rat TAO1, is most likely the rodent orthologue of human SULU3 (Hutchinson, M. et al. J. Biol. Chem 273:28625-28632, 1998). TAO1 activates MEK3, 4 and 6 in vitro, while in transfected cells it associates and activates only MEK3, resulting in phosphorylation and activation of p38. These results implicate TAO1 (SULU3) in the regulation of the p38 containing stress-responsive MAP kinase pathway. [0434]
  • Human SULU1 is weakly expressed in hematopoietic sources whereas SULU3 is found in B-cells and TH1-restricted T cells. These mammalian SULU STKs display strong homology to the [0435] C. elegans SULU kinase. The role that this kinase plays in nematode development is unknown. The strong sequence homology between the catalytic domain of mammalian SULU kinases and other STE20-subfamily kinases such as SOK-1 (human STE20) and KHS2 suggests that the mammalian kinases may participate in the stress-response pathway. The potential coiled-coil domains found at the C-terminus of the SULU kinases may play a role in the regulation of this kinase.
  • Murine LOK (lymphocyte-oriented kinase) is a constitutively activated STK of approximately 130,000 daltons that is predominantly expressed in spleen, thymus and bone marrow (Kuramochi, S. et al (1997) J. Biol. Chem. 272: 22679-22684) as well as in meiotic testicular and primordial germ cells. The LOK1 gene is located in [0436] chromosome 11 of the mouse near the wr locus, a region that is associated with reproductive and neurological defects (Yanagisawa, M. et al (1996) Mol. Reprod. and Dev. 45:411-420). LOK does not activate any of the known MAPK pathways (ERK, JNK and p38) nor the NFκB pathway. The upstream signaling elements of LOK as well as the extracellular stimuli that utilize this kinase to elicit a biological response are also unknown (Kuramochi, S. et al (1997) J. Biol. Chem. 272: 22679-22684).
  • Human GEK2 is highly related to murine LOK, but based on sequence divergence in the non-catalytic domain, it appears to be a distinct member of this STE20-subfamily. GEK2 may signal through a pathway that remains to be defined. The presence of potential coiled-coil regions at the C-terminus of GEK2 could play a key role in regulating the functions of this kinase. [0437]
  • Human PAK4 and PAK5 [0438]
  • The p21 activated protein kinases (PAK) are a closely related subgroup of the STE20 family of serine/threonine kinases. Extensive genetic and biochemical analysis of the budding yeast STE20 has shown the critical role this serine/threonine kinase plays at the juncture of several important intracellular pathways required to appropriately respond to extracellular signals. STE20 links the transcriptional response by mediating the activation of the appropriate downstream MAPK pathway as well as coupling changes in cellular morphology via its control of the actin cytoskeleton. [0439]
  • A hallmark of the PAK subgroup is their small G protein-binding domain (PBD) that confers G protein-dependent activation upon this group of kinases. Via the PBD, PAKs bind to activated small G proteins resulting in the derepression of the PAK's intrinsic kinase activity. [0440]
  • Until recently, there were three known PAK kinases: PAK1, a 68 kD protein whose expression is restricted expression to brain, muscle, and spleen; PAK2 (PAK1, PAK65), a 62 kD protein whose expression is ubiquitous; and PAK3, a 65kD protein whose expression is restricted to the brain. Similar to STE20, the mammalian PAKs (1,2, and 3) have been shown to respond to extracellular signals (growth factors, mitogens, cytokines and a variety of cellular stresses) (Bagrodia, et al. (1995). J. Biol. Chem. 270: 22731-22737; Zhang, S., et al. (1995). J. Biol. Chem. 270: 23934-23936, Frost, J. et al. (1998) J. Biol. Chem. 273: 28191-28198; Galisteo, M. et al. (1996) J. Biol. Chem. 271: 20997-21000), and are linked to TCR activation (Yablonski, D., et al. (1998) EMBO J. 17: 5647-5657), and heterotrimeric G protein-coupled receptors (Knaus, U. et al. (1995) Science 269: 221-223). [0441]
  • The PAKs were originally identified as effectors for members of the Rho family of small G proteins (such as Rac and Cdc42), hence their name, p21-activated kinases (PAK) (Manser et al Nature 367:40-46). The recruitment of the PAKs to the appropriate intracellular location is critical to their function. Attempts to elucidate the role played by PAKs in intracellular signaling and morphological changes is complicated due to the complex interactions by which they can be recruited by such factors as activated small G proteins (rac, cdc42), adaptors (nck) and exchange proteins (PIX, Cool). [0442]
  • The adaptor molecule, Nck, is constitutively bound via its SH3 domain to the proline-rich motif in the N-terminal portion of PAK1. Binding of the Nck-PAK complex to activated growth factor receptors in response to growth factor stimulation provides a mechanism to link growth factor-stimulated and stress-response pathways (Galisteo, M. et al. (1996) J. Biol. Chem. 271:20997-21000). [0443]
  • The PBD found at the N-terminus of PAK1 is responsible for its high-affinity interaction with the GTP-bound forms of Cdc42 and Rac (Burbelo, P. et al. (1995) J. Biol. Chem. 270:29071-29074). The exact mechanism through which the small GTPases activate PAKs may involve, in part, association of the kinase with activated growth factor receptors through guanine nucleotide exchange factors (GEFs). GEFs activate small GTPases by catalyzing the formation of their GTP-bound state, thereby promoting their association with, and activation of, PAKs. The known mammalian PAK kinases, as well as [0444] Drosophila and C. elegans PAKs, all conserve an N-terminal extracatalytic motif responsible for a high-affinity interaction with the GEF, PIX. The PAK-Cdc42 interaction and subsequent PAKs occurs as a PIX/PAK complex (Manser, E. et al. (1998) Molecular Cell, 1, 183-192).
  • PAK signaling stimulated by heterotrimeric G proteins is mediated through the interaction between a short conserved amino acid region located at the C-terminus of PAK1 with the G-protein β-subunit (Leeuw, T. et al.(1998) Nature, 391: 191-195). [0445]
  • A variety of studies have indicated that the human PAKs are involved in mediating the activation of stress-activated protein kinase pathways (JNK and to lesser extent p38). PAKs are also potential mediators in the crosstalk between the pathways regulated by the Rho family of small G proteins and the signaling pathways directly downstream of Ras leading to the activation of the ERK pathway (Bagrodia, et al. (1995). J. Biol. Chem. 270: 22731-22737; Zhang, S., et al. (1995). J. Biol. Chem. 270: 23934-23936; Brown, J., et al. (1996) Curr Biol. 6:598-60596; Frost, J., et al. (1996). Mol. Cell. Biol. 16: 3707-3713). [0446]
  • PAK1 has been implicated in phosphorylating a regulatory site in MEK1 that is necessary for MEK1's ability to interact with Raf1 (Frost, et al. (1997) EMBO J. 16:6426-6438). PAK3 has been shown to phosphorylate Raf1 on a site that is important for Raf1 activity (King, A., et al. (1998). Nature 396: 180-183). [0447]
  • PAKs play an important role in controlling morphological changes in cell shape mediated by the actin cytoskeleton. Such morphological changes are required for cellular functions ranging from cell division and proliferation to cell motility and vesicle transport. PAK activity has been implicated in the localized assembly (leading edge) and disassembly (retracting edge) of focal adhesions necessary for cell motility (Frost J. et al (1998) J. Biol. Chem. 273:28191-28198). [0448]
  • PAK2 may have a role in the morphological changes induced during apoptosis (Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. (Rudel, T. (1997) Science. 276:1571-4)), and PAK1 maybe important in preventing apoptosis (Faure S, et al. (1997) EMBO J. (1997) 16:5550-61). In addition to overcoming mitogen- and anchorage-independent growth, tumor cells need to escape the programmed cell death that accompanies deregulated cell growth. Thus, inhibition of PAKs may be effective in triggering apoptosis in tumors. [0449]
  • A direct requirement for PAKs in the transformation of mammalian cells has been shown for PAK1 and PAK2. Kinase-dead alleles of PAK1 block ras transformation of RAT1 and Schwann cells (Tang, Y., et al. (1997) Mol. Cell. Biol. 17, 4454-4464). Dominant-negative alleles of PAK2 have been shown to interfere with ras-mediated transformation of mammalian cells (Osada, S., (1997) FEBS Lett 404:227-233) [0450]
  • Mutations in PAK3 have been implicated in nonsyndromic X-linked mental retardation suggesting a role for PAK3 in cognitive function (Allen, K. et al. (1998) Nat. Genet. 20: 25-30). PAK1 has been implicated in neurite outgrowth in PC12 cells (Daniels, R. et al. (1998) EMBO J. 17: 754-764; Nikolic, M. et al. (1998) Nature 395:194-198). [0451]
  • Finally, PAK-like STKs may also play a role in AIDS pathogenesis since the myristoylated 27kD membrane-associated HIV Nef gene product directly interacts with and activates these kinases via cdc42 and Rac. The Nef-mediated activation of PAK-like STKs correlates with the induction of high viral titers and the development of AIDS in infected hosts (Cullen, B. R. (1996) Curr. Biol. 6:1557-1559). [0452]
  • Our results show that PAK4 is expressed in thymus, dendrocytes, mast cells, monocytes, as well as in T cells (TH2-restricted cells and MOLT4) and the B cell line RPMI. PAK5 is found in mast cells and in the T cell line MOLT4. These data suggest potential roles for PAK4 and PAK5 in the immune system. [0453]
  • PAK4 and PAK5 share with the known PAKs a potential cdc42-binding motif at their N-termini. Both PAK4 and PAK5 display sequence homology in their C-termini to a motif responsible for an interaction between PAK1 and the β-subunit of heterotrimic G-proteins (amino acid residues 665-676 in [0454] PAK 4, and amino acid residues 386-398 in PAK5). Consequently, PAK4, and possibly PAK5, could mediate signaling events originating from growth factors as well as from ligands that stimulate G-protein-linked receptors.
  • PAK4 conserves a leucine (leu 44), that when mutated to a phenylalanine renders the kinase activity of human PAK1 constitutively active, bypassing its cdc42-binding requirement for activation (Brown J. et al (1996) Current Biol. 6:598-605). PAK5 contains an isoleucine at the equivalent position. Therefore, the mechanism by which cdc42 potentially activates human PAK1, PAK4, and possibly PAK5, may be very similar. [0455]
  • PAK4 and PAK5 however, lack the PIX-binding motif, and consequently cdc42-activating GEFs other than PIX (for example Db1 and Cool) must be responsible for the activation of these kinases. Alternatively, PAK4 and PAK5 may be activated by another GTPase, such as Rac1 which uses the Tiam1 GEF for its activation to the GTP-bound state. [0456]
  • PAK4 and PAK5 also lack the PxxP (SEQ ID NO: 148) motif responsible for the Nck-PAK1 association. Between the PBD or cdc42-binding N-terminal motifs and the putative GEF-binding regions, PAK4 and PAK5 have long insertions (185 and 123 amino acids for PAK4 and PAK5, respectively) relative to PAK1. This region probably confers different binding characteristics to adaptor molecules and/or GEFs from those exhibited by known mammalian PAKs. [0457]
  • PAKs have been shown to be upstream in pathways leading to activation of both the JNK (Bagrodia, S., et al. (1995) J. Biol. Chem. 270: 22731-22737) and ERK kinase pathways (Brown, J., et al. (1996). Curr Biol. 6:598-605). PAK1 was shown to synergize with ras in activation of the ERK pathway through phosphorylation of MEK1 (Frost, J. et al. (1997). EMBO J. 16:6426-6438). Our data shows that MEK1 serves as an in vitro substrate for PAK4, suggesting a potential role for PAK4 in the activation of the ERK pathway and mitogenesis. [0458]
  • PAK5 may also have a mitogenic role, and be linked to cancer, based on its expression profile (elevated RNA and protein levels in a wide variety of tumor cell lines), its interaction with cdc42 via its PBD, and the ability of a kinase-dead allele (Lys350, 351 Ala) to block ras transformation of NIH3T3 cells. Thus, a screen for small molecule inhibitors of PAK5 kinase activity may yield compounds with therapeutic potential for intervention in cancer derived from a wide variety of tissue types. [0459]
  • PAK4 and PAK5 may also play a role in HIV pathogenesis as potential mediators of Nef signaling, since none of the known PAKs correspond to the PAK-like kinase shown to interact with, and be activated by, the HIV nef protein (Lu, X. et al. (1996) Current Biology 6:1677-1684) [0460]
  • The 3′ untranslated region of PAK4 contains a CA repeat that is prone to undergo expansion. CA dinucleotide repeat instability has been associated with disease (Toren, M. Z. et al (1998) Am. J. Hematol. 57: 148-152), and expansion of such repeat in the 3′ untranslated region of PAK4 could implicate this kinase in as yet unknown pathologies. [0461]
  • Clinical Applications [0462]
  • Human STLK2, STLK3, STLK4, STLK5, STLK6, and STLK7 [0463]
  • STLK3, STLK5, STLK6 and STLK7, as well as other homologues of the STLK subfamily of STE20 protein kinases such as STLK4, may play an important role as mediators of the immune response. Thus, they are targets for the development of specific small molecule inhibitors to treat immunological diseases, including, but not limited to, rheumatoid arthritis, chronic inflammatory bowel diseases (e.g. Crohn's disease), chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis and autoimmunity, as well as in organ transplantation. Other diseases include cardiovascular diseases. [0464]
  • The human STLKs may also play an important role in cell growth regulation. Thus, they are targets for developing small molecule kinase inhibitors for the treatment of cancer and metastases. STLK5 maps to a chromosomal region frequently amplified in a variety of tumors including those from non-small cell lung cancer, breast cancer and peripheral nerve tumors. This suggests that STLK5 could play a role in the development, maintenance, or progression of human tumors. [0465]
  • The potential role of [0466] human STLKs 2,3, and 4 in mediating oxidative stress strongly suggests that drugs targeting these kinases could prove useful in the treatment of myocardial infarction, arrhythmia and other cardiomyopathies, stroke, renal failure, oxidative stress-related neurodegenerative disorders such amyotrophic lateral sclerosis, Parkinson's disease and Leigh syndrome, a necrotizing mitochondrial encephalopathy, as well.
  • Human ZC1, ZC2, ZC3, and ZC4 [0467]
  • ZC1 may be a component of the CD28-signaling pathway and therefore important in T cell activation. As such, ZC1 as well as other ZC subfamily kinases, are targets for the development of specific small molecule inhibitors to treat immunological diseases, including, but not limited to, rheumatoid arthritis, chronic inflammatory bowel diseases (e.g. Crohn's disease), chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis and autoimmunity, as well as organ transplantation. Other diseases include cardiovascular diseases. [0468]
  • ZC1 and ZC2 are also implicated in cell growth regulation. Thus, ZC subfamily kinases are targets for developing small molecule inhibitors for the treatment of cancer and metastases. ZC2 maps to a chromosomal region frequently amplified in a variety of tumors including those from non-small cell lung cancer, small cell lung cancer, and cervical cancer. This suggests that ZC2 could play a role in the development, maintenance, or progression of human tumors. [0469]
  • The role of human ZC1, ZC2, ZC3, and ZC4 in the inflammatory and stress-response pathways, strongly suggests that drugs targeting these kinases could have strong immunosuppressive actions. These drugs can prove valuable for the treatment of rheumatoid arthritis, artherosclerosis, autoimmune disorders and organ transplantation among others. At least one very important class of immunosuppresants, corticosteroids, functions by blocking SAPK activation at an as yet undefined site on this pathway (Swantek, J. L. et al (1997) Mol. Cell. Biol. (1997) 6274-6282). Other immunosuppresive drugs like the pyridinyl imidazoles specifically target the p38 kinases (Kumar, S. et al (1997) Biochem. Biophys. Res. Commun. 235: 533-528). Drug targeting of the MAPK and p38 pathways could lead to the development of novel immunosuppresants. [0470]
  • Human SULU and GEK [0471]
  • The potential role of these novel STE20-related protein kinases in the control of mitosis strongly suggests that agents that specifically inhibit these kinases could be useful for cancer and metastases treatment. [0472]
  • The close homology of human STLK5, GEK2, SULU1 and SULU3 to STE20-subfamily kinases involved in the stress and oxidative response pathway strongly suggests that drugs targeting these kinases may also be useful as immunosuppressants as well as to treat ischemic disorders. [0473]
  • Human KHS2 [0474]
  • The role of human KHS2 in the inflammatory and stress-response pathways, strongly suggests that drugs targeting this and related kinases could have strong immunosuppressive actions. These drugs can prove valuable for the treatment of rheumatoid arthritis, artherosclerosis, autoimmune disorders and organ transplantation among others. At least one very important class of immunosuppresants, corticosteroids, functions by blocking SAPK activation at an as yet undefined site on this pathway (Swantek, J. L. et al (1997) Mol. Cell. Biol. (1997) 6274-6282). Other immunosuppresive drugs like the pyridinyl imidazoles specifically target the p38 kinases (Kumar, S. et al (1997) Biochem. Biophys. Res. Commun. 235: 533-528). Drug targeting of the MAPK and p38 pathways could lead to the development of novel immunosuppressants. [0475]
  • Human PAK Family [0476]
  • PAK5 has a role in cancer based on its expression profile (elevated RNA and protein levels in wide variety of tumor lines), its interaction with Cdc42 via its PBD, and the ability of the kinase-dead allele of PAK5 (Lys350, 351Ala) to block ras transformation of NIH3T3 cells. Thus, a screen for small molecule inhibitors of PAK5 kinase activity may yield compounds with therapeutic potential for intervention in cancers and metastases derived from a wide range of tissue types. [0477]
  • PAK5 maps to a chromosomal region frequently amplified in a variety of tumors including those from non-small cell lung cancer, and small cell lung cancer. These findings suggest that PAK5 could play a role in the development, maintenance, or progression of human tumors and/or metastases. [0478]
  • The role of human PAK4, and PAK5 in the inflammatory and stress-response pathways also strongly suggests that drugs targeting these kinases could have strong immunosuppressive actions. These drugs can prove valuable for the treatment of rheumatoid arthritis, artherosclerosis, autoimmune disorders and organ transplantation among others. At least one very important class of immunosuppresants, corticosteroids, functions by blocking SAPK activation at an as yet undefined site on this pathway (Swantek, J. L. et al (1997) Mol. Cell. Biol. (1997) 6274-6282). Other immunosuppresive drugs like the pyridinyl imidazoles specifically target the p38 kinases (Kumar, S. et al (1997) Biochem. Biophys. Res. Commun. 235: 533-528). Drug targeting of the MAPK and p38 pathways could lead to the development of novel immunosuppresants. In addition, drugs targeting PAK4 or PAK5 could prove useful as immunosuppresants as well as in AIDS treatment. [0479]
  • VIII. Transgenic Animals. [0480]
  • A variety of methods are available for the production of transgenic animals associated with this invention. DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442, 1985). Embryos can be infected with viruses, especially retroviruses, modified to carry inorganic-ion receptor nucleotide sequences of the invention. [0481]
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention. A transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc. [0482]
  • The procedures for manipulation of the rodent embryo and for microinjection of DNA into the pronucleus of the zygote are well known to those of ordinary skill in the art (Hogan et al., supra). Microinjection procedures for fish, amphibian eggs and birds are detailed in Houdebine and Chourrout (Experientia 47: 897-905, 1991). Other procedures for introduction of DNA into tissues of animals are described in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990). [0483]
  • By way of example only, to prepare a transgenic mouse, female mice are induced to superovulate. Females are placed with males, and the mated females are sacrificed by CO[0484] 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice (Hammer et al., Cell 63:1099-1112, 1990).
  • Methods for the culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection also are well known to those of ordinary skill in the art (Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., IRL Press, 1987). [0485]
  • In cases involving random gene integration, a clone containing the sequence(s) of the invention is co-transfected with a gene encoding resistance. Alternatively, the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention. Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E. J. Robertson, supra). [0486]
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination (Capecchi, Science 244: 1288-1292, 1989). Methods for positive selection of the recombination event (i.e., neo resistance) and dual positive-negative selection (i.e., neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et al. (Nature 338: 153-156, 1989), the teachings of which are incorporated herein in their entirety including any drawings. The final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females. The resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene. Procedures for the production of non-rodent mammals and other animals have been discussed by others (Houdebine and Chourrout, supra; Pursel et al., Science 244:1281-1288, 1989; and Simms et al., Bio/Technology 6:179-183, 1988). [0487]
  • Thus, the invention provides transgenic, nonhuman mammals containing a transgene encoding a kinase of the invention or a gene effecting the expression of the kinase. Such transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introduction of a kinase, or regulating the expression of a kinase (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes). [0488]
  • A “transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. The transgenic DNA may encode human STE20-related kinases. Native expression in an animal may be reduced by providing an amount of anti-sense RNA or DNA effective to reduce expression of the receptor. [0489]
  • IX. Gene Therapy [0490]
  • STE20-related kinases or their genetic sequences will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460, 1992). Miller states that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results. The basic science of gene therapy is described in Mulligan (Science 260:926-931, 1993). [0491]
  • In one preferred embodiment, an expression vector containing STE20-related kinase coding sequence is inserted into cells, the cells are grown in vitro and then infused in large numbers into patients. In another preferred embodiment, a DNA segment containing a promoter of choice (for example a strong promoter) is transferred into cells containing an endogenous gene encoding kinases of the invention in such a manner that the promoter segment enhances expression of the endogenous kinase gene (for example, the promoter segment is transferred to the cell such that it becomes directly linked to the endogenous kinase gene). [0492]
  • The gene therapy may involve the use of an adenovirus containing kinase cDNA targeted to a tumor, systemic kinase increase by implantation of engineered cells, injection with kinase-encoding virus, or injection of naked kinase DNA into appropriate tissues. [0493]
  • Target cell populations may be modified by introducing altered forms of one or more components of the protein complexes in order to modulate the activity of such complexes. For example, by reducing or inhibiting a complex component activity within target cells, an abnormal signal transduction event(s) leading to a condition may be decreased, inhibited, or reversed. Deletion or missense mutants of a component, that retain the ability to interact with other components of the protein complexes but cannot function in signal transduction may be used to inhibit an abnormal, deleterious signal transduction event. [0494]
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA) encoding recombinant kinase of the invention protein into the targeted cell population (e.g., tumor cells). Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., 1989). Alternatively, recombinant nucleic acid molecules encoding protein sequences can be used as naked DNA or in a reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (e.g., Felgner et al., Nature 337:387-8, 1989). Several other methods for the direct transfer of plasmid DNA into cells exist for use in human gene therapy and involve targeting the DNA to receptors on cells by complexing the plasmid DNA to proteins (Miller, supra). [0495]
  • In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection (Capecchi, Cell 22:479-88, 1980). Once recombinant genes are introduced into a cell, they can be recognized by the cell's normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been attempted for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with CaPO[0496] 4 and taken into cells by pinocytosis (Chen et al., Mol. Cell Biol. 7:2745-52, 1987); electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane (Chu et al., Nucleic Acids Res. 15:1311-26, 1987); lipofection/liposome fusion, wherein DNA is packaged into lipophilic vesicles which fuse with a target cell (Felgner et al., Proc. Natl. Acad. Sci. USA. 84:7413-7417, 1987); and particle bombardment using DNA bound to small projectiles (Yang et al., Proc. Natl. Acad. Sci. 87:9568-9572, 1990). Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins.
  • It has also been shown that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. The admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene (Curiel et al., Am. J. Respir. Cell. Mol. Biol., 6:247-52, 1992). [0497]
  • As used herein “gene transfer” means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene. The product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cytoplasm from the plasma membrane or endosome. Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription. [0498]
  • As used herein “gene therapy” is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient. [0499]
  • In another preferred embodiment, a vector having nucleic acid sequences encoding a STE20-related kinase polypeptide is provided in which the nucleic acid sequence is expressed only in specific tissue. Methods of achieving tissue-specific gene expression are set forth in International Publication No. WO 93/09236, filed Nov. 3, 1992 and published May 13, 1993. [0500]
  • In all of the preceding vectors set forth above, a further aspect of the invention is that the nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above. [0501]
  • In another preferred embodiment, a method of gene replacement is set forth. “Gene replacement” as used herein means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal. [0502]
  • X. Administration of Substances [0503]
  • Methods of determining the dosages of compounds to be administered to a patient and modes of administering compounds to an organism are disclosed in U.S. application Ser. No. 08/702,282, filed Aug. 23, 1996 and International patent publication number WO 96/22976, published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings, figures, or tables. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it. [0504]
  • The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used, and the size and physiological condition of the patient. Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC[0505] 50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
  • Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors, and major organs can be also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder. Such measurements can be carried out. For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan, and MRI. Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model. [0506]
  • Toxicity studies can also be carried out by measuring the blood cell composition. For example, toxicity studies can be carried out in a suitable animal model as follows: 1) the compound is administered to mice (an untreated control mouse should also be used); 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition, and the percent of lymphocytes versus polymorphonuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present. [0507]
  • At the termination of each toxicity study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Euthanasia, [0508] Journal of American Veterinary Medical Assoc., 202:229-249, 1993). Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness, or toxicity. Gross abnormalities in tissue are noted, and tissues are examined histologically. Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs. In general, the greater the adverse effect the less preferred the compound.
  • For the treatment of cancers the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness. [0509]
  • Plasma levels should reflect the potency of the drug. Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy. [0510]
    • EXAMPLES
  • The examples below are not limiting and are merely representative of various aspects and features of the present invention. The examples below demonstrate the isolation and characterization of the STE20-related kinases of the invention. [0511]
    • Example 1 Isolation of cDNAs Encoding Mammalian STE20-Related Protein Kinases Materials and Methods
  • Identification of novel clones [0512]
  • Total RNAs were isolated using the Guanidine Salts/Phenol extraction protocol of Chomczynski and Sacchi (P. Chomczynski and N. Sacchi, Anal. Biochem. 162, 156 (1987)) from primary human tumors, normal and tumor cell lines, normal human tissues, and sorted human hematopoietic cells. These RNAs were used to generate single-stranded cDNA using the Superscript Preamplification System (GIBCO BRL, Gaithersburg, MD; Gerard, GF et al. (1989), [0513] FOCUS 11, 66) under conditions recommended by the manufacturer. A typical reaction used 10 μg total RNA with 1.5 μg oligo(dT)12-18 in a reaction volume of 60 μL. The product was treated with RNaseH and diluted to 100 μL with H2O. For subsequent PCR amplification, 1-4 μL of this sscDNA was used in each reaction.
  • Degenerate oligonucleotides were synthesized on an Applied Biosystems 3948 DNA synthesizer using established phosphoramidite chemistry, precipitated with ethanol and used unpurified for PCR. The sequence of some of the degenerate oligonucleotide primers and the amino acid motif they encode is as follows: [0514]
    TRK1 5′-CTGAATTCGGNGCNTTYGGNAAR (SEQ ID NO:32)
    GT-3′
    GAFGKV (sense) (SEQ ID NO:37)
    TRK4 5′-GCTGGATCCYTCNGGNGGCATCC (SEQ ID NO:33)
    A-3′
    WMPPE (antisense) (SEQ ID NO:38)
    ROS1 5′-GCNTTYGGNGARGTNTAYGARG (SEQ ID NO:34)
    G-3 ′
    AFGEVYEG (sense) (SEQ ID NO:39)
    CCK4b 5′-GCTGGATCCYTCNGGNSWCATC (SEQ ID NO:35)
    CA-3′
    WMSPE (antisense) (SEQ ID NO:40)
    CCK4c 5′-GAGTTYGGNGARGTNTTYYTNG (SEQ ID NO:36)
    C-3′
    EFGEVYEG (sense) (SEQ ID NO:41)
  • These primers were derived from the sense and antisense strands of conserved motifs within the catalytic domain of several protein kinases. Degenerate nucleotide residue designations are: N=A, C, G, or T; R=A or G; Y=C or T; H=A, C or T not G; D=A, G or T not C; S=C or G; and W=A or T. [0515]
  • PCR reactions were performed using degenerate primers applied to multiple single-stranded cDNAs. The primers were added at a final concentration of 5 μM each to a mixture containing 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl[0516] 2, 200 μM each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 1-4 μL cDNA. Following 3 min denaturation at 95° C., the cycling conditions were 94° C. for 30 s, 50° C. for 1 min, and 72° C. for 1 min 45 s for 35 cycles. PCR fragments migrating between 300-350 bp were isolated from 2% agarose gels using the GeneClean Kit (Bio101), and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's protocol.
  • Colonies were selected for mini plasmid DNA-preparations using Qiagen columns and the plasmid DNA was sequenced using a cycle sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA). Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer, and analyzed using the BLAST alignment algorithm (Altschul, S. F. et al., J. Mol. Biol. 215: 403-10). [0517]
  • Additional PCR strategies were employed to connect various PCR fragments or ESTs using exact or near exact oligonucleotide primers as detailed in the results section for each cDNA. PCR conditions were as described above except the annealing temperatures were calculated for each oligo pair using the formula: Tm=4(G+C)+2(A+T). [0518]
  • Isolation of cDNA Clones: [0519]
  • Human cDNA libraries were probed with PCR or EST fragments corresponding to STE20-related genes. Probes were [0520] 32P-labeled by random priming and used at 2x 106 cpm/mL following standard techniques for library screening. Pre-hybridization (3 h) and hybridization (overnight) were conducted at 42° C. in 5× SSC, 5X Denhart's solution, 2.5% dextran sulfate, 50 mM Na2PO4/NaHPO4, pH 7.0, 50% formamide with 100 mg/mL denatured salmon sperm DNA. Stringent washes were performed at 65° C. in 0.1× SSC and 0.1% SDS. DNA sequencing was carried out on both strands using a cycle sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA). Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer.
  • Makegene Bioinformatics EST Assembler [0521]
  • The EST reports were downloaded from National Institute for Biotechnology Information. After uncompressing the files, the program ‘report2est’ was scripted to extract the following information: 1) EST names, 2) GenBank Accession numbers, 3) GenBank gi numbers, 4) Clone Id numbers, 5) the nucleotide sequences of the ESTs 6) the organism, 7) the library name, 8) the name of the lab, and 9) the institution. The output of ‘report2est’ is a file in FASTA format with all of the information listed above in the first line of each entry except the sequence, which is listed in the second line of each entry. The resulting file is formatted for BLAST using ‘pressdb’ (available as part of the ncbi tool kit). [0522]
  • To build a gene or part of a gene from ESTs, the program ‘makegene’ was developed. Input to this program is a query sequence and the organism/species for which a gene is to be built. An initial search of the formatted EST database described above is performed using BLAST (blastn). Any results that contain warnings, such as polyA tails or other repeat elements, are eliminated from future queries. The program ‘blast_parse_reports’ was developed to extract the FASTA header line from the search results and the output is then filtered to extract only FASTA header lines for the desired species. [0523]
  • The initial results, having been filtered for warnings and species, go into a loop in which searches against the database are repeated until no new ESTs are found. The loop consists of the following steps: 1) when possible the names of both ends of the ESTs are extracted from the database by searching using the ‘Clone Id’ field or the part of the ‘EST name’ field before the .r or s postscript, 2) any ESTs that have been used as queries in previous loops are removed from the current query by the program ‘subtract’, 3) the resulting list of ESTs is used to extract the sequences from the database by the program batch_parse_fasta, 4) BLAST is run against the database using each sequence, 5) the output files from BLAST containing warnings are removed, 6) the results are filtered by species, and 7) the loop is reentered if there were new ESTs found in the previous pass through the loop. [0524]
  • The ESTs chosen by ‘makegene’ are used as input for the program ‘mpd2_cluster’ (Hide, W., Burke, J, and Davison, D. U. of Houston, unpublished) which clusters overlapping sequences. The programs ‘contig’ (Kerlavage, T., TIGR, unpublished), ‘gde2mult’ and ‘gde2sing’ (Smith, S. W., et al., [0525] CABIOS 10, 671-675 (1994)), are used to make an alignment and consensus sequence of the overlapping ESTs.
  • Results [0526]
  • cDNA Cloning and Characterization of STLK2 [0527]
  • The human STLK2 cDNA sequence is composed of two overlapping EST fragments, AA191319 and W16504, that were identified using a Smith-Waterman search of the EST database with STLK1 (MST3 GB:AF024636) as a query. The complete sequence of both clones was determined and used to generate the full-length human STL2 sequence. [0528]
  • EST clone AA191319 contains a 1327 bp insert and an ORF of 1146 bp (382 amino acids). EST clone W16504 contains a 2474 bp insert (not including the poly-A tail) and an ORF of 687 bp (382 amino acids). [0529]
  • The full-length human STLK2 cDNA (SEQ ID NO. 1) is 3268 bp long. AA191319 spans positions 1-1327 and W16504 positions 743-3216. The overlap between these two [0530] clones exhibits 100% sequence identity. The human STLK2 cDNA constains a 1248 bp ORF flanked by a 181 bp 5′ UTR (1-181) and a 1784 bp 3′ UTR (1433-3216) that is followed by a 52 nucleotide polyadenylated region. A polyadenylation signal (AATAAA) is found at positions 3193-3198. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for STLK2. Furthermore, human STLK2, and the related SOK-1 and MST3 proteins, conserve the amino acid sequence immediately following this presumed initiating methionine.
  • Several EST fragments span the complete STLK2 sequence with AA191319 at the 5′ end and W16504 at the 3′ end. [0531]
  • All searches against the public nucleic acid database (NRN) and protein database (NRP) were conducted using the Smith-Waterman gap alignment program ((Smith, T F and Waterman, M S (1981) J. Mol. Biol, 147, 195-197).) with the PAM100 matrix and gap open -and extension penalties of 14:1, respectively. [0532]
  • cDNA Cloning and Characterization of STLK3 [0533]
  • A mammalian STLK3 clone, 135-31-19, was first identified from a PCR screen with the degenerate oligos, TRK1 and TRK4, applied to a sscDNA generated from adult rat brain substantia nigra. Sequence analysis of the 457 bp insert indicated that it represented a novel member of the STE20-subfamily of STKs. [0534]
  • A Smith-Waterman search of the EST database with the rat STLK3 fragment and human STLK1 (MST3 GB:AF024636) as queries identified several overlapping ESTs spanning most of the human STLK3 cDNA sequence. A Makegene analysis generated a 3037 bp contig from approximately 44 EST sequences. Since the 3′ ESTs were not commercially available, a pair of primers (5′-CACAGAAACGGTCAGATTCAC-3′(SEQ ID NO: 42) and 5′-GATCAGGGTGACATCAAGGGAC-3′(SEQ ID NO: 43)) were derived from this region to generate PCR clone 3R21-20-6 from human fetal liver sscDNA. This clone and EST AA278967 were fully sequenced to generate the full-length STLK2 cDNA sequence. [0535]
  • AA278967 is a 837 bp EST isolated by the IMAGE consortium from cDNA made from CD20+/IgD-germinal center B cells sorted from human tonsillar cells. [0536]
  • PCR clone 3R21-20-6 was isolated from human fetal sscDNA and contains a 1116 bp insert, including a 1086 bp ORF encoding the 362 C-terminal amino acids of STLK3. [0537]
  • The full-length human STLK3 cDNA (SEQ ID NO. 2) is 3030 bp long. AA278967 spans positions 1-814 and 3R21-20-6 spans positions 464-1579. The overlap between these two [0538] clones exhibits 100% sequence identity. The remaining 1452 bp of 3′ UTR is derived from an assembly of multiple unconfirmed EST fragments.
  • The near full-length human STLK3 cDNA (SEQ ID NO.2) is 3030 bp long and consists of a 1548 bp ORF flanked by a 1476 [0539] bp 3′ UTR (1550-3025) and a 5 nucleotide polyadenylated region. A polyadenylation signal (AATAAA) begins at position 3004. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. Six copies of a “GGCCCC” repeat were observed in positions 21-67. Five independent ESTs (AA150838, AA286879, AA251679, AA252004, AA278967) showed the same repeat, suggesting that this sequence may be an integral region of the human STLK3 gene. Stronger evidence for this being the case is provided by the sequence of the murine orthologue of STLK3 represented by a 876 bp EST W20737.
  • Multiple EST fragments span the complete STLK3 sequence with AA278967 at the 5′ end and AA628477 and others at the 3′ end. [0540]
  • cDNA Cloning and Characterization of STLK4 [0541]
  • The human STLK4 cDNA sequence is composed of two overlapping EST fragments, AA297759 and AA100484, that were identified using a Smith-Waterman search of the EST database with STLK1 (MST3 GB:AF024636) as a query. The complete sequence of both clones was determined and used to generate the near full-length human STLK4 sequence. [0542]
  • AA100484 is an IMAGE consortium cDNA clone isolated from the T-84 colonic epithelium cell line. It has an insert of 3694 bp and a coding region of 1146 bp (382 amino acids). A Smith-Waterman sequence alignment against the NRN database showed this EST to be 71.4% identical to the human STE20-like kinase (GB:X99325). [0543]
  • W16504 is an IMAGE consortium clone isolated from a human fetal heart cDNA library. It has an insert length of 2474 bp (not including the poly-A tail) and a coding region of 687 bp (229 amino acids). A Smith-Waterman sequence alignment of W16504 against the NRN database showed this EST to be 69.2% identical to the human STE20-like kinase (GB:X99325). [0544]
  • The full-length human STLK2 cDNA (SEQ ID NO. 1) is 3268 bp long. AA191319 spans positions 1-1327, and W16504 positions 743-3216. The overlap between these two clones is 585 bp long with 100% sequence identity. [0545]
  • AA100484 is an IMAGE consortium cDNA clone isolated from the T-84 colonic epithelium cell line. AA100484 covers the bulk of Human STLK4 with its 3694 bp, which spans positions 146-3839 of SEQ ID NO:3. A second EST, AA297759, isolated from a Jurkat T cell cDNA library, spans positions 1-271 of the human STLK4 contig. The two ESTs overlap over a 126 bp stretch that has only one nucleotide discrepancy at position 149 (G in AA297759 and T in AA100484). A T at this position was chosen for the SEQ ID NO:3 based on sequence data generated from A100484. The 5′ 145 bp of human STLK4 contains three sequencing ambiguities (N's in SEQ ID NO:3) arising from sequence errors in the GenBank entry for AA297759. Three amino acid sequence ambiguities in the N-terminus of human STLK4 are present also in SEQ ID NO:7 as a consequence of the sequence inaccuracies from the EST entry. [0546]
  • The coding region of human STLK4 is 1242 bp long (2-1243), capable of encoding a 414 amino acid polypeptide, and is followed by a 2596 [0547] nucleotide 3′ UTR (1244-3839). Human STLK4 ends in a polyadenylated stretch that has 18 adenines (3840-3857). A polyadenylation signal (AATAAA) is found between positions 3822-3827. Targeted-PCR cloning identified one rat orthologue of human STLK4, clone 135-31-19. In addition, one murine orthologue of human STLK4 was recognized in the EST database as AA 117483. None of these orthologues add additional N-terminal sequence to the human STLK4.
  • The near full-length human STLK4 cDNA (SEQ ID NO.3) is 3857 bp long and consists of a 1242 bp ORF flanked by a 2596 [0548] bp 3′ UTR (1244-3839) and an 18 nucleotide polyadenylated region. Polyadenylation signals (AATAAA) begin at positions 2181 and 3822. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. A near full-length murine STLK4 cDNA is represented in the 1773 bp EST AA117438. It extends an additional 21 nucleotides 5′ of the human STLK4 consensus, but since its coding region is open throughout the 5′ extent of the sequence, this is also probably a partial cDNA clone lacking the N-terminal start methionine.
  • Several EST fragments span the complete STLK3 sequence with AA297759 at the 5′ end and AA100484 and others at the 3′ end. [0549]
  • cDNA Cloning and Characterization of STLK5 [0550]
  • The human STLK5 cDNA sequence is composed of four overlapping sequences, A1418298, 2R96-13-1, 3R25-45-3 and R46685. A human STLK5 clone, F07734, was first identified using a Smith-Waterman search of the EST database with SPS_sc (U33057) as a query. [0551]
  • AI418298 is an IMAGE consortium cDNA clone with an 895 bp insert. [0552]
  • PCR clone 2R96-13-1 was isolated from human brain [0553] sscDNA using primers 5′-CTCATCTGTACACACTTCATGG(SEQ ID NO:44) and 5′-GATTCCCACACTGTAGATGTC(SEQ ID NO:45) derived from F07734. 2R96-13-1 contains a 330 bp insert and an ORF of 330 bp (110 amino acids).
  • EST clone R46685 was identified using a Smith-Waterman search of the EST database with the C-terminus of SPS_sc (GB:U33057) as query. Sequence analysis of the 1047 bp insert identified this EST to contain an ORF of 285 bp (95 amino acids) encoding the C-terminus of human STLK5. [0554]
  • PCR clone 3R25-45-3 was isolated from human fetal brain [0555] sscDNA using primers 5′-GGCCCTCGACTACATCCACCACAT(SEQ ID NO:46) and 5′-CAACGAAACTAACACAGCATAAGG(SEQ ID NO:47) derived from 2R96-13-1 and R46685, respectively. 3R25-45-3 contains a 330 bp insert and an ORF of 750 bp (250 amino acids).
  • The full-length human STLK5 cDNA (SEQ ID NO:96) is 2110 bp long and consists of a 1119 bp ORF flanked by a 229 [0556] bp 5′ UTR and a 762 bp 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus (supra) for an initiating methionine, and is believed to be the translational start site for STLK5.
  • Several EST fragments span the complete STLK5 sequence with AA297059 and F07734 at the 5′ end and R46686 and F03423 and others at the 3′ end. [0557]
  • STLK5 displays a 100% match over a 41 bp stretch (position 2-42, SEQ ID NO. 97) to a human CpG island repeat (Z61277). [0558]
  • cDNA Cloning and Characterization of STLK6 [0559]
  • Human STLK6 was first identified in the translated EST database (AA219667) as a novel serine threonine kinase. [0560]
  • The partial human STLK6 cDNA (SEQ ID NO:98) is 2,001 bp long and consists of a 1,254 bp ORF flanked by a 75 [0561] bp 5′ UTR and a 673 bp 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiating methionine, and is believed to be the translational start site for STLK6.
  • At the time of filing, inventors believe that STLK6 does not have any significant match in the nucleic acid database. [0562]
  • cDNA Cloning and Characterization of STLK7 [0563]
  • Human STLK7 was first identified in the translated EST database (AA988954) as a novel serine threonine kinase. The original clone was not available through public sources, so a PCR fragment amplified from the sequence of AA988954 yielded 5R54-21-2. [0564]
  • The partial human STLK7 cDNA (SEQ ID NO: 100) is 311 bp long and consists of a 309 bp ORF. Since the coding region is open throughout the 5′ and 3′ extent of this sequence, this appears to be a partial cDNA clone lacking the N-terminal start methionine and C-terminal stop codon. [0565]
  • STLK7 shares 80% sequence identity to human SPAK (AF099989) over a 167 bp region and 50% nucleotide sequence identity to SLTK7 (SEQ ID NO. 101) over 391 nucleotides. [0566]
  • cDNA Cloning and Characterization of ZC1 [0567]
  • The human ZC1 cDNA sequence is composed of two overlapping PCR clones, 3R25-24-2 and R65-12-2. [0568]
  • A human ZC1 clone, 125-33-5, was first identified from a PCR screen with degenerate oligos, TRK1 and TRK4, applied to sscDNA generated from human small airway epithelial cells (Clontech). Sequence analysis of the 503 bp insert identified a 501 bp ORF (167 amino acids) with the potential to encode a novel human STK related to the [0569] C. elegans ZC504.4 gene product.
  • PCR clone 3R25-24-2 was isolated from human SNB19 glioblastoma [0570] sscDNA using primers 5′-ATGGCGAACGACTCTCCCGCGAA(SEQ ID NO:48) and 5′-ACACCAAAATCAACAAGTTTCACCTC(SEQ ID NO:49) derived from the N-terminus of a murine orthologue of ZC1 (NIK, GB:U88984) and the original human ZC1 clone 125-33-5, respectively. 3R25-24-2 contains a 527 bp insert and an ORF of 519 bp (173 amino acids).
  • PCR clone R65-12-2 was isolated as follows: A Smith-Waterman search of the EST database with the [0571] C. elegans ZC504.4 gene (GB:Z50029) as a query identified a human EST (W81656) whose ORF is related to the C. elegans gene and terminates in an identical residue (Trp). A primer was designed 3′ to this stop codon (5′-AGTTACAAGGAATTCCAAGTTCT(SEQ ID NO:50)) and used in a PCR reaction with a primer derived from the original human ZC1 clone 125-33-5 (5′-ATGAAGAGGAAGAAATCAAACTG(SEQ ID NO:51)) using sscDNA from human SNB19 glioblastoma as a template. PCR clone R65-12-2 was identified and was found to contain a 3611 bp insert with a 3534 bp ORF encoding the C-terminal portion of human ZC1 (1178 amino acids).
  • The full-length human ZC1 cDNA (SEQ ID NO. 9) is 3798 bp long. Clone 3R25-24-2 spans positions 1-527, and clone R65-12-2 spans positions 188-3798. The overlap between these two [0572] clones exhibits 100% sequence identity. The human ZC1 contains a 3717 bp ORF (17-3723) flanked by a 6 bp 5′ UTR and a 75 bp (3724-3798) 3′ UTR. No polyadenylation signal (AATAAA) or polyadenylated region are present in the 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human ZC1.
  • Multiple EST fragments (W81656) match the 3′ end of the human ZC1 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0573]
  • cDNA Cloning and Characterization of ZC2 [0574]
  • The human ZC2 cDNA sequence is composed of four overlapping PCR clones, G75-31-17, R65-24-6, 2R28-8-1, and R99-6-10. [0575]
  • A human ZC2 clone, G75-31-17, was first identified from a PCR screen with degenerate oligos, ROS 1(5′-GCNTTYGGNGARGTNTAYGARGG(SEQ ID NO:34)) and CCK4b (5′-GCTGGATCCYTCNGGNSWCATCCA(SEQ ID NO:35)), applied to sscDNA generated from the human HLT383 primary non-small cell lung cancer tissue. Sequence analysis of the 492 bp insert identified a 492 ORF (164 amino acids) with the potential to encode a novel human STK related to the [0576] C. elegans ZC504.4 gene product.
  • PCR clone R99-6-10 was isolated as follows: A Smith-Waterman search of the EST database with [0577] C. elegans ZC504.4 gene (GB:Z50029) as a query identified two overlapping human EST fragments (AA115844 and R51245) whose ORFs were related to the C. elegans gene and terminate in an identical residue (Trp). A primer was designed 3′ to the stop codon found in R51245 (5′-AGATGGACTGTACTGGGAGG(SEQ ID NO:52)) and used in a PCR reaction with a primer derived from AA115844 (5′-ACTTTGTGCAGCTCTGTGGG(SEQ ID NO:53)) using human fetal brain sscDNA as a template. PCR clone R99-6-10 was identified and was found to contain a 1095 bp insert with a 930 bp ORF encoding the C-terminal portion of human ZC2 (310 amino acids).
  • PCR clone R65-24-6 was isolated from human HT29 colon cancer cell line [0578] sscDNA using primers 5′-AAGGTTATGGATGTCACAGGG(SEQ ID NO:54) and 5′-AGATGGACTGTACTGGGAGG(SEQ ID NO:52) derived from G75-31-17 and R51245, respectively. The 3′ primer used in this PCR reaction misprimed between positions 1634-1653 of this gene leading to the formation of a truncated product. R65-24-6 contains a 1593 bp insert and an ORF of 1593 bp (531 amino acids).
  • PCR clone 2R28-8-1 was isolated from human colon cancer cell line HT29 [0579] sscDNA using primers 5′-CTCACAAGGTTGCCAACAGG(SEQ ID NO:55) and 5′-AGTCCCCACCAGAAGGTTTAC(SEQ ID NO:56) derived from R65-24-6 and R99-6-10, respectively. 2R28-8-1 contains a 1538 bp insert and an ORF of 1536 bp (512 amino acids).
  • The partial human ZC2 cDNA (SEQ ID NO. 10) is 4055 bp long. Clone G75-31-17 spans positions 1-492, clone R65-24-6 spans positions 58-1650, clone 2R28-8-1 spans positions 1466-3003 and clone R99-6-10 spans positions 2961-4055. The overlaping regions between these clones exhibit 100% sequence identity except for a single guanine (G75-31-17) to adenosine (R65-24-6) mismatch at position 280 resulting in a Glu to Lys change. Based on the presence of an acidic residue in this position in human ZC1 and ZC3 and [0580] C. elegans ZC504.4, the sequence encoding the Glu is probably correct. The human ZC2 gene contains a 3891 bp ORF (1-3891) flanked by 164 bp (3892-4055) 3′ UTR. No polyadenylation signal (AATAAA) or polyadenylated region is present in the 3′ UTR.
  • Multiple EST fragments (R51245) match the 3′ end of the human ZC2 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0581]
  • cDNA Cloning and Characterization of ZC3 [0582]
  • The human ZC3 cDNA sequence is composed of four overlapping PCR clones, G75-30-30, 3R33-5-3, 3R19-17-6, and R99-43-11. [0583]
  • A human ZC3 clone, G75-30-30, was first identified from a PCR screen with degenerate oligos, ROS1 and CCK4b, applied to sscDNA generated from a human HLT370 primary non-small cell lung cancer tissue. Sequence analysis of the 492 bp insert identified a 492 ORF (164 amino acids) with the potential to encode a novel human STK related to the [0584] C. elegans ZC504.4 gene product.
  • PCR clone R99-43-11 was isolated as follows: A Smith-Waterman search of the EST database with the [0585] C. elegans ZC504.4 gene (GB:Z50029) as a query identified a human EST (R54563) whose ORF is related to the C. elegans gene and terminates in an identical residue (Trp). A primer was designed 3′ to the stop codon found in R54563 (5′-TCAGGGGTCAGAGGTCACG(SEQ ID NO:57)) and used in a PCR reaction with a primer derived from the 5′ end of R54563 (5′-CCCAAACCCTACCACAAATTC(SEQ ID NO:58)) using sscDNA from human fetal brain as a template. PCR clone R99-43-11 was identified and was found to contain a 719 bp insert with a 564 bp ORF encoding the C-terminal portion of human ZC3 (188 amino acids).
  • PCR clone 3R19-17-6 was isolated from human A549 lung cancer cell line [0586] sscDNA using primers 5′-CCCCCGGGAAACGATGACCA and 5′-AGCCGCTGCCCCTCCTCTACTGT derived from G75-30-30 and R99-43-11, respectively. The 3′ primer used in this PCR reaction misprimed leading to the formation of a truncated product. 3R19-17-6 contains a 1172 bp insert and an ORF of 1170 bp (390 amino acids).
  • PCR clone 3R33-5-3 was isolated from human A549 lung cancer cell line [0587] sscDNA using primers 5′-ACCGCAACATCGCCACCTACTAC(SEQ ID NO:61) and 5′-CTCGACGTCGTGGACCACC(SEQ ID NO:62) derived from G75-30-30 and 3R19-17-6, respectively. 3R33-5-3 contains a 2465 bp insert and an ORF of 2463 bp (821 amino acids).
  • The full-length human ZC3 cDNA (SEQ ID NO. 11) is 4133 bp long. Clone G75-30-30 spans positions 1-483, clone 3R33-5-3 spans positions 134-2598, clone 3R19-17-6 spans positions 2356-3512 and clone R99-43-11 spans positions 3415-4133. The overlaps between these clones exhibit 100% sequence identity. The human ZC3 gene contains a 3978 bp ORF (1-3978) flanked by a 152 [0588] bp 3′ UTR (3979-4133). No polyadenylation signal (AATAAA) or polyadenylated region is present in the 3′ UTR.
  • Multiple EST fragments (R54563) match the 3′ end of the human ZC3 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0589]
  • cDNA Cloning and Characterization of ZC4 [0590]
  • The human ZC4 cDNA sequence, represented by PCR fragment 3R25-27-1, was first identified in the human genomic cosmid 82J11 (GB:Z833850) containing exon sequences that displayed strong homology to the ZC504.4 [0591] C. elegans gene.
  • PCR clone 3R25-27-1 was isolated from human fetal liver sscDNA and [0592] primers 5′-CAATGTTAACCCACTCTATGTCTC(SEQ ID NO:63) and 5′-AGTTTGCCGATGTTTTTCTTTTC(SEQ ID NO:64) derived from a potential ORF (positions 25729-25852) from the 82J11 cosmid and from an EST (R98571) encoding the C-terminus of the human ZC4 gene, respectively.
  • The partial human ZC4 cDNA (SEQ ID NO.12) is 1459 bp long and consists of a 1047 bp ORF (2-1048) flanked by a 411 bp (1049-1459) 3′ UTR region. No polyadenylation signal (AATAAA) or polyadenylated region is present in the 3′ UTR. [0593]
  • The N-terminal coding sequence for ZC4_h was extended by building a contiguous DNA sequence of 233,137 bp containing Z83850 and four other sequences: cU84B10 and cU230B10 (from the Sanger Human Genome Sequencing Project) and Z97356 and Z69734 (available from the National Institute for Biotechnology Information. The position of each sequence in the contig is represented in the table below. [0594]
    Accession Length Start End
    cUS4B10 43273 0 43273
    Z97356 21848 43171 65018
    Z69734 37077 63073 100149
    cU230B10 11841 88416 100256
    Z83850 132981 100156 233137
  • Sequences in ZC4 genomic contig. [0595]
  • The 233,137 bp contig was analyzed for exons using the programs FGENES 1.5 and FGENESH, human gene structure prediction software available from The Sanger Centre. [0596]
  • The resulting human ZC4 coding sequence (SEQ ID NO:104) is 3,681 bp long (excluding the stop codon) and encodes for a STE20 kinase of 1227 amino acids. [0597]
  • cDNA Cloning and Characterization of KHS2 [0598]
  • The human KHS2 cDNA sequence is composed of four overlapping clones, 3R25-51-2, 3R16-34-2, 3R16-31-2, and T79916. [0599]
  • A human KHS2 clone, AA250855, was first identified using a Smith-Waterman search of the EST database with KHS1 (GB:U77129) as a query. Sequence analysis of the 1112 bp insert identified a 618 bp ORF (206 amino acids) with the potential to encode a novel STK related to the human KHS1 gene product. Using AA250855 as a query, a second EST (AA446022) was found whose sequence was shown to contain the initiator methionine for human KHS2 based on a comparison with KHS1. [0600]
  • PCR clone 3R25-51-2 was isolated from human testicular cancer [0601] sscDNA using primers 5′-CCGCCATGAACCCCGGCTT(SEQ ID NO:65) and 5′-CGATTGCCAAAGACCGTGTCA(SEQ ID NO:66) derived from AA446022 and AA250855, respectively. 3R25-51-2 contains an 850 bp insert and an ORF of 849 bp (283 amino acids).
  • EST clone, T79916, was identified using a Smith-Waterman search of the EST database with the C-terminus of KHS1 (GB:U77129) as a query. Sequence analysis of the 2107 bp insert identified this EST to contain an ORF of 345 bp (115 amino acids disrupted by a single stop codon) encoding the C-terminus of human KHS2, followed by 1762 [0602] bp 3′ UTR.
  • PCR clone 3R16-34-2 was isolated from human testis [0603] sscDNA using primers 5′-AGAAGTTGCAGCTGTTGAGAGGA(SEQ ID NO:67) and 5′-TATGGCCCGTGTAAGGATTTC(SEQ ID NO:68) derived from AA250885 and T79916, respectively. 3R16-34-2 contains an 1516 bp insert and an ORF of 1128 bp (376 amino acids).
  • PCR clone 3R16-31-2 was isolated from normal human colon [0604] sscDNA using primers 5′-GTGCCAGAAGTGTTGTGTTGTAA(SEQ ID NO:69) and 5′-TATGGCCCGTGTAAGGATTTC(SEQ ID NO:68) derived from EST T79916. 3R16-31-2 contains a 728 bp insert and an ORF of 669 bp (223 amino acids). This clone lacked the stop codon present within EST T79916 (postion 2662 in the KHS2 sequence).
  • The full-length human KHS2 cDNA (SEQ ID NO.17) is 4023 bp long. Clone 3R25-51-2 spans positions 1-855, clone AA250885 spans positions 336-923, clone 3R16-34-2 spans positions 545-2061, and clone T79916 spans positions 1917-4023. The overlaping regions between these clones exhibit 100% sequence identity, except for 4 nucleotide differences, two of which are silent, a third corrects the internal stop codon at position 2662, and the fourth at position 247 (T to C change) results in a Pro to Leu change. The human KHS2 cDNA contains a 2682 bp ORF (6-2687) flanked by a 5 bp (1-5) 5′UTR and a 1336 bp (2688-4023) 3′ UTR. A potential polyadenylation signal (AATAAA) is found at positions 4008-4013. No polyadenylated region is present in the 3′ UTR. The sequence flanking the first ATG is in a poor context for translational initiation, however, a 134 [0605] bp 5′UTR sequence from EST AA446022 did not reveal any additional ATG's and displayed two in-frame stop codons 5′ to the putative start ATG for human KHS2.
  • Multiple EST fragments match the 5′end (AA446022) as well as the 3′ end (R37625) of the human KHS2 gene. [0606]
  • cDNA Cloning and Characterization of SULU1 [0607]
  • The human SULU1 cDNA sequence is composed of three overlapping clones, N40091, 2R90-1-1 and R90907. [0608]
  • A human SULU1 clone, N40091, was first identified using a Smith-Waterman search of the EST database with the [0609] C. elegans SULU gene (GB: U32275) as a query. Sequence analysis of the 1321 bp insert identified a 906 bp ORF (302 amino acids) with the potential to encode a novel human STK related to the C. elegans SULU gene product.
  • EST clone R90907 was first identified using a Smith-Waterman search of the EST database with the 3′ end of the [0610] C. elegans SULU gene (GB: U32275) as a query. Sequence analysis of the 1647 bp insert identified a 578 bp ORF (192 amino acids) with the potential to encode the C-terminus of the human SULU1 gene product.
  • PCR clone 2R90-1-1 was isolated from human HT29 colon cancer cell [0611] sscDNA using primers 5′-TATTGAATTGGCGGAACGGAAG(SEQ ID NO:70) and 5′-TTGTTTTGTGCTCATTCTTTGGAG(SEQ ID NO:71) derived from N40091 and R90907, respectively. 2R90-1-1 contains a 1625 bp insert and an ORF of 1623 bp (541 amino acids).
  • The full-length human SULU1 cDNA (SEQ ID NO.19) is 4177 bp long Clone N40091 spans positions 1-1321, clone 2R90-1-1 spans positions 1048-2671, and clone R90907 spans positions 2531-4177. The overlaping regions between these clones exhibit 100% sequence identity. The human SULU1 cDNA contains a 2694 bp ORF (415-3108) flanked by a 414 bp (1-414) 5′UTR and a 1069 bp (3109-4177) 3′ UTR followed by a 19 nucleotide polydenylated region. A potential polyadenylation signal (AATAAA) is found at positions 4164-4169. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human SULU1. [0612]
  • Multiple EST fragments match the 5′end (N27153) as well as the 3′ end (R90908) of the human SULU1 gene. [0613]
  • cDNA Cloning and Characterization of Murine SULU3 [0614]
  • The murine SULU3 cDNA sequence is represented by PCR fragment 2R92-1-6. [0615]
  • A murine SULU3 clone, G83-4-5, was first identified from a PCR screen with degenerate oligos, CCK4c and CCK4b, applied to sscDNA generated from murine day-12 embryos. Sequence analysis of the 473 bp insert identified a 471 ORF (157 amino acids) with the potential to encode a novel human STK related to the [0616] C. elegans SULU gene (GB: U32275) product. The antisense strand of G83-4-5 is identical at the nucleic acid level to the 5′UTR of the murine etsl protooncogenic transcription factor (GB:X53953). This homology is likely the result of a cloning artifact attached to the 5′-end of the database entry for murine ets1.
  • PCR clone 3R19-17-6 was isolated from human A549 cell [0617] sscDNA using primers 5′-CCCCCGGGAAACGATGACCA(SEQ ID NP:59) and 5′-AGCCGCTGCCCCTCCTCTACTGT(SEQ ID NO:60) derived from G75-30-30 and R99-43-11, respectively. The 3′ primer used in this PCR reaction misprimed leading to the formation of a truncated product. 3R19-17-6 contains a 1172 bp insert and an ORF of 1170 bp (390 amino acids).
  • PCR clone 2R92-1-6 was isolated from murine d8 embryo [0618] sscDNA using primers 5′-ACCGCAACATCGCCACCTACTAC(SEQ ID NO:61) and 5′-GATTGCTTTGTGCTCATTCTTTGG(SEQ ID NO:72) derived from the 5′ UTR of the etsl gene and the human EST AA234623, respectively. The latter (shown herein) encodes the C-terminus of human SULU3. 2R92-1-6 contains a 2249 bp insert and an ORF of 2244 bp (748 amino acids).
  • The partial murine SULU3 cDNA (SEQ ID NO.21) is 2249 bp long and consists of a 2244 bp ORF (6-2249) flanked by a 5 bp (1-5) 5′UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for murine SULU3. [0619]
  • One EST fragment (AA446022) matches the 3′ end of the partial murine SULU3 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0620]
  • cDNA Cloning and Characterization of Human SULU3 [0621]
  • The human SULU3 cDNA sequence is composed of two overlapping clones, 2R90-22-1 and AA234623. [0622]
  • A human SULU3 clone, AA234623, was first identified using a Smith-Waterman search of the EST database with the [0623] C. elegans SULU gene (GB: U32275) as a query. Sequence analysis of the 2652 bp insert identified a 1185 bp ORF (395 amino acids) with the potential to encode the C-terminus of a novel human STK related to the C. elegans SULU gene product.
  • PCR clone 2R90-22-1 was isolated from human SKMel128 melanoma cell line [0624] sscDNA using primers 5′-TATTGAATTGGCGGAACGGAAG(SEQ ID NO:70) and 5′-TTGTTCTAAGAGTGCCCTCCG(SEQ ID NO:73) derived from the murine SULU3 2R92-1-6 clone and from AA234623, respectively. 2R92-1-6 contains a 1897 bp insert and an ORF of 1896 bp (632 amino acids).
  • The partial human SULU3 cDNA (SEQ ID NO.20) is 3824 bp long. Clone 2R90-22-1 spans positions 1-1897 and clone AA234623 spans [0625] positions 1173. The overlaping region between these clones exhibits 100% sequence identity. The human SULU3 cDNA contains a 2358 bp ORF (2-2359) flanked by a 1465 bp (2360-3824) 3′ UTR followed by a 19 nucleotide polydenylated region. A potential polyadenylation signal (AATAAA) is found at positions 2602-2607. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine.
  • Multiple EST fragments (R02283) match the 3′ end of the human SULU3 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0626]
  • cDNA Cloning and Characterization of GEK2 [0627]
  • The human GEK2 cDNA sequence is composed of three overlapping clones, AA459448, 3R25-48-1 and [0628] GEK2_h#3.
  • A human GEK2 clone, AA459448, was first identified using a Smith-Waterman search of the EST database with the human SLK gene (GB: AB002804) as a query. Sequence analysis of the 1286 bp insert identified a 1227 bp ORF (409 amino acids) with the potential to encode the N-terminus of a novel human STK related to the human SLK gene product. An additional Smith-Waterman search using the C-terminus of the SLK gene as a query yielded three additional EST's, AA323687, AA380492 and AA168869, that encode the C-terminal region of human GEK2. [0629]
  • PCR clone 2R98-41-17 was isolated from human testis [0630] sscDNA using primers 5′-AAGACCATGCCGTGCGCCG(SEQ ID NO:74) and 5′-ATTCCTTCAGGTTCTGGTTATGG(SEQ ID NO:75) derived from AA323687 and from AA380492, respectively. 2R98-41-17 contains a 851 bp insert and an ORF of 849 bp (283 amino acids).
  • PCR [0631] clone GEK2_h#3 was isolated from human sscDNA made from the H23 tumor cell line using primers 5′-GCAGCAAGTGGAGAAGATGG(SEQ ID NO: 109) and 5′-GGAAGCATCCCCAGAGCTGTAG(SEQ ID NO: 110) derived from the sequence of clone 3R25-48-1 and from the 3′ end of murine LOK (GB:D89728), respectively. GEK2_h#3 contains a 1042bp insert and an ORF of 1041 bp (347 amino acids).
  • The full-length human GEK2 cDNA (SEQ ID NO:106) is 2962 bp long. Clone AA459448 spans positions 1-1286, clone 3R25-48-1 spans positions 1100-2449 and clone [0632] GEK2_h#3 spans positions 1920-2962. The overlapping regions between these clones exhibit 100% sequence identity.
  • The human GEK2 cDNA contains a 2904 bp ORF (59-2962) flanked by a 58 bp (1-58) 5′UTR. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human GEK2. [0633]
  • Multiple EST fragments (AA465671) match the 5′end of the sequence, but only one (AA380492) matches the 3′ end of the human GEK2 gene. [0634]
  • cDNA Cloning and Characterization of PAK4 [0635]
  • The human PAK4 cDNA sequence is represented by [0636] clone SNB2#1.
  • A human PAK4 clone, R88460, was first identified using a Smith-Waterman search of the EST database with the human PAK gene (GB: U24152) as a query. Sequence analysis of the 2332 bp insert identified a 930 bp ORF (310 amino acids) with the potential to encode the C-terminus of a novel human STK related to the human PAK gene product. [0637]
  • cDNA [0638] clone SNB2#1 was isolated from human glioblastoma cell line SNB75 cDNA library using a probe derived from R88460. SNB2#1 contains a 3604 bp insert and an ORF of 2043 bp (681 amino acids).
  • The full-length human PAK4 cDNA (SEQ ID NO.27) is 3604 bp long and consists of a 2043 bp ORF (143-2185) flanked by a 142 bp (1-142) 5′UTR and a 1419 3′ UTR followed by a 22 nucleotide polydenylated region. A potential polyadenylation signal (AATTAAA) is found at positions 3582-3588. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for human PAK4. The 3′ UTR of the PAK4 gene contains a GT dinucleotide repeat prone to undergo expansion based on the number of repeats found in [0639] clones SNB#1 and R88460, 32 and 23, respectively. Several neurologic disorders have been correlated with the expansion of di- or tri-nucleotide repeats similar to those found in the PAK4 sequence, suggesting PAK 4 may also be a disease target and that this repeat in its 3′ UTR may serve as a diagnostic marker.
  • Multiple EST fragments (AA535791) match the 3′end of the human PAK4 gene, but at the time of filing, the inventors believe that none exist in GenBank or the EST database that match its 5′ end. [0640]
  • cDNA Cloning and Characterization of PAK5 [0641]
  • The full-length human PAK5 cDNA sequence is composed of two overlapping clones, H450#1-1 and [0642] SNB8#5.
  • A human PAK5 clone, RI 8825, was first identified using a Smith-Waterman search of the EST database with the human PAK4 gene as a query. Sequence analysis of the 1248 bp insert identified a 420 bp ORF (140 amino acids) with the potential to encode the C-terminus of a novel human STK related to the human PAK4 gene product. [0643]
  • cDNA [0644] clone SNB8#5 was isolated from human SNB75 cDNA library using a probe derived from R18825. SNB2#1 contains a 2028 bp insert and an ORF of 1194 bp (398 amino acids).
  • The partial human PAK5 cDNA (SEQ ID NO.28) is 2028 bp long and consists of a 1194 bp ORF (2-1195) flanked by an 833 bp (1196-2028) 3′ UTR followed by a 22 nucleotide polydenylated region. A potential polyadenylation signal (AATTAAA) is found at positions 2004-2010. Since the coding region is open throughout the 5′ extent of this sequence, this is apparently a partial cDNA clone lacking the N-terminal start methionine. [0645]
  • Clone H460#1-1 was isolated from a human lung H460 cDNA library using a probe derived from the [0646] partial SNB2#1 cDNA clone described above. Sequence analysis of the 2526 bp insert identified a 1773 bp ORF (592 amino acids) with the potential to encode a full-length PAK5.
  • The human PAK5 cDNA (SEQ ID NO:102) is 2,806 bp long and consists of a 1,773 bp ORF flanked by a 201 [0647] bp 5′ UTR and a 833 bp 3′ UTR. The sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiating methionine, and is believed to be the translational start site for PAK5.
  • PAK5 shares 99% sequence identity over 2795 bp to a recent database entry, AF005046. These sequences are presumed to be from the same gene, with minor polymorphic variations. [0648]
    • Example 2 Expression Analysis of Mammalian STE20-Related Protein Kinases
  • Materials and Methods [0649]
  • Northern blot analysis [0650]
  • Northern blots were prepared by running 10 μg total RNA isolated from 60 human tumor cell lines (HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, SNB-19, SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, K-562, MOLT-4, HL-60, RPMI 8226, SR, DU-145, PC-3, HT-29, HCC-2998, HCT-116, SW620, Colo 205, HTC15, KM-12, UO-31, SN12C, A498, CaKil, RXF-393, ACHN, 786-0, TK-10, LOX IMVI, Malme-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, MCF-7, MCF-7/ADR RES, Hs578T, MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D), from 22 human adult tissues (thymus, lung, duodenum, colon, testis, brain, cerebellum, cortex, salivary gland, liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle, placenta, mammary gland, bladder, lymph node, adipose tissue), and 2 human fetal normal tissues (fetal liver, fetal brain), on a denaturing formaldehyde 1.2% agarose gel and transferring to nylon membranes. [0651]
  • Filters were hybridized with random primed [α[0652] 32P]dCTP-labeled probes synthesized from the inserts of several of the STE20-related kinase genes. Hybridization was performed at 42° C. overnight in 6X SSC, 0.1% SDS, 1× Denhardt's solution, 100 μg/mL denatured herring sperm DNA with 1-2 x 106 cpm/mL of 32P-labeled DNA probes. The filters were washed in 0.1× SSC/0.1% SDS, 65° C., and exposed on a Molecular Dynamics phosphorimager.
  • Quantitative PCR Analysis [0653]
  • RNA was isolated from a variety of normal human tissues and cell lines. Single stranded cDNA was synthesized from 10 □g of each RNA as described above using the Superscript Preamplification System (GibcoBRL). These single strand templates were then used in a 25 cycle PCR reaction with primers specific to each clone. Reaction products were electrophoresed on 2% agarose gels, stained with ethidium bromide and photographed on a UV light box. The relative intensity of the STK-specific bands were estimated for each sample. [0654]
  • DNA Array Based Expression Analysis [0655]
  • Plasmid DNA array blots were prepared by loading 0.5 □g denatured plasmid for each STE20-related kinase on a nylon membrane. The [α[0656] 32P]dCTP labeled single stranded DNA probes were synthesized from the total RNA isolated from several human immune tissue sources or tumor cells (thymus, dendrocytes, mast cells, monocytes, B cells (primary, Jurkat, RPMI8226, SR), T cells (CD8/CD4+, TH 1, TH2, CEM, MOLT4), K562 (megakaryocytes). Hybridization was performed at 42° C. for 16 hours in 6× SSC, 0.1% SDS, 1× Denhardt's solution, 100 μg/mL denatured herring sperm DNA with 106 cpm/mL of [α32P]dCTP labeled single stranded probe. The filters were washed in 0.1× SSC/0.1% SDS, 65° C., and exposed for quantitative analysis on a Molecular Dynamics phosphorimager.
  • Results [0657]
  • Distribution of STE20-Related Gene Transcripts in Normal Tissues and Tumor Cell Lines [0658]
  • ZC1, ZC2, and ZC3 RNA expression was analyzed by quantitative PCR from multiple human normal tissues, cultured primary epithelial and endothelial cells, and tumor cell lines. The results are summarized in Tables 1 and 2, with relative expression values ranging from 0 (undetectable) to 23 (very strong). An “x” refers to sample not tested. ZC1, ZC2, and ZC3 were all expressed at very low levels in most normal human tissues, however ZC1 and ZC2 were more abundant in cultured epithelial cells and ZC3 in normal kidney and breast tissue. [0659]
  • Expression of these 3 genes was also examined in a panel of human tumor cell lines representing a diverse sampling of tumor types (Table 2). ZC1 and ZC2 showed strong expression in cell lines from most melanomas and renal tumors and from some non-small cell lung cancers and colon tumors. ZC3 expression was consistently lower in the tumor cell lines except for high expression in most breast cancers and leukemias. The robust overexpression ZC1, ZC2, and ZC3 in tumor cells versus normal tissues may provide an attractive target for oncology drug development. [0660]
  • Expression of all the novel STE20-related kinases was examined in a panel of human immune tissues/cells by hybridization to a DNA array blot containing plasmids encoding each of these genes. STLK2 was broadly expressed in all 14 immune samples, whereas STLK4 and PAK4 were highly expressed in a subset of 6-7 of the samples (Table 3). Several other kinases (SULU3, ZC4, KHS2) had more restricted expression, while others were expressed in only a single immune source (STLK3, thymus; ZC1, dendrocytes; ZC3, monocytes; PAK5, mast cells and MOLT4), and several more were absent from all the immune sources assayed (GEK2, SULU1, ZC2, STLK5). These expression patterns were quite distinct among members of the same subfamily (i.e., ZC1, ZC2, ZC3 and ZC4, or PAK1, PAK2, PAK3, PAK4, PAK5). This analysis suggests that some of these kinases may be candidate targets for various immune disorders, and that some, which are more broadly expressed, may mediate functions vital to the basic biology of most proliferating cells. [0661]
    TABLE 1
    ZC1, ZC2 and ZC3 Expression
    in Normal Human Tissues and Cells
    Sample ZC1 ZC2 ZC3
    NORMAL
    Brain Tiss 2.8 0.6 0.9
    Duod Tiss 3.8 1.5 0.3
    Heart Tiss 1.2 0.3 0.0
    Kidney Tiss 0.7 0.0 7.0
    Lung Tiss 1.6 0.2 0.0
    Pancreas Tiss 2.0 0.4 2.5
    Placenta Tiss 1.4 0.0 0.0
    Sal gl. Tiss 3.0 0.3 3.2
    Sk mus. Tiss 2.3 0.1 0.1
    Spleen Tiss 0.4 0.0 x
    Stomach Tiss 0.8 0.0 0.0
    Thymus Tiss 3.5 0.4 1.5
    Cereb Tiss 2.8 1.1 4.4
    Liver Tiss 1.8 0.0 0.4
    Uterus Tiss 1.6 0.0 1.4
    Prostate Tiss 1.4 0.0 1.6
    Testis Tiss x x 5.8
    f Brain Tiss x x 3.1
    Mam gl Tiss x x 7.2
    HCAEC ENDO 1.0 0.0 0.0
    HMVEC-d ENDO 0.7 0.0 0.4
    HMVEC-L ENDO 2.2 1.6 1.8
    HPAEC ENDO 9.3 5.3 6.4
    HMEC EPI 4.1 2.3 1.9
    RPTEC EPI 3.6 2.2 0.2
    HRCE EPI 5.3 3.5 1.3
    HSAE EPI 0.9 3.3 4.8
  • [0662]
    TABLE 2
    ZC1, ZC2 and ZC3 Expression in Tumor Cell 1Lnes
    Sample Origin ZC1 ZC2 ZC3 Sample Origin ZC1 ZC2 ZC3
    HOP-92 Lung 9.3 7.2 3.3 HCC-2998 Colon 2.4 3.8 3.0
    EKVX Lung 10.7 3.7 3.5 HCT 116 Colon 2.2 2.1 5.4
    NCI-H23 Lung 5.8 6.3 4.1 SW-620 Colon 7.8 12.1 3.1
    NCI-H226 Lung 6.5 6.8 3.3 COLO 205 Colon 9.1 16.2 3.0
    NCI-H322M Lung 3.5 5.8 4.9 HCT-15 Colon 13.8 4.9 2.5
    NCI-H460 Lung 4.5 3.7 2.9 KM-12 Colon 7.0 13.2 3.1
    NCI-H522 Lung 4.7 3.3 4.6 UO-31 Colon 10.4 10.6 0.9
    A549/ATCC Lung 3.8 3.6 4.1 SN12C Renal 8.1 3.4 2.8
    HOP-62 Lung 4.3 3.8 4.2 A498 Renal 6.2 3.1 2.9
    OVCAR-3 Ovary 2.9 3.1 1.5 Caki-1 Renal 9.2 14.4 2.3
    OVCAR-4 Ovary 3.3 1.0 3.8 RXF 393 Renal 10.6 4.8 2.8
    OVCAR-5 Ovary 2.6 3.6 2.2 ACHN Renal 9.3 6.0 3.9
    OVCAR-8 Ovary 3.6 2.0 4.7 786-0 Renal 8.8 15.6 5.6
    IGROV1 Ovary 3.8 1.7 3.2 TK-10 Renal 20.9 21.2 5.0
    SK-OV-3 Ovary 4.9 0.0 3.5 LOX IMVI Mel 2.3 2.4 3.3
    SNB-19 CNS 5.1 5.4 4.2 Malme-3M Mel x x 2.2
    SNB-75 CNS 2.5 0.9 0.7 SK-MEL-2 Mel 15.7 14.1 2.9
    U251 CNS 1.5 1.2 0.6 SK-MEL-5 Mel 7.9 7.0 0.0
    SF-268 CNS 5.8 2.7 3.0 SK-MEL-28 Mel 16.5 23.1 0.0
    SF-295 CNS 6.4 1.1 3.2 UACC-62 Mel 12.1 18.3 5.3
    SF-539 CNS 5.1 2.9 4.3 UACC-257 Mel 10.8 9.4 6.2
    CCRF-CEM Leuk 3.4 2.7 3.1 M14 Mel 4.4 0.9 7.9
    K-562 Leuk 4.1 6.3 4.3 MCF7 Breast 4.8 1.3 7.7
    MOLT-4 Leuk 7.1 3.4 4.2 MCF-7/ADR Breast 8.8 3.4 7.7
    HL-60 Leuk x x 0.4 Hs 578T Breast 6.9 2.6 5.7
    RPMI 8226 Leuk 0.5 0.2 1.4 MDA-MB-231 Breast 5.7 1.9 6.4
    SR Leuk 3.5 7.2 5.4 MDA-MB-435 Breast 4.8 6.7 9.1
    DU-145 Pro x x 3.4 MDA-N Breast 7.3 6.3 9.1
    PC-3 Pro x x 3.4 BT-549 Breast 3.6 1.9 8.0
    HT-29 Colon 2.4 5.9 6.6 T-47D Breast 0.4 12.3 9.3
  • [0663]
    TABLE 3
    STE20-related kinase expression in a human immune panel
    Mast Mono- B CD8+
    KINASE thymus Dendrocytes cells cytes cells CD4+ TH1 TH2
    GEK2 350 350 350 350 350 350 350 350
    SULU1 350 350 350 350 350 350 350 350
    SULU3 350 350 350 350 12149 350 5115 350
    STLK2 117770 13771 27620 92036 18305 39109 5408 3564
    STLK3 8624 350 350 350 350 350 350 350
    STLK4 8524 350 350 350 350 8685 5642 350
    STLK5 xxx xxx xxx xxx 350 350 350 xxx
    ZC1 350 3377 350 350 350 350 350 350
    ZC2 350 350 350 350 350 350 350 350
    ZC3 350 350 350 20156 350 350 350 350
    ZC4 xxx xxx xxx xxx 350 350 350 xxx
    KHS2 8766 2508 350 56575 350 350 350 350
    PAK4 32658 7684 3729 100948 350 350 350 1604
    PAK5 350 350 4905 350 350 350 350 350
    CEM MOLT4 JURKAT RPMI8226 SR K562
    KINASE (T cell) (T cell) (B cell) (B cell) (B cell) (MO)
    GEK2 350 350 350 350 350 350
    SULU1 350 350 350 350 350 350
    SULU3 350 350 350 350 350 350
    STLK2 47236 53262 47605 22560 65936 30390
    STLK3 350 350 350 350 350 350
    STLK4 3648 350 26772 1570 350 350
    STLK5 350 350 350 xxx 350 350
    ZC1 350 350 350 350 350 350
    ZC2 350 350 350 350 350 350
    ZC3 350 350 350 350 350 350
    ZC4 1094 7813 14945 xxx 350 6385
    KHS2 350 350 350 350 350 350
    PAK4 350 10246 350 3229 350 350
    PAK5 350 12672 350 350 350 350
  • Transcript Size from Northern Data [0664]
    Kinase (kb)
    STLK2 3.8
    STLK4 5.0
    ZC1 6.9/4.7
    ZC2 6.0/8.0
    ZC4 5
    KHS2 4.4
    SULU1 4.5
    SULU3 10.0
    GEK2 5.5
    PAK4 4.8
    PAK5 3.5
  • STLK2is widely expressed; the highest expression levels were found in placenta, spleen and PBL. [0665]
  • STLK4 is also widely expressed in normal tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood lymphocytes. STLK4 was also detected in Jurkat T cells. [0666]
  • ZC1 is highly overexpressed in the following human cancer cell lines: HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62 (lung); OVCAR-3, OVCAR-4, OVCAR-5 (ovary); SNB-19, U251, SF-268, SF-295, SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3 (prostate); HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12 (colon); UO-31, CAKi-1, RXF-393, 786-0, TK-10 (renal); LOXIMVI, Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); and MCF-7, MCF-7/ADR, HIS 578T, MDA-MB-231, MDA-MB-431, MDA-N, BT-549, T-47D (breast). [0667]
  • ZC2 is expressed in brain and testis. It is highly overexpressed in the following human cancer cell lines: TK-10 (renal); SK-MEL-28, UACC-62 (melanoma); T47D (breast). [0668]
  • Moderate expression in HOP92 (lung); OVCAR4, IGROVI (ovary); DNB75, U251 (brain); K-562 (leukemia); and COL0205 (colon). [0669]
  • SULU1 is overexpressed in the following human cancer cell lines: HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62 (lung); OVCAR-3, OVCAR-4, OVCAR-5, SK-OV-3 (ovary); SNB-19, U251, SF-268, SF-295, SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3 (prostate); HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12 (colon); UO-31, CAKi-1, RXF-393, 786-0, TK-10 (renal); LOX, IMVI, Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); MCF-7, MCF-7/ADR, HIS 578T, MDA-MB-231, MDA-MB-431, MDA-N, BT-549, T-47D (breast) [0670]
  • SULU3 showed a broad pattern of expression in the normal tissue panel of RNAs. [0671]
  • GEK2 was expressed in spleen, thymus and testis. Expression was high in the cell lines RBL-2H3 and H441. [0672]
  • PAK4 was expressed in the normal tissues: brain, testis and prostate, and in the human cancer cell lines: HNCI-H23 (lung); OVCAR-3 (ovary); SNB-19, U251 (CNS); RPMI-8226 (leukemia); DU-145 (prostate); COLO-205, HCT-15 (colon). [0673]
  • PAK5 showed weak expression levels in the normal tissues: brain, testes, bladder, colon, adrenal medulla, spleen, fetal liver, breast, cerebral cortex, cerebellum, thymus, salivary gland, lung, stomach, duodenum, uterus, prostate, skeletal muscle and placenta. PAK5 was overexpressed in the human cancer cell lines: HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H522, A549, HOP-62 (lung); OVCAR-3, OVCAR-4, OVCAR-5, SK-OV-3 (ovary); SNB-19, U251, SF-268, SF-295, SF-539 (CNS); K-562, RPMI-8226 (leukemia); DU-145, PC-3 (prostate); HT-29, HCC-2998, HCT-116, SW620, COLO-205, HCT-15, KM-12 (colon); UO-31, CAKi-1, RXF-393, 786-0, TK-10 (renal); LOXIMVI, Malme-3M, SK-MEL-2, SK-MEL-28, UACC-62, UACC-257, M14 (melanoma); MCF-7, MCF-7/ADR, HIS 578T, MDA-MB-231, MDA-MB-431, MDA-N, BT-549, T-47D (breast). [0674]
    • Example 3 STE20-related Protein Kinase Gene Expression Vector Construction
  • Materials and Methods [0675]
  • Expression Vector Construction [0676]
  • Several expression constructs were generated for some of the human STE20-related cDNAs including: a) full-length clones in a pCDNA expression vector; b) a GST-fusion construct containing the catalytic domain of the novel STE20-related kinase fused to the C-terminal end of a GST expression cassette; and c) a full-length clone containing a Lys to Ala (K to A) mutation at the predicted ATP binding site within the kinase domain, inserted in the pCDNA vector. [0677]
  • The “K to A”mutants of the STE20-related kinase might function as dominant negative constructs, and will be used to elucidate the function of these novel STKs. [0678]
  • Results [0679]
  • Constructs for ZC1, ZC2, ZC3, SULU1, SULU3, PAK4 and PAK5 have been generated. [0680]
  • Numerous additional constructs have been generated for the various STE20-subfamily kinases, including full length, kinase inactive and tagged versions. In addition, the following three constructs were designed for specific applications based on their unique domain structure: [0681]
  • Construct 1: SULU1-Coiled-Coil2 [0682]
  • Vector: pGEX-4T [0683]
  • Insert: Coiled-coil2 [0684]
  • Sequence: Amino acids 752-898 [0685]
  • Purpose: phage display [0686]
  • Result: Interacts with [0687] GEK2 CC 1
  • Construct 2: SULU3-Coiled-Coil2 [0688]
  • Vector: pGEX4T [0689]
  • Insert: coiled-[0690] coil 2 domain fused to GST
  • Sequence range of insert: amino acids 802-898 of SEQ [0691]
  • Purpose: phage display [0692]
  • Result: Interacts with coiled-coiled region of human SLK [0693]
  • Construct 3: PAK5 Dominant Negative [0694]
  • Vector: pCAN5 [0695]
  • Insert: Full length coding sequence of human PAK5 containing the following mutation: [0696]
  • K350,351A (Lys at [0697] aa positions 350 and 351 changed to Ala).
  • Purpose: to determine role of human PAK5 kinase activity in cell growth and transformation. [0698]
  • Result: Interferes with Ras transformation. [0699]
    • Example 4 Generation of Specific Immunoreagents to STE20-Related Protein Kinases
  • Materials and Methods [0700]
  • Specific immunoreagents were raised in rabbits against KLH- or MAP-conjugated synthetic peptides corresponding to the human STE20-related kinases. C-terminal peptides were conjugated to KLH with glutaraldehyde, leaving a free C-terminus. Internal peptides were MAP-conjugated with a blocked N-terminus. Additional immunoreagents can also be generated by immunizing rabbits with the bacterially expressed GST-fusion proteins containing the cytoplasmic domains of each novel STK. [0701]
  • The various immune sera are first tested for reactivity and selectivity to recombinant protein, prior to testing for endogenous sources. [0702]
  • Western blots [0703]
  • Proteins in SDS PAGE are transferred to immobilon membrane. The washing buffer is PBST (standard phosphate-buffered saline pH 7.4+0.1% triton x 100). Blocking and antibody incubation buffer is PBST +5% milk. Antibody dilutions varied from 1:1000 to 1:2000. [0704]
  • Results [0705]
  • Three SULU1 antisera (against both 539A (SEQ ID NO: 79) and 540A (SEQ ID NO: 78)) and two SULU3 antisera (542A) (SEQ ID NO: 81) reacted specifically with the peptide antigens. Antisera binding was competable with peptide. Experiments with extracts from cells transfected with epitope-tagged SULU1 and SULU3 genes are underway. [0706]
  • Antisera against the PAK4 C-terminal peptide 554A (SEQ ID NO: 82) reacted with purified Gst-PAK4 and detected a protein of the correct molecular weight from tissue culture cells. Specific immunoprecipitation experiments are ongoing to determine the reactivity with native protein. [0707]
  • Similar immunization and antisera testing experiments are underway for each of the other novel STE20-kinases. [0708]
  • STE20-related protein kinase peptide immunogens and their specificity in recognizing endogenous protein by Western blots or immunoprecipitations. [0709]
    Aa
    Protein Sequence positions Conj West. IP
    STLK2 EKFQKCSADESP 405-416 KLH Y Y
    (SEQ ID No: 111)
    STLK4 SISNSELFPTTDPVGT 252-267 KLH Y Y
    (SEQ ID NO: 112)
    SULU1 LDFPKEDYR 890-898 KLH Y Y
    (SEQ ID NO: 113)
    SULU1 HGDPRPEPRPTQ 409-420 KLH Y Y
    (SEQ ID NO: 114)
    SULU3 PSTNRAGSLKDPEC  2-14 KLH N ND
    (SEQ ID NO: 115)
    SULU3 DPRTRASDPQSPPQVS 411-429 KLH ND ND
    RHK
    (SEQ ID NO: 116)
    PAK4 CLVPLIQLYRKQTSTC 666-680 KLH ND Y
    (SEQ ID NO: 117)
    PAK5 PLMRQNRTR 390-398 KLH Y Y
    (SEQ ID NO: 118)
    PAK5 SGDRRRAGPEKRPKSS 148-163 KLH Y Y
    (SEQ ID NO: 119)
    PAK5 (C) RRKSLVGTPYWM 471-485 KLH Y ND
    APE
    (SEQ ID NO: 120)
  • STE20-related protein kinase GST fusion protein immunogens and their specificity in recognizing endogenous protein by Western blots or immunoprecipitations. [0710]
    Protein domain Aa positions West. IP
    ZC1 Coiled-coil/pro/B/C 350-867 Y Y
    ZC1 B 615-732 Y Y
    ZC2 Coiled-coil/pro/B 348-762 ND ND
    ZC2 B 658-762 Y Y
    PAK4 Nterm 252-426 ND ND
    PAK4 Kinase/Cterm 350-681 ND Y
    PAK5 A/Nterm  53-330 ND ND
    PAK5 A/Nterm  53-309 ND ND
  • The 50 kD STLK2 protein was expressed highly in several hematopoietic cell lines including Jurkat, pGL10, Ramos, A20, WEHI-231, K562, HEL and freshly isolated thyrnocytes from C57/BL6 mice. High levels of STLK2 expression were also detected in several tumor cell lines including Calu6, Colo205, [0711] LS 180, MDAM231 and A549.
  • The 160 kD ZC1 protein was detected in Jurkat T cells, Colo205, HCT116, RIE-1, 293T, MDAMB231, and SK-MEL28. [0712]
  • The 170 kD ZC2 protein was detected in SK-Mel28 and UACC-62. [0713]
  • Elevated levels of the 64 kD PAK5 protein were confirmed in the breast cancer cell lines MDA-231 and MCF-7, and in the lung cancer cell line A549. [0714]
    • Example 5 Recombinant Expression and Biological Assays for STE20-Related Protein Kinases
  • Materials and Methods [0715]
  • Transient Expression of the Ste20-Related Kinases in Mammalian Cells [0716]
  • The pcDNA expression plasmids (10 μg DNA/100 mm plate) containing the STE20-related kinase constructs are introduced into 293 cells with lipofectamine (Gibco BRL). After 72 hours, the cells are harvested in 0.5 mL solubilization buffer (20 mM HEPES, pH 7.35, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl[0717] 2, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin). Sample aliquots were resolved by SDS polyacrylamide gel electrophoresis (PAGE) on 6% acrylamide/0.5% bis-acrylamide gels and electrophoretically transferred to nitrocellulose. Non-specific binding was blocked by preincubating blots in Blotto (phosphate buffered saline containing 5% w/v non-fat dried milk and 0.2% v/v nonidet P-40 (Sigma)), and recombinant protein was detected using the various anti-peptide or anti-GST-fusion specific antisera.
  • In Vitro Kinase Assays [0718]
  • Three days after transfection with the STE20-related kinase expression contructs, a 10 cm plate of 293 cells was washed with PBS and solubilized on ice with 2 mL PBSTDS containing phosphatase inhibitors (10 mM NaHPO[0719] 4, pH 7.25, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 0.2% sodium azide, 1 mM NaF, 1 mM EGTA, 4 mM sodium orthovanadate, 1% aprotinin, 5 μg/mL leupeptin). Cell debris was removed by centrifugation (12000× g, 15 min, 4° C.) and the lysate was precleared by two successive incubations with 50 μL of a 1:1 slurry of protein A sepharose for 1 hour each. One-half mL of the cleared supernatant was reacted with 10 μL of protein A purified kinase-specific antisera (generated from the GST fusion protein or antipeptide antisera) plus 50 μL of a 1:1 slurry of protein A-sepharose for 2 hr at 4° C. The beads were then washed 2 times in PBSTDS, and 2 times in HNTG (20 mM HEPES, pH 7.5/150 mM NaCl, 0,1% Triton X-100, 10% glycerol).
  • The immunopurified kinases on sepharose beads were resuspended in 20 gL HNTG plus 30 mM MgCl[0720] 2, 10 mM MnCl2, and 20 μCi [α32P]ATP (3000 Ci/mmol). The kinase reactions were run for 30 min at room temperature, and stopped by addition of HNTG supplemented with 50 mM EDTA. The samples were washed 6 times in HNTG, boiled 5 min in SDS sample buffer and analyzed by 6% SDS-PAGE followed by autoradiography. Phosphoamino acid analysis was performed by standard 2D methods on 32P-labeled bands excised from the SDS-PAGE gel.
  • Similar assays were performed on bacterially expressed GST-fusion constructs of the kinases. [0721]
  • ZC1 Assay buffer: 20 mM Tris pH 7.4, 200 mM NaCl, 0.5 mM DTT, 3 mM MgCl2, 0.3 mM MnCl2, 100 μM [0722] 32PγATP.
  • Substrates: myelin basic protein (MBP) at 0.28 mg/mL and phosphorylated ZC1 peptide RTVGRRNTFIG[0723] T-PPYWMAPE(SEQ ID NO: 121) at 17 μM (bold underlined residue shows site of phosphorylation).
  • At higher concentrations of MgCl[0724] 2 (3 mM), the activity of ZC1 (both full-length and recombinant kinase domain) is up to 10-fold greater towards exogenous substrate MBP. In contrast, the autophosphorylation and the phosphorylation of the activation loop peptide substrate are both inhibited. Mn++ does not inhibit the autophosphorylation and the peptide phosphorylation by the truncated kinase domain form. However, both the MBP phosphorylation, Mn++-preferring activity AND the autophosphorylating, Mg++-preferring activity are eliminated with mutation of the ATP-binding lysine in ZC1 (Lys54Ala) indicating that both activities are attributable to the ZC1 kinase domain.
  • SULU1 Assay buffer: This buffer is identical to that for ZC1, except for 5 mM MgCl2. Under these conditions, other STE20 family members (PAK4, ZC1) were inhibited for autophosphorylation and required reducing the [Mn] to <0.3 mM for an efficient autophosphorylation reaction. [0725]
  • Substrates: MBP, phosvitin, or α-casein at 0.28 mg/mL. [0726]
  • PAK4, PAK5 Assay Buffer: 20 mM Hepes pH 7.2, 130 mM KCl, 10 mM MgCl2, 1 mM NaF, 20 mM B-glycerolphosphate, 0.5 mM DTT, 50 μM ATP, 0.5 μCi [0727] 32PγATP.
  • Substrates: MBP at 0.28 mg/mL and peptide substrates derived from PAK5 activation loop at 2.5 μM. [0728]
  • STLK2 Assay buffer: Similar to that described above, except for the inclusion of 5 mM MgCl[0729] 2, 5 mM MnCl2 and 5 μCi 32PγATP.
  • Transformation (PAK Experiments) [0730]
  • Low-passage NIH3T3 fibroblasts displaying normal morphology (flat, non-refractile cellular morphology), as well as low rates of spontaneous transformation, were used in transformation assays. NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL) and kept in an humidified incubator at 37° C. and 5% CO[0731] 2.
  • Cells were transfected with DNA-lipid complexes. As per manufacturer instructions, lipofectamine was utilized to transfect NIH3T3 cells. All transfections were with equal amounts of plasmid DNA (DNA from the appropriate expression vector without insert was used to give equivalent amounts of DNA per transfection). 1 μg of activated allele of H-Ras was co-transfected with increasing amounts of various alleles of PAK5. [0732]
  • Foci were scored after 3 weeks by fixing 10 min in 10% methanol, 10% acetic acid for 10 min, followed by staining with 0.4% (w/v) crystal violet in 10% methanol for 10 min, and washing with deionized water and drying at room temperature. [0733]
  • Transfections Stimulations, and Luciferase Assays (ZC1 Experiments) [0734]
  • Cells (10[0735] 7) were transiently transfected by electroporation using a Gene Pulser (Bio-Rad Labs) with the setting of 960_F and 250 V. 20-40 hours later, transfected cells (about 105) were stimulated with various stimuli. After a 6-hour stimulation, cells were lysed, and luciferase activities were measured using the MicroLumatPlus (EG&G Berthold). (J. Exp. Med. 183:611-620, 1996, hereby incorporated by reference herein in its entirety including any drawings, tables, or figures.)
  • Results [0736]
  • Protein expression and kinase activity of novel STE20-related protein kinases [0737]
    Endogenous
    Observed size Predicted In vitro Kinase Kinase
    Protein (kD) Size(kD) activity activity
    STLK2
    50 46 y y
    STLK4
    55 50 y ND
    ZC1
    160 140 y y
    ZC2 170 150 y y
    KHS2 ND 101 ND ND
    SULU1 119 105 y y
    SULU3
    140 115 ND Y
    PAK4 80 75 y y
    PAK5
    64 64 y y
  • ZC1: Regulation of Kinase Activity [0738]
  • ZC1 is constitutively active as a full-length kinase when expressed either in vitro (TNT rabbit reticulocyte system) or in NIH 3T3, 293T, or H1299 tissue culture cells. The endogenously expressed kinase is also active when immunoprecipitated from carcinoma cell lines. [0739]
  • ZC1 Signaling Pathways [0740]
  • Using human leukemic T cell line Jurkat as a model system, the impact of cotransfected wild-type ZC1 on the activation of two reporter genes, RE/AP-luciferase and NFκB luciferase, was examined. RE/AP is a composite in the IL-2 gene promoter containing both a NFκB-like site and an AP-1 site. [0741]
  • Optimal activation of both RE/AP-luciferase and NFκB-luciferase reporter genes in Jurkat T cells requires signals generated from stimulation of both T cell receptor and the costimulator receptor CD28. Cotransfection of wild-type ZC1 with either the RE/AP-luciferase or the NFκB-luciferase reporter results in the activation of RE/AP or NFκB when costimulated with the anti-T cell receptor monoclonal antibody or the pharmacological reagents PMA and ionomycin that bypass proximal T cell receptor. No activation was seen when costimulated with an anti-CD28 monoclonal antibody. [0742]
  • These results suggest that wild-type ZC1, when overexpressed, was replacing a CD28-specific signal to activate RE/AP or NFκB. These results imply that ZC1 is involved in the CD28 signaling pathway. Since NFκB is one of the major pathways also activated by the pro-inflammatory cytokine TNF-α signaling, it is also likely that ZC1 may be a component in the TNF-α signaling pathways. [0743]
  • PAK5: Design of Specific Peptide Substrates [0744]
  • To aid in the development of in vitro kinase assays for screening small molecule libraries to identify specific inhibitors, the search for specific peptide substrates for PAK5 was undertaken. [0745]
  • The rationale used to design such peptides is based on the hypothesis that upon binding activated small G protein, PAK5 undergoes a conformational change that results in derepression of its kinase activity followed by autophosphorylation on the activation loop resulting in a fully active kinase. The site of autophosphorylation for related family members has been identified by biochemical and/or genetic means (e.g. Wu, C, et al. J. Biol. Chem 270:15984-15992 and Szczepanowska, et al. Proc. Natl. Acad. Sci 94, 8503-8508, 1997). Specific peptide substrates for PAK5 were designed from the sequence of the activation loop of this kinase. [0746]
  • An activation loop PAK5 peptide phosphorylated on the Thr residue of the TPY motif served as a high-affinity substrate for PAK5. [0747]
  • PAK5 Activation Loop Peptides as Kinase Substrates [0748]
    SEQ
    Peptide # Kinase Sequence Aa ID Kinase substrate
    1 PAK5 (C)RRKSLVGTPYWMA 471-485 120 PAK5 yes
    PE
    2 PAK5 (C)RRKSLVG T PYWMA 471-485 120 PAK5 yes
    PE
    3 PAK5 (C)RRK S LVGTPYWMA 471-485 120 PAK5 no
    PE
    4 KHS1 KRKSFIGTPYWMAPE 171-185 122 PAK5 yes
    5 STLK2 KRNTFVGTPFWMA 175-189 123 PAK5 poor
    PE
    6 SULU1 PANSFVGTPYWMAPE 174-188 124 PAK5 poor
    7 ZC1 RRNTFIGTPYWMAPE 184-198 125 PAK5 poor
    8 ZC1 RRNTFIG T PYWMAPE 184-198 126 PAK5 poor
    9 STLK4 RNKVRKTFVGTPCWM 66-83 127 PAK5 poor
    APE
    10 PAK5 (C)RRKSLVG T PYWMA 471-485 120 PAK4 yes
    PE
  • [0749]
    Peptide # Kinase Notes
    1 PAK5 Equally well as MBB
    2 PAK5 High Km for PAK5 (1-10 μM)
    3 PAK5 S is the site of phosphorylation
    4 KHS1 Similar to peptide 1
    5 STLK2
    6 SULU1
    7 ZC1
    8 ZC1 Better than 7
    9 STLK4
    10 PAK5 Same Km as phosph. by PAK5
  • PAK5: Transformation [0750]
  • Transformation of low-passage NIH3T3 cells by ras in the presence or absence of various alleles of PAK5 showed that the dominant negative, kinase-dead allele of PAK5 was able to block ras transformation of NIH3T3 cells. Thus, PAK5 activity is required for ras transformation of NIH3T3 cells. Inhibition of PAK5 activity may have therapeutic value as an anti-proliferative agent for treating cancer. [0751]
  • PAK4 and PAK5: Interaction with Cdc42 [0752]
  • [0753] PAK 4 interacts with CDC42 small G-protein but not Rac, RhoA, or Ras as determined by co-transfection of recombinant genes and detection by kinase assays. PAK5 also interacts with Cdc42. Coding sequences of activated alleles of small G proteins (ras, Cdc42, Rac, Rho) tagged with a Myc epitope were transiently expressed in 293T cells, various alleles of 35S-labeled PAK5 tagged with HA epitope were expressed in vitro with the reticulocyte (TNT) system.
    • Example 6 Chromosomal Localization of Ste20-Related Protein Kinases
  • Materials And Methods [0754]
  • STE20 protein kinases STLK3, STLK4, ZC1, ZC2, ZC3, KHS2, SULU1, PAK4, and PAK5 were mapped using the [0755] GeneBridge 4 Radiation Hybrid Panel, RH02.05 (Research Genetics). The GeneBridge 4 Panel consists of 91 hybrid panel samples, in addition to one human positive control (HFL), and one hamster negative control (A23). The standard reaction conditions used to test and conduct PCR reactions using the GeneBridge 4 Panel are available from Research Genetics.
  • Oligonucleotide sequences (all 5′ to 3′) used for PCR mapping were: [0756]
    STLK3: CTCCCATTTCCTAGCAAAATCA, (SEQ ID NO:128)
    AGAGGCAGTATTGTCAGATGTA (SEQ ID NO:129)
    STLK4: CCACACATGCGTATCTCTGTTG, (SEQ ID NO:130)
    TTGCTAGAATTCACATCAGGTACA (SEQ ID NO:131)
    ZC1: ATCCCTGGATCACACTGCTTCT, (SEQ ID NO:132)
    CAAGGTGTTCTTTGCCTCTGTT (SEQ ID NO:133)
    ZC2: AGATGGACTGTACTGGGAGGG, (SEQ ID NO:134)
    AGAAGAGCACTTGGCACTTATC (SEQ ID NO:135)
    ZC3: CATCATGAACTGGTGACGGG, (SEQ ID NO:136)
    CCAGTGAAATCAAACCAGTAAAA (SEQ ID NO:137)
    SULU1: CAAAACCTGGCCGTCTCTTCTATT, (SEQ ID NO:138)
    ATTTGTGCTACTGGGATTCTGTG (SEQ ID NO:139)
    KHS2: GAATAGCGGTACCATGATAGAATA, (SEQ ID NO:140)
    TACCAAAAAGAGCCAAAAGTGTG (SEQ ID NO:141)
    PAK4: CTCAGTATTCTCTCCAAAGATTG, (SEQ ID NO:142)
    GATGTTCTCTCCATTCTGTAAAG (SEQ ID NO:143)
    PAK5: CATCACTGGAAGTCTGCAGTG, (SEQ ID NO:144)
    CAGGTGCAGTAGTCATTTGC (SEQ ID NO:145)
  • Positive reactions were assigned a score of “1”, negative reactions are assigned a score of “0”, and ambiguous reactions are assigned a score of “2”. Results were submitted to the Whitehead Institute (www@genome.wi.mit.edu) for position analysis. Chromosomal localizations for ZC4, SULU3, STLK2, STLK5 and STLK6 were available publicly (for example, from Unigene). The chromosomal locations of GEK2 and STLK7 have not been determined. [0757]
    STLK2_h Xq25-27.1 (Public)
    STLK3 2q31.3 (Sugen)
    STLK4_h 3p22.3-p22.2 (Sugen)
    STLK5_h 17q23.2-24.2 (Public)
    STLK6_h 2q32.2-q33.3 (Public)
    STLK7_h NA
    ZC1_h 2p11.2 (Sugen)
    ZC2_h 3q26.31-3q26.32 (Sugen)
    ZC3_h 17p13.2-13.3 (Sugen)
    ZC4_h Xq22 (Public)
    KHS2_h 2p22-2p22.2 (Sugen)
    SULU1_h 12q24.21 (Sugen)
    SULU3_h 17p11.2 (Public)
    GEK2_h NA
    PAK4_h 15q14 (Sugen)
    PAK5_h 19q13.2-q13.3 (Sugen)
  • Many of the [0758] STE 20 kinases were mapped to regions associated with various human cancers, as shown below.
  • The regions were also cross-checked with the Mendalian Iheritance in Man database, which tracks genetic information for many human diseases, including cancer. References for association of the mapped sites with chromosomal abnormalities found in human cancer can be found in: Knuutila, et al., Am J Pathol, 1998, 152:1107-1123, hereby incorporated herein be reference in its entirety including any figures, tables, or drawings. Association of these mapped regions with other diseases is documented in the Online Mendalian Inheritance in Man (OMIM). [0759]
  • STLK2_h, Xq25-27.1, (Public) [0760]
  • Osteosarcoma, Xq25-qter, 2 of 31. [0761]
  • Lymphoproliferative syndrome, X-linked (OMIM No. 308240) [0762]
  • human STLK3, 2q31.3, (Sugen) [0763]
  • Squamous cell carcinoma of Head and Neck, 3 of 30. [0764]
  • STLK4_h, 3p22.3-p22.2, (Sugen) [0765]
  • Mantle cell lymphoma 3p 14-[0766] p22 1 of 27
  • Squamous cell carcinoma of Head and Neck 3p22-[0767] p24 1 of 14
  • Cardiomyopathy, dilated (OMIM 601154) [0768]
  • STLK5_h, 17q23.2-24.2, (Public) [0769]
  • Cervical cancer, 17q, 1 of 30 [0770]
  • Gastroesophageal junction adenocarcinoma xenograft, 17q, 1 of 5 [0771]
  • Breast carcinoma, 17q12-qter, 1 of 16 [0772]
  • Bladder carcinoma, 17q22-q23, 1 of 14 [0773]
  • Breast carcinoma, 17q22-q25, 8 of 101 [0774]
  • Non-small cell lung cancer, 17q24-q25, 6 of 50 [0775]
  • Testis, 17q24-qter, 2 of 11 [0776]
  • Malignant peripheral nerve sheath tumors, 17q24-qter, 5 of 7 [0777]
  • Alzheimer disease, susceptibility to (OMIM 106180) [0778]
  • STLK6_h, 2q32.2-q33.3, (Public) [0779]
  • Non-small cell lung cancer, 2q31-q32, 1 of 50 [0780]
  • Squamous cell carcinoma of Head and Neck, 2q31-q33, 3 of 30 [0781]
  • Small cell lung cancer, 2q32-q35, 1 of 22 [0782]
  • ZC1_h, 2p11.2, (Sugen) [0783]
  • non-small cell lung cancer, 2pter-q13, 1 of 10 [0784]
  • non-small cell lung cancer, 2pter-q21, 1 of 10 [0785]
  • Pulmonary alveolar proteinosis, congenital (OMIM 178640). [0786]
  • ZC2_h, 3q26.31-3q26.32, (Sugen) [0787]
  • Non-small cell lung cancer, 3q26.1-q26.3, 26 of 103 [0788]
  • Cervical cancer, 3q26.1-q27, 4 of 30 [0789]
  • Small cell lung cancer, 3q26.3-qter, 3 of 35 [0790]
  • Squamous cell carcinoma of Head and Neck, 3q26.3-qter, 3 of 13 [0791]
  • Marginal zone B-cell lymphoma, 3q26-q27, 1 of 25 [0792]
  • Parosteal osteosarcoma, 3q26-q28, 1 of 1 [0793]
  • Gastrointestinal stromal tumor, 3q26-q29, 1 of 16 [0794]
  • Mantle cell lymphoma, 3q26-q29, 1 of 5 [0795]
  • ZC3_h 17pl3.2-13.3 (Sugen) [0796]
  • Malignant fibrous histiocytoma of soft tissue, 17p, 2 of 58 [0797]
  • Leiomyosarcoma, 17p, 7 of 29 [0798]
  • Non-small cell lung cancer, 17p, 1 of 50 [0799]
  • ZC4_h, Xq22, (Public) [0800]
  • Diffuse large cell lymphoma, Xq22-ter, 1 of 32 [0801]
  • Deafness, [0802] X-linked 1, progressive. (OMIM 304700).
  • KHS2_h, 2p22-2p22.2, (Sugen) [0803]
  • Synovial sarcoma, 2p21-q14, 1_of[0804] —67
  • Follicular lymphoma, 2p22-p24, 1_of[0805] —46
  • Colorectal cancer, hereditary, nonpolyposis, [0806] type 1, Ovarian cancer (MSH2, COCA1, FCC1). (OMIM 120435).
  • SULU1_h, 12q24.21 (Sugen) [0807]
  • Neuroglial tumors, 12q22-qter, [0808] 1_of —15
  • Gastroesophageal junction adenocarcinoma, 12q23-qter, 1 of 5. [0809]
  • Non-small cell lung cancer, 12q24.1-24.3, 2 of 50. [0810]
  • SULU3_h 17p 11.2 (Public) [0811]
  • Malignant fibrous histiocytoma of soft tissue, 17p, [0812] 2_of —58
  • Leiomyosarcoma, 17p, [0813] 7_of —29
  • non-small cell lung cancer, 17p, [0814] 1_of —50
  • Diffuse large cell lymphoma, 17p11.2, 1_of[0815] —32
  • Osteosarcoma, 17p11.2-p12, 4_of[0816] —31
  • PAK4_h: 15q14 (Sugen) [0817]
  • Schizophrenia, (OMIM 118511). [0818]
  • PAK5_h: 19q13.2-q13.3 (Sugen) [0819]
  • Follicular lymphoma, 19q13, 1 of 46* [0820]
  • Mantle cell lymphoma, 19q13, 1 of 5 [0821]
  • Hepatocellular carcinoma, 19q13.1, 2 of 50 [0822]
  • Small cell lung cancer, 19q13.1, 10 of 35 [0823]
  • Breast carcinoma, 19q13.1-qter, 1 of 33 [0824]
  • cervical cancer, 19q13.1-qter, 1 of 30 [0825]
  • Testis, 19q13.1-qter, 1 of 11 [0826]
  • Chondrosarcoma, 19q13.2, 1 of 29 [0827]
  • Malignant fibrous histiocytoma of soft tissue, 19q13.2-qter, 2 of 58 [0828]
  • Non-small cell lung cancer, 19qcen-q13.3, 6 of 104 [0829]
    • Example 7 Demonstration Of Gene Amplification By Southern Blotting
  • Materials and Methods [0830]
  • Nylon membranes were purchased from Boehringer Mannheim. Denaturing solution contains 0.4 M NaOH and 0.6 M NaCl. Neutralization solution contains 0.5 M Tris-HCL, pH 7.5 and 1.5 M NaCl. Hybridization solution contains 50% formamide, 6× SSPE, 2.5× Denhardt's solution, 0.2 mg/mL denatured salmon DNA, 0.1 mg/mL yeast tRNA, and 0.2% sodium dodecyl sulfate. Restriction enzymes were purchased from Boehringer Mannheim. Radiolabeled probes were prepared using the Prime-it 11 kit by Stratagene. The beta actin DNA fragment used for a probe template was purchased from Clontech. [0831]
  • Genomic DNA was isolated from 20 different tumor cell lines: MCF-7, MDA-MB-231, Calu-6, A549, HCT-15, HT-29, [0832] Colo 205, LS-180, DLD-1, HCT-116, PC3, CAPAN-2, MIA-PaCa-2, PANC-1, AsPc-1, BxPC-3, OVCAR-3, SKOV3, SW 626 and PA-1, and from two normal cell lines: human mammary epithelial cells and human umbilical vein endothelial cells.
  • A 10 μg aliquot of each genomic DNA sample was digested with EcoR I restriction enzyme and a separate 10 μg sample was digested with Hind III restriction enzyme. The restriction-digested DNA samples were loaded onto a 0.7% agarose gel and, following electrophoretic separation, the DNA was capillary-transferred to a nylon membrane by standard methods (Sambrook, J. et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory). [0833]
  • PAK5 Amplicon: [0834]
  • A 600 base pair fragment (EcoR I-Sac I) of the PAK5 gene was used as a template for a radiolabeled DNA probe which was hybridized to the blots at 42° C. for 48 hours in hybridization solution using standard methods (supra). The blots were exposed to a phosphorimager screen for 4 days, then scanned and analyzed using a [0835] Molecular Dynamics Storm 840 phosphorimager. The relative mass and gene copy number values of the PAK5 DNA fragments were calculated from the band density values obtained. The blots were re-hybridized with a radiolabeled probe copied from a fragment of human beta actin DNA and developed as above to confirm the sample mass loading equivalency.
  • Results [0836]
  • The PAK5 gene was determined to exhibit 3-fold amplification compared to the normal DNA copy number in PANC-1 (pancreatic epithelioid carcinoma) and OVCAR-3 (ovarian adenocarcinoma) human cell lines, and approximately 2 times the normal copy number in the BxPC-3 (primary pancreatic adenocarcinoma) human cell line. [0837]
  • Similar Southern analyses can be performed for other STE20 kinases. [0838]
    • Example 8 Detection Of Protein-Protein Interaction Through Phage Display
  • Materials And Methods [0839]
  • Phage display provides a method for isolating molecular interactions based on affinity for a desired bait. cDNA fragments cloned as fusions to phage coat proteins are displayed on the surface of the phage. Phage(s) interacting with a bait are enriched by affinity purification and the insert DNA from individual clones is analyzed. [0840]
  • T7 Phage Display Libraries [0841]
  • All libraries were constructed in the T7Select1-1b vector (Novagen) according to the manufacturer's directions. [0842]
  • Bait Presentation [0843]
  • Protein domains to be used as baits were generated as C-terminal fusions to GST and expressed in [0844] E. coli. Peptides were chemically synthesized and biotinylated at the N-terminus using a long chain spacer biotin reagent.
  • Selection [0845]
  • Aliquots of refreshed libraries (10[0846] 10-1012 pfu) supplemented with PanMix and a cocktail of E. coli inhibitors (Sigma P-8465) were incubated for 1-2 hrs at room temperature with the immobilized baits. Unbound phage was extensively washed (at least 4 times) with wash buffer.
  • After 3-4 rounds of selection, bound phage was eluted in 100 μL of 1% SDS and plated on agarose plates to obtain single plaques. [0847]
  • Identification of Insert DNAs [0848]
  • Individual plaques were picked into 25 μL of 10 mM EDTA and the phage was disrupted by heating at 70° C. for 10 min. 2 μL of the disrupted phage were added to 50 μL PCR reaction mix. The insert DNA was amplified by 35 rounds of thermal cycling (94° C., 50sec; 50° C., 1 min; 72° C., 1 min). [0849]
  • Composition of Buffer [0850]
  • 10× PanMix [0851]
  • 5% Triton X100 [0852]
  • 10% non-fat dry milk (Carnation) [0853]
  • 10 mM EGTA [0854]
  • 250 mM NaF [0855]
  • 250 μg/mL Heparin (sigma) [0856]
  • 250 μg/mL sheared, boiled salmon sperm DNA (sigma) [0857]
  • 0.05% Na azide [0858]
  • Prepared in PBS [0859]
  • Wash Buffer [0860]
  • PBS supplemented with: [0861]
  • 0.5% NP-40 [0862]
  • 25 μg/mL heparin [0863]
  • PCR Reaction Mix [0864]
  • 1.0 [0865] mL 10× PCR buffer (Perkin-Elmer, with 15 mM Mg)
  • 0.2 mL each dNTPs (10 mM stock) [0866]
  • 0.1 mLT7UP primer (15 μmol/μL) GGAGCTGTCGTATTCCAGTC [0867]
  • 0.1 mLT7DN primer (15 μmol/μL) AACCCCTCAAGACCCGTTTAG [0868]
  • 0.2 mL25 mM MgCl[0869] 2 or MgSO4 to compensate for EDTA
  • Q.S. to 10 mL with distilled water [0870]
  • Add 1 unit of Taq polymerase per 50 μL reaction [0871]
  • PCR Reaction Mix [0872]
  • 1.0 [0873] mL 10× PCR buffer (Perkin-Elmer, with 15 mM Mg)
  • 0.2 mL each dNTPs (10 mM stock) [0874]
  • 0.1 mLT7UP primer (15 pmol/μL) GGAGCTGTCGTATTCCAGTC(SEQ ID NO:146) [0875]
  • 0.1 mLT7DN primer (15 pmol/μL) AACCCCTCAAGACCCGTTTAG(SEQ ID NO:147) [0876]
  • 0.2 mL25 mM MgCl[0877] 2 or MgSO4 to compensate for EDTA
  • Q.S. to 10 mL with distilled water [0878]
  • Add 1 unit of Taq polymerase per 50 μL reaction [0879]
  • LIBRARY: T7 Select1-H441 [0880]
  • Results [0881]
  • Phage Display Baits and Interactors [0882]
    Sequence
    Patent CDNA Range
    Bait Domain Aa SEQ ID library Interactor & SEQ ID
    SULU1 Coiled-coil2 752-898 22 H441 GEK2 cc dom (1) 677-820
    SEQ #26
    SULU3 Coiled-coil2 755-898 23 H441 SLK isoform M83780
  • The phage display data suggest potential interactions of SULU3 with SLK and SULU1 with GEK2 through their coiled-coil domains. Therefore two members of the SULU subfamily of STE20 kinases interact with two members of a separate STE20 family, the prototype being SLK. [0883]
  • These results suggest a specificity in the interaction, and imply that these STE20 kinases may interact with each other through homo- and hetero-dimerization. Alternatively SULU-related kinases could act immediately up- or down-stream of the SLK-related kinases in a signaling cascade. [0884]
  • One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The molecular complexes and the methods, procedures, treatments, molecules, specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. [0885]
  • It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. [0886]
  • All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. [0887]
  • The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. [0888]
  • In particular, although some formulations described herein have been identified by the excipients added to the formulations, the invention is meant to also cover the final formulation formed by the combination of these excipients. Specifically, the invention includes formulations in which one to all of the added excipients undergo a reaction during formulation and are no longer present in the final formulation, or are present in modified forms. [0889]
  • In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. [0890]
  • Other embodiments are within the following claims. [0891]
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 155
    <210> SEQ ID NO 1
    <211> LENGTH: 3268
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 1
    taacagccca cctcctagcc ccgggctacg cgccgccagc ccagtaaccc cacttttgtg 60
    tgtcctccca ggccccgatc gaaaagcctg ggagggccgc cgaactaccc ccggagggag 120
    gagccagtcc gaacccaagg cgccaccgcc gcagaagcgg agcgaggcag cattcgcctc 180
    catggcccac tcgccggtgg ctgtccaagt gcctgggatg cagaataaca tagctgatcc 240
    agaagaactg ttcacaaaat tagagcgcat tgggaaaggc tcatttgggg aagttttcaa 300
    aggaattgat aaccgtaccc agcaagtcgt tgctattaaa atcatagacc ttgaggaagc 360
    cgaagatgaa atagaagaca ttcagcaaga aataactgtc ttgagtcaat gtgacagctc 420
    atatgtaaca aaatactatg ggtcatattt aaaggggtct aaattatgga taataatgga 480
    atacctgggc ggtggttcag cactggatct tcttcgagct ggtccatttg atgagttcca 540
    gattgctacc atgctaaagg aaattttaaa aggtctggac tatctgcatt cagaaaagaa 600
    aattcaccga gacataaaag ctgccaatgt cttgctctca gaacaaggag atgttaaact 660
    tgctgatttt ggagttgctg gtcagctgac agatacacag attaaaagaa atacctttgt 720
    gggaactcca ttttggatgg ctcctgaagt tattcaacag tcagcttatg actcaaaagc 780
    tgacatttgg tcattgggaa ttactgctat tgaactagcc aagggagagc cacctaactc 840
    cgatatgcat ccaatgagag ttctgtttct tattcccaaa aacaatcctc caactcttgt 900
    tggagacttt actaagtctt ttaaggagtt tattgatgct tgcctgaaca aagatccatc 960
    atttcgtcct acagcaaaag aacttctgaa acacaaattc attgtaaaaa attcaaagaa 1020
    gacttcttat ctgactgaac tgatagatcg ttttaagaga tggaaggcag aaggacacag 1080
    tgatgatgaa tctgattccg agggctctga ttcggaatct accagcaggg aaaacaatac 1140
    tcatcctgaa tggagcttta ccaccgtacg aaagaagcct gatccaaaga aagtacagaa 1200
    tggggcagag caagatcttg tgcaaaccct gagttgtttg tctatgataa tcacacctgc 1260
    atttgctgaa cttaaacagc aggacgagaa taacgctagc aggaatcagg cgattgaaga 1320
    actcgagaaa agtattgctg tggctgaagc cgcctgtccc ggcatcacag ataaaatggt 1380
    gaagaaacta attgaaaaat ttcaaaagtg ttcagcagac gaatccccct aagaaactta 1440
    ttattggctt ctgtttcata tggacccaga gagccccacc aaacctacgt caagattaac 1500
    aatgcttaac ccatgagctc catgtgcctt ttggatcttt gcaacactga agatttggaa 1560
    gaagctatta aactattttg tgatggcgtt tatcatttta tattttgaaa ggattatttt 1620
    gtaaggaata acttttaata ctatagtttc acctgtattc tagtaaatgt tgagacaccg 1680
    ttttgctttt aagtatccct atttcttaag ttacgaggat gaataccttt cacattttga 1740
    tctttagttg actctacagt catgaaacat acaggtcttt caaagtcatt ctcaatattc 1800
    agcttttgta aattatcaag cttcaaaaag ctttttttta aaaaaaaaaa catgcatatt 1860
    ctaaaaatga ctattggtgg ggaggtgtaa ataagtcata ccttcttaaa acagaaaatt 1920
    taagtaaagt cttttaaatg aaacctgtaa aagtattgac tcttctacca agttggtatg 1980
    atattccagg cagctcaatg attatcacat ttgagaccct gtgtttgaag catttacagg 2040
    caatgtacag caacagaggt acctcttggt gtatagtatt tacattctct tttaggtaga 2100
    agaggcaatt ttacccttat ttcacatggt tagaaattta aagcaagatc atttacccaa 2160
    ggataggtgt ttggtaatgt tgaaggagtt agtctggctt catgttttac atcttcaact 2220
    aaaatcccat actatctgct tggatttgga gagccaaaaa ataaagctga ttgtcatgtg 2280
    attaaatatc tgatcaacag gtatgaatat aacttaaatc agcatatttt tgccatggta 2340
    ataaattgtc ctataaacta tttatatatt tttgttcttc ataattatca ctaataagca 2400
    tcagtttgtt gtttttaaaa ggatatttaa gtgagcattt tctagttcat atgaaaataa 2460
    ccatagtaca ggatgatttc tgtccacaca aaggttaaat tagattgcac agttaatttt 2520
    cacttatatt tatggtacta ttatgtgggt gatgcctttt tcttttaagc ccagtacata 2580
    tattatgcct gcctaagttc tgaactgggg ctgtatttca gtagttgtag aattattgat 2640
    atttagtttt gatagctaat gtttaattgt ttggatctgc acagtttggt ttttgcacaa 2700
    aagtcattta aaaaaatctg agtaattgtc aaatattaaa agaaagatat tcttcctgta 2760
    aggaatacag tttttagtca aagtggccat tacatcctct ttttaattta cataatacag 2820
    atacttgaga aagttgttgt ggtgttgtat gccaagaaaa ttctttttat tggtgcctat 2880
    attgtaacaa ttatttttaa tgcattgtat tttgaagtaa cggttcagtt aaatttttca 2940
    cctgctgtgt aactgaaaca caattacagt ttataatcat ctgtagaagt ctggagataa 3000
    ttttgcaact catgttatgg gttaaatgaa tatttttgta aaagtaaaag caacaaattt 3060
    ataaattgat tatttgaaac tttacaacac aattgcatcc caaatacaaa ttgtattgct 3120
    tattcattat agctattcgt cctgtaatct gtttctaggt gaagcatact ccagtgtttt 3180
    aggggttttg aaaataaata tttaaatttc acagtcaaaa aaaaaaaaaa aaaaaaaaaa 3240
    aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3268
    <210> SEQ ID NO 2
    <211> LENGTH: 3030
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 2
    gacagcagcg ccggccccgg cagctcccgc ggccccggcc ccggccccgg ccccggcccc 60
    ggcggcacag gctgtcggct ggcccatctg cagggacgcg tacgagctgc aggaggttat 120
    cggcagtgga gctactgctg tggttcaggc agccctatgc aaacccaggc aagaacgtgt 180
    agcaataaaa cggatcaact tggaaaaatg ccagaccagt atggatgaac tattaaaaga 240
    aattcaagcc atgagtcagt gcagccatcc caacgtagtg acctattaca cctcttttgt 300
    ggtcaaagat gaactttggc tggtcatgaa attactaagt ggaggttcaa tgttggatat 360
    cataaaatac attgtcaacc gaggagaaca caagaatgga gttctggaag aggcaataat 420
    agcaacaatt cttaaagagg ttttggaagg cttagactat ctacacagaa acggtcagat 480
    tcacagggat ttgaaagctg gtaatattct tctgggtgag gatggttcag tacaaatagc 540
    agattttggg gtaagtgcgt tcctagcaac agggggtgat gttacccgaa ataaagtaag 600
    aaaaacattc gttggcaccc catgttggat ggctcctgaa gtcatggaac aggtgagagg 660
    ctatgacttc aaggctgaca tgtggagttt tggaataact gccattgaat tagcaacagg 720
    agcagcgcct tatcacaaat atcctcccat gaaagtgtta atgttgactt tgcaaaatga 780
    tccacccact ttggaaacag gggtagagga taaagaaatg atgaaaaagt acggcaagtc 840
    ctttagaaaa ttactttcac tgtgtcttca gaaagatcct tccaaaaggc ccacagcagc 900
    agaactttta aaatgcaaat tcttccagaa agccaagaac agagagtacc tgattgagaa 960
    gctgcttaca agaacaccag acatagccca aagagccaaa aaggtaagaa gagttcctgg 1020
    gtcaagtggt caccttcata aaaccgaaga cggggactgg gagtggagtg acgacgagat 1080
    ggatgagaag agcgaagaag ggaaagcagc tttttctcag gaaaagtcac gaagagtaaa 1140
    agaagaaaat ccagagattg cagtgagtgc cagcaccatc cccgaacaaa tacagtccct 1200
    ctctgtgcac gactctcagg gcccacccaa tgctaatgaa gactacagag aagcttcttc 1260
    ttgtgccgtg aacctcgttt tgagattaag aaactccaga aaggaactta atgacatacg 1320
    atttgagttt actccaggaa gagatacagc agatggtgta tctcaggagc tcttctctgc 1380
    tggcttggtg gatggtcacg atgtagttat agtggctgct aatttacaga agattgtaga 1440
    tgatcccaaa gctttaaaaa cattgacatt taagttggct tctggctgtg atgggtcgga 1500
    gattcctgat gaagtgaagc tgattgggtt tgctcagttg agtgtcagct gatgtatgtc 1560
    ccttgatgtc accctgatct gtcatgcccc accgccaccc ctactccctt caaccctccc 1620
    tctttctgcc catttcctcc caccccctca ctcccatttc ctagcaaaat cagaagattg 1680
    tgaagaggcc ggcttcaaca aaatgggata aaaaaataat tttttaaaac ttacaacact 1740
    ccgagttctg ctttattctc tagcaatcca cagtacaaga acaagcaaat gccacagctg 1800
    cacgactgtt gctcattttt ccaaaagcta tttaatattc ttagcaatca atttggatat 1860
    cccttaagtg aaaagaatct gaaatacact caggtggtct tatttattgg caacaaaagg 1920
    aattttctat ccagaagcct atttctcctt tcattgttgt tatttctgtt ataatacttt 1980
    aattgtacat ctgacaatac tgcctctttt atgttgtatt tagaaattaa tatacttata 2040
    aaattaagat ttattagcca aacttgaatt ctagttttaa aactgactgt gaattttatt 2100
    tttcatatat ttatgcatta cacaccttag ctataagaaa aaaagggttt tgattatatg 2160
    cttcttgcag ttaatctcgt tatttaaaca aaaagttttg ggtctatctt tggagtattt 2220
    gtaacttcta aattttgaaa tgactgaatt aggaatttgg atgcttattc ttttagtctg 2280
    tttgcctaaa aaccaattta caatctgact gtctcttggg agagggaggt gccttgcaaa 2340
    ctttcacatt aagaatgtgc ctgaggctgc tttactctgg aatagtctca gatctaaaat 2400
    ttcctctata taaggtggca tatgttaagt tttgcttcat tggaccgttt agaatgctat 2460
    gtaaaatgtt gccattctgt tagattgcta actatatacc catctctgat ttggctctcc 2520
    ttaagtgata ggatttgtta ttctaaaggt gataaacttg aaaatatcag aatctgagtt 2580
    ttacttgaaa ttttgcagaa tacccaggtg gagtgaaaat tggaagggtt ttgtgcaatg 2640
    actaaaaggt aaaacgctgt taaggttcaa gaatcaatac tttcaaccca agtagccctc 2700
    tgcttgactg tatattatgg aactagtaaa ccttaggatt ttgaaaattg gagtctaatc 2760
    tttcaaggag gtgggctccc aggatggtac cattgctctt tcctagctaa ccctagatat 2820
    ggcagctctt taatgtactt caaaaagcaa atatatatta ctaaggaaaa aaagttattt 2880
    ataattgcct tgtcataatt gttaaggtgt tctagagcca tttgcataca atttaatgta 2940
    atttcattcc attctattgt ttacacaacg attactcgaa gatgactgca aaggtaaaag 3000
    gaaaataaaa gtgtattgca caatgaaaaa 3030
    <210> SEQ ID NO 3
    <211> LENGTH: 3857
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (22)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (81)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (122)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <400> SEQUENCE: 3
    caaaagtgga gtcctagatg antctaccat tgctacgata ctccgagaag tactggaagg 60
    gctggaatat ctgcataaaa ntggacagat ccacagagat gtgaaagctg gaaacattct 120
    tnttggagaa gatggctcag tacagatttc agactttggg gttagtgctt ttttagcaac 180
    tggtggtgat attacccgaa ataaagtgag aaagaccttt gttggcaccc cttgttggat 240
    ggcacctgaa gttatggaac aggtccgtgg ttatgatttc aaagctgata tttggagttt 300
    tggaattaca gcaattgaat tggctacagg ggcggctcct tatcataaat atccaccaat 360
    gaaggtttta atgctgacac tgcagaacga tcctccttct ttggaaactg gtgttcaaga 420
    taaagaaatg ctgaaaaaat atggaaaatc atttagaaaa atgatttcat tgtgccttca 480
    aaaagatcca gaaaaaagac caacagcagc agaactatta aggcacaaat ttttccagaa 540
    agcaaagaat aaagaatttc ttcaagaaaa aacattgcag agagcaccaa ccatttctga 600
    aagagcaaaa aaggttcgga gagtaccagg ttccagtggg cgtcttcata agacagagga 660
    tggaggctgg gagtggagtg atgatgaatt tgatgaagaa agtgaggaag ggaaagcagc 720
    aatttcacaa ctcaggtctc cccgagtgaa agaatcaata tcaaattctg agctctttcc 780
    aacaactgat cctgtgggta ctttgctcca agttccagaa cagatctctg ctcatctacc 840
    tcagccagct gggcagattg ctacacagcc aactcaagtc tctctcccac ccaccgcaga 900
    gccagcaaaa acagctcagg ctttgtcttc aggatcaggt tcacaagaaa ccaagatccc 960
    aatcagtcta gtactaagat taaggaattc caaaaaagaa ctaaatgata ttcgatttga 1020
    atttactcct gggagagata cagcagaggg tgtctctcag gaactcattt ctgctggcct 1080
    ggtcgacgga agggatttag taatagtggc agctaatttg cagaaaattg tggaagaacc 1140
    tcagtcaaat cgatctgtca ctttcaaact ggcatctggt gtcgaaggct cagatattcc 1200
    tgatgatggt aaactgatag gatttgccca gctcagcatc agctaaacca caaccctgga 1260
    agaggcggcc taaggagatt ccacacatgc gtatctctgt tgcttctatt ggcctaaacc 1320
    cactactgcc aaagaaccca gcaacaaacc tcccggctag gagctttaga agtctttatg 1380
    ttcttcctgc catcattcct ccttttccca cagggaaaga aaagttggat cactagtggc 1440
    cagcatcccc agagttccgt tagtaaactt acttcatatg tcccctgtct tcctccatct 1500
    gagaagtggc ccatgtgctt caaggcccag gagggagatc tgtcagctca ttcttgcctt 1560
    actccaatga tggcccaggt ggaaaagtag cagctgtatc gggcttcctc atcctgcctg 1620
    ttcccccaca cctgccagga tatggacatc ttgggatatc tctttaccac tgaagtagaa 1680
    ttgattgttc agctggagcc cagagaattt aatttaatgt tttttctttg tacctgatgt 1740
    gaattctagc aacctttgtt aggaaaaagc acagcctcag atggaggcag cctaaactgt 1800
    gttcttgttt tgttcatggt gtttctaagc gttttgctga agctgctctc aggcaccccc 1860
    ttcttcattg ctctctccag aaagggttgc tagccttaac ttcagctggt gcaaaacatc 1920
    tgactgtagc cgaacttcag ccatcagatc cttcaaagtg gaactttgga ttgtttttac 1980
    agacaacatc gagtaatggc ttgtaaatgt gaattttgcc agaggtggtt tttgaacagg 2040
    aaaatcataa ttcatatcat tggagaagta tttattttca aatatcaaat tgaagaaaaa 2100
    ctcaatcctc ccatgaaaat cagttcgcct ggcctccaag tcgtgaggaa atgggtatgc 2160
    aaggctgaga tttctacagc aataaaggag acacacactg ggccagagag gcctgccttc 2220
    tgcctgctct cctgcactga ccctttggag ggggtctctg tgtgctgaag ctaactcaag 2280
    atggaaagtg aaaccacatg tgccgtgacc tttaggtttt atgagtagac agtgttcatt 2340
    tgattttcta cagaaataat ataaattatt ctttaggttt aaaaaagagc actcataatg 2400
    caatatgtga ataatcagtg aggttgattt ttcttttttc ctaccgtttc atagtctttg 2460
    tctaactgct agtaacccta ccgagtttta tatatgagtg ggatactcaa tctggcctta 2520
    aaaagataca caaagatggg ctgtgggtcc ctggaaaggg ggagagttgc cctttacaga 2580
    atcactcgag ccctttccag cactgttggt ctgatgaaca aggttgtttt accttatttt 2640
    ctcttggaac atatctgaaa accttcccca caaataactt gtcacacctt ttgtttcatt 2700
    ctgagtcttt agttttagtc atgggctttc ttcacctgct ctaggtgcaa aggcatgttg 2760
    ggaaagagat ggatgttggg gaggaagaga ggagatggat ttcagttggg agttaggagg 2820
    agagtaggtg agatgatcag acaccggagt tcaacgtccc agcagtcttg gtaaaaggag 2880
    ggagcctgct gagccaggag ggagaaaaga agattgacca gcttgctaga aaaatactta 2940
    gcttttcttt ttcttttttt gtggaggggg gacggagagg aacaaggatg gggaggtagg 3000
    aatgaggtat agaaaagaga tagcatcttc tttggcacaa gactagtggc ttaccgctta 3060
    ccttagagtt ttgttttttt tttttcaaac ccatcaaaat ctacttattt atgaatccaa 3120
    ggggtggcag catcactctg ttctagcatt ctttgtggag atggtctggt gcctagctgg 3180
    gagtgagcag cagcccatcc cctgttcact ttctctagcc catcattacc tgtgaactgc 3240
    agtggggcag tcatggcaaa tagaattggg ctggggtttc tccttctttt cagttcattg 3300
    tttgccctgc taggaattag aagacagaca ccatgtccca ggacagtgtt acttcttctg 3360
    catgatgtgt ggtagactcc ctttgctggc ttgtgcagtg atactgagaa aatacatgaa 3420
    cagaaactgc ccaggtggaa cagcacgtaa cctagtgagt gactgtactc ctttctagga 3480
    atgctgattc agagtgcacc tctttgacta ggtcccagga tccccttgtc cctggagtag 3540
    ggactaacta tagcacaaag taatatgtgc caatgctatt tgtgaaatgt ttggtctttc 3600
    taaacgacta aaggatttgt tgggtttttg cttaagtttt gaaccaaatc ctagagccag 3660
    ctgataatat ttaataatct ggaggagaga ataatgatgt accaataagt ggagattcct 3720
    ccttatgatg tatgctaggt tatggaagat gtaaaatatt caactttttc ctcctttttt 3780
    tggactttgt attttactgc atgttttctt catttttaat caataaagag taaattgtca 3840
    aaaaaaaaaa aaaaaaa 3857
    <210> SEQ ID NO 4
    <211> LENGTH: 1584
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 4
    ctcatctgta cacacttcat ggatggcatg aatgagctgg cgattgctta catcctgcag 60
    ggggtgctga aggccctcga ctacatccac cacatgggat atgtacacag gagtgtcaaa 120
    gccagccaca tcctgatctc tgtggatggg aaggtctacc tgtctggttt gcgcagcaac 180
    ctcagcatga taagccatgg gcagcggcag cgagtggtcc acgattttcc caagtacagt 240
    gtcaaggttc tgccgtggct cagccccgag gtcctccagc agaatctcca gggttatgat 300
    gccaagtctg acatctacag tgtgggaatc acagcctgtg aactggccaa cggccatgtc 360
    ccctttaagg atatgcctgc cacccagatg ctgctagaga aactgaacgg cacagtgccc 420
    tgcctgttgg ataccagcac catccccgct gaggagctga ccatgagccc ttcgcgctca 480
    gtggccaact ctggcctgag tgacagcctg accaccagca ccccccggcc ctccaacggt 540
    gactcgccct cccaccccta ccaccgaacc ttctcccccc acttccacca ctttgtggag 600
    cagtgccttc agcgcaaccc ggatgccagg cccagtgcca gcaccctcct gaaccactct 660
    ttcttcaagc agatcaagcg acgtgcctca gaggctttgc ccgaattgct tcgtcctgtc 720
    acccccatca ccaattttga gggcagccag tctcaggacc acagtggaat ctttggcctg 780
    gtaacaaacc tggaagagct ggaggtggac gattgggagt tctgagcctc tgcaaactgt 840
    gcgcattctc cagccaggga tgcagaggcc acccagaggc ccttcctgag ggccggccac 900
    attcccgccc tcctgggcag attgggtaga aaggacattc ttccaggaaa gttgactgct 960
    gactgattgg gaaagaaaat cctggagaga tacttcactg ctccaaggct tttgagacac 1020
    aagggaatct caacaaccag ggatcaggag ggtccaaagc cgacattccc agtcctgtga 1080
    gctcaggtga cctcctccgc agaagagaga tgctgctctg gccctgggag ctgaattcca 1140
    agcccagggt ttggctcctt aaacccgagg accgccacct cttcccagtg cttgcgacca 1200
    gcctcattct atttaacttt gctctcagat gcctcagatg ctataggtca gtgaaagggc 1260
    aagtagtaag ctgcctgcct cccttccctc agacctctcc ctcataattc cagagaaggg 1320
    catttctgtc tttttaagca cagactaagg ctggaacagt ccatccttat ccctcttctg 1380
    gcttgggccc tgacacctaa gtctttccca cggtttatgt gtgtgcctca ttcctttccc 1440
    accaagaatc catcttagcg cctcctgcca gctgccctgg tgctttctcc aagggccatc 1500
    agtgtcttgc ctagcttgag ggcttaagtc cttatgctgt gttagtttcg ttgtcagaac 1560
    aaattaaaat tttcagagac gctg 1584
    <210> SEQ ID NO 5
    <211> LENGTH: 416
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 5
    Met Ala His Ser Pro Val Ala Val Gln Val Pro Gly Met Gln Asn Asn
    1 5 10 15
    Ile Ala Asp Pro Glu Glu Leu Phe Thr Lys Leu Glu Arg Ile Gly Lys
    20 25 30
    Gly Ser Phe Gly Glu Val Phe Lys Gly Ile Asp Asn Arg Thr Gln Gln
    35 40 45
    Val Val Ala Ile Lys Ile Ile Asp Leu Glu Glu Ala Glu Asp Glu Ile
    50 55 60
    Glu Asp Ile Gln Gln Glu Ile Thr Val Leu Ser Gln Cys Asp Ser Ser
    65 70 75 80
    Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Leu Lys Gly Ser Lys Leu Trp
    85 90 95
    Ile Ile Met Glu Tyr Leu Gly Gly Gly Ser Ala Leu Asp Leu Leu Arg
    100 105 110
    Ala Gly Pro Phe Asp Glu Phe Gln Ile Ala Thr Met Leu Lys Glu Ile
    115 120 125
    Leu Lys Gly Leu Asp Tyr Leu His Ser Glu Lys Lys Ile His Arg Asp
    130 135 140
    Ile Lys Ala Ala Asn Val Leu Leu Ser Glu Gln Gly Asp Val Lys Leu
    145 150 155 160
    Ala Asp Phe Gly Val Ala Gly Gln Leu Thr Asp Thr Gln Ile Lys Arg
    165 170 175
    Asn Thr Phe Val Gly Thr Pro Phe Trp Met Ala Pro Glu Val Ile Gln
    180 185 190
    Gln Ser Ala Tyr Asp Ser Lys Ala Asp Ile Trp Ser Leu Gly Ile Thr
    195 200 205
    Ala Ile Glu Leu Ala Lys Gly Glu Pro Pro Asn Ser Asp Met His Pro
    210 215 220
    Met Arg Val Leu Phe Leu Ile Pro Lys Asn Asn Pro Pro Thr Leu Val
    225 230 235 240
    Gly Asp Phe Thr Lys Ser Phe Lys Glu Phe Ile Asp Ala Cys Leu Asn
    245 250 255
    Lys Asp Pro Ser Phe Arg Pro Thr Ala Lys Glu Leu Leu Lys His Lys
    260 265 270
    Phe Ile Val Lys Asn Ser Lys Lys Thr Ser Tyr Leu Thr Glu Leu Ile
    275 280 285
    Asp Arg Phe Lys Arg Trp Lys Ala Glu Gly His Ser Asp Asp Glu Ser
    290 295 300
    Asp Ser Glu Gly Ser Asp Ser Glu Ser Thr Ser Arg Glu Asn Asn Thr
    305 310 315 320
    His Pro Glu Trp Ser Phe Thr Thr Val Arg Lys Lys Pro Asp Pro Lys
    325 330 335
    Lys Val Gln Asn Gly Ala Glu Gln Asp Leu Val Gln Thr Leu Ser Cys
    340 345 350
    Leu Ser Met Ile Ile Thr Pro Ala Phe Ala Glu Leu Lys Gln Gln Asp
    355 360 365
    Glu Asn Asn Ala Ser Arg Asn Gln Ala Ile Glu Glu Leu Glu Lys Ser
    370 375 380
    Ile Ala Val Ala Glu Ala Ala Cys Pro Gly Ile Thr Asp Lys Met Val
    385 390 395 400
    Lys Lys Leu Ile Glu Lys Phe Gln Lys Cys Ser Ala Asp Glu Ser Pro
    405 410 415
    <210> SEQ ID NO 6
    <211> LENGTH: 516
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 6
    Thr Ala Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Pro
    1 5 10 15
    Ala Pro Ala Pro Ala Ala Gln Ala Val Gly Trp Pro Ile Cys Arg Asp
    20 25 30
    Ala Tyr Glu Leu Gln Glu Val Ile Gly Ser Gly Ala Thr Ala Val Val
    35 40 45
    Gln Ala Ala Leu Cys Lys Pro Arg Gln Glu Arg Val Ala Ile Lys Arg
    50 55 60
    Ile Asn Leu Glu Lys Cys Gln Thr Ser Met Asp Glu Leu Leu Lys Glu
    65 70 75 80
    Ile Gln Ala Met Ser Gln Cys Ser His Pro Asn Val Val Thr Tyr Tyr
    85 90 95
    Thr Ser Phe Val Val Lys Asp Glu Leu Trp Leu Val Met Lys Leu Leu
    100 105 110
    Ser Gly Gly Ser Met Leu Asp Ile Ile Lys Tyr Ile Val Asn Arg Gly
    115 120 125
    Glu His Lys Asn Gly Val Leu Glu Glu Ala Ile Ile Ala Thr Ile Leu
    130 135 140
    Lys Glu Val Leu Glu Gly Leu Asp Tyr Leu His Arg Asn Gly Gln Ile
    145 150 155 160
    His Arg Asp Leu Lys Ala Gly Asn Ile Leu Leu Gly Glu Asp Gly Ser
    165 170 175
    Val Gln Ile Ala Asp Phe Gly Val Ser Ala Phe Leu Ala Thr Gly Gly
    180 185 190
    Asp Val Thr Arg Asn Lys Val Arg Lys Thr Phe Val Gly Thr Pro Cys
    195 200 205
    Trp Met Ala Pro Glu Val Met Glu Gln Val Arg Gly Tyr Asp Phe Lys
    210 215 220
    Ala Asp Met Trp Ser Phe Gly Ile Thr Ala Ile Glu Leu Ala Thr Gly
    225 230 235 240
    Ala Ala Pro Tyr His Lys Tyr Pro Pro Met Lys Val Leu Met Leu Thr
    245 250 255
    Leu Gln Asn Asp Pro Pro Thr Leu Glu Thr Gly Val Glu Asp Lys Glu
    260 265 270
    Met Met Lys Lys Tyr Gly Lys Ser Phe Arg Lys Leu Leu Ser Leu Cys
    275 280 285
    Leu Gln Lys Asp Pro Ser Lys Arg Pro Thr Ala Ala Glu Leu Leu Lys
    290 295 300
    Cys Lys Phe Phe Gln Lys Ala Lys Asn Arg Glu Tyr Leu Ile Glu Lys
    305 310 315 320
    Leu Leu Thr Arg Thr Pro Asp Ile Ala Gln Arg Ala Lys Lys Val Arg
    325 330 335
    Arg Val Pro Gly Ser Ser Gly His Leu His Lys Thr Glu Asp Gly Asp
    340 345 350
    Trp Glu Trp Ser Asp Asp Glu Met Asp Glu Lys Ser Glu Glu Gly Lys
    355 360 365
    Ala Ala Phe Ser Gln Glu Lys Ser Arg Arg Val Lys Glu Glu Asn Pro
    370 375 380
    Glu Ile Ala Val Ser Ala Ser Thr Ile Pro Glu Gln Ile Gln Ser Leu
    385 390 395 400
    Ser Val His Asp Ser Gln Gly Pro Pro Asn Ala Asn Glu Asp Tyr Arg
    405 410 415
    Glu Ala Ser Ser Cys Ala Val Asn Leu Val Leu Arg Leu Arg Asn Ser
    420 425 430
    Arg Lys Glu Leu Asn Asp Ile Arg Phe Glu Phe Thr Pro Gly Arg Asp
    435 440 445
    Thr Ala Asp Gly Val Ser Gln Glu Leu Phe Ser Ala Gly Leu Val Asp
    450 455 460
    Gly His Asp Val Val Ile Val Ala Ala Asn Leu Gln Lys Ile Val Asp
    465 470 475 480
    Asp Pro Lys Ala Leu Lys Thr Leu Thr Phe Lys Leu Ala Ser Gly Cys
    485 490 495
    Asp Gly Ser Glu Ile Pro Asp Glu Val Lys Leu Ile Gly Phe Ala Gln
    500 505 510
    Leu Ser Val Ser
    515
    <210> SEQ ID NO 7
    <211> LENGTH: 414
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (7)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (27)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (41)
    <223> OTHER INFORMATION: Any amino acid
    <400> SEQUENCE: 7
    Lys Ser Gly Val Leu Asp Xaa Ser Thr Ile Ala Thr Ile Leu Arg Glu
    1 5 10 15
    Val Leu Glu Gly Leu Glu Tyr Leu His Lys Xaa Gly Gln Ile His Arg
    20 25 30
    Asp Val Lys Ala Gly Asn Ile Leu Xaa Gly Glu Asp Gly Ser Val Gln
    35 40 45
    Ile Ala Asp Phe Gly Val Ser Ala Phe Leu Ala Thr Gly Gly Asp Ile
    50 55 60
    Thr Arg Asn Lys Val Arg Lys Thr Phe Val Gly Thr Pro Cys Trp Met
    65 70 75 80
    Ala Pro Glu Val Met Glu Gln Val Arg Gly Tyr Asp Phe Lys Ala Asp
    85 90 95
    Ile Trp Ser Phe Gly Ile Thr Ala Ile Glu Leu Ala Thr Gly Ala Ala
    100 105 110
    Pro Tyr His Lys Tyr Pro Pro Met Lys Val Leu Met Leu Thr Leu Gln
    115 120 125
    Asn Asp Pro Pro Ser Leu Glu Thr Gly Val Gln Asp Lys Glu Met Leu
    130 135 140
    Lys Lys Tyr Gly Lys Ser Phe Arg Lys Met Ile Ser Leu Cys Leu Gln
    145 150 155 160
    Lys Asp Pro Glu Lys Arg Pro Thr Ala Ala Glu Leu Leu Arg His Lys
    165 170 175
    Phe Phe Gln Lys Ala Lys Asn Lys Glu Phe Leu Gln Glu Lys Thr Leu
    180 185 190
    Gln Arg Ala Pro Thr Ile Ser Glu Arg Ala Lys Lys Val Arg Arg Val
    195 200 205
    Pro Gly Ser Ser Gly Arg Leu His Lys Thr Glu Asp Gly Gly Trp Glu
    210 215 220
    Trp Ser Asp Asp Glu Phe Asp Glu Glu Ser Glu Glu Gly Lys Ala Ala
    225 230 235 240
    Ile Ser Gln Leu Arg Ser Pro Arg Val Lys Glu Ser Ile Ser Asn Ser
    245 250 255
    Glu Leu Phe Pro Thr Thr Asp Pro Val Gly Thr Leu Leu Gln Val Pro
    260 265 270
    Glu Gln Ile Ser Ala His Leu Pro Gln Pro Ala Gly Gln Ile Ala Thr
    275 280 285
    Gln Pro Thr Gln Val Ser Leu Pro Pro Thr Ala Glu Pro Ala Lys Thr
    290 295 300
    Ala Gln Ala Leu Ser Ser Gly Ser Gly Ser Gln Glu Thr Lys Ile Pro
    305 310 315 320
    Ile Ser Leu Val Leu Arg Leu Arg Asn Ser Lys Lys Glu Leu Asn Asp
    325 330 335
    Ile Arg Phe Glu Phe Thr Pro Gly Arg Asp Thr Ala Glu Gly Val Ser
    340 345 350
    Gln Glu Leu Ile Ser Ala Gly Leu Val Asp Gly Arg Asp Leu Val Ile
    355 360 365
    Val Ala Ala Asn Leu Gln Lys Ile Val Glu Glu Pro Gln Ser Asn Arg
    370 375 380
    Ser Val Thr Phe Lys Leu Ala Ser Gly Val Glu Gly Ser Asp Ile Pro
    385 390 395 400
    Asp Asp Gly Lys Leu Ile Gly Phe Ala Gln Leu Ser Ile Ser
    405 410
    <210> SEQ ID NO 8
    <211> LENGTH: 274
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 8
    Leu Ile Cys Thr His Phe Met Asp Gly Met Asn Glu Leu Ala Ile Ala
    1 5 10 15
    Tyr Ile Leu Gln Gly Val Leu Lys Ala Leu Asp Tyr Ile His His Met
    20 25 30
    Gly Tyr Val His Arg Ser Val Lys Ala Ser His Ile Leu Ile Ser Val
    35 40 45
    Asp Gly Lys Val Tyr Leu Ser Gly Leu Arg Ser Asn Leu Ser Met Ile
    50 55 60
    Ser His Gly Gln Arg Gln Arg Val Val His Asp Phe Pro Lys Tyr Ser
    65 70 75 80
    Val Lys Val Leu Pro Trp Leu Ser Pro Glu Val Leu Gln Gln Asn Leu
    85 90 95
    Gln Gly Tyr Asp Ala Lys Ser Asp Ile Tyr Ser Val Gly Ile Thr Ala
    100 105 110
    Cys Glu Leu Ala Asn Gly His Val Pro Phe Lys Asp Met Pro Ala Thr
    115 120 125
    Gln Met Leu Leu Glu Lys Leu Asn Gly Thr Val Pro Cys Leu Leu Asp
    130 135 140
    Thr Ser Thr Ile Pro Ala Glu Glu Leu Thr Met Ser Pro Ser Arg Ser
    145 150 155 160
    Val Ala Asn Ser Gly Leu Ser Asp Ser Leu Thr Thr Ser Thr Pro Arg
    165 170 175
    Pro Ser Asn Gly Asp Ser Pro Ser His Pro Tyr His Arg Thr Phe Ser
    180 185 190
    Pro His Phe His His Phe Val Glu Gln Cys Leu Gln Arg Asn Pro Asp
    195 200 205
    Ala Arg Pro Ser Ala Ser Thr Leu Leu Asn His Ser Phe Phe Lys Gln
    210 215 220
    Ile Lys Arg Arg Ala Ser Glu Ala Leu Pro Glu Leu Leu Arg Pro Val
    225 230 235 240
    Thr Pro Ile Thr Asn Phe Glu Gly Ser Gln Ser Gln Asp His Ser Gly
    245 250 255
    Ile Phe Gly Leu Val Thr Asn Leu Glu Glu Leu Glu Val Asp Asp Trp
    260 265 270
    Glu Phe
    <210> SEQ ID NO 9
    <211> LENGTH: 3798
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 9
    gagaccatgg cgaacgactc tcccgcgaaa agtctggtgg acatcgacct ctcctccctg 60
    cgggatcctg ctgggatttt tgagctggtg gaagtggttg gaaatggcac ctatggacaa 120
    gtctataagg gtcgacatgt taaaacgggt cagttggcag ccatcaaagt tatggatgtc 180
    actgaggatg aagaggaaga aatcaaactg gagataaata tgctaaagaa atactctcat 240
    cacagaaaca ttgcaacata ttatggtgct ttcatcaaaa agagccctcc aggacatgat 300
    gaccaactct ggcttgttat ggagttctgt ggggctgggt ccattacaga ccttgtgaag 360
    aacaccaaag ggaacacact caaagaagac tggatcgctt acatctccag agaaatcctg 420
    aggggactgg cacatcttca cattcatcat gtgattcacc gggatatcaa gggccagaat 480
    gtgttgctga ctgagaatgc agaggtgaaa cttgttgact ttggtgtgag tgctcagctg 540
    gacaggactg tggggcggag aaatacgttc ataggcactc cctactggat ggctcctgag 600
    gtcatcgcct gtgatgagaa cccagatgcc acctatgatt acagaagtga tctttggtct 660
    tgtggcatta cagccattga gatggcagaa ggtgctcccc ctctctgtga catgcatcca 720
    atgagagcac tgtttctcat tcccagaaac cctcctcccc ggctgaagtc aaaaaaatgg 780
    tcgaagaagt tttttagttt tatagaaggg tgcctggtga agaattacat gcagcggccc 840
    tctacagagc agcttttgaa acatcctttt ataagggatc agccaaatga aaggcaagtt 900
    agaatccagc ttaaggatca tatagatcgt accaggaaga agagaggcga gaaagatgaa 960
    actgagtatg agtacagtgg gagtgaggaa gaagaggagg aagtgcctga acaggaagga 1020
    gagccaagtt ccattgtgaa cgtgcctggt gagtctactc ttcgccgaga tttcctgaga 1080
    ctgcagcagg agaacaagga acgttccgag gctcttcgga gacaacagtt actacaggag 1140
    caacagctcc gggagcagga agaatataaa aggcaactgc tggcagagag acagaagcgg 1200
    attgagcagc agaaagaaca gaggcgacgg ctagaagagc aacaaaggag agagcgggaa 1260
    gctagaaggc agcaggaacg tgaacagcga aggagagaac aagaagaaaa gaggcgtcta 1320
    gaggagttgg agagaaggcg caaagaagaa gaggagagga gacgggcaga agaagaaaag 1380
    aggagagttg aaagagaaca ggagtatatc aggcgacagc tagaagagga gcagcggcac 1440
    ttggaagtcc ttcagcagca gctgctccag gagcaggcca tgttactgga gtgccgatgg 1500
    cgggagatgg aggagcaccg gcaggcagag aggctccaga ggcagttgca acaagaacaa 1560
    gcatatctcc tgtctctaca gcatgaccat aggaggccgc acccgcagca ctcgcagcag 1620
    ccgccaccac cgcagcagga aaggagcaag ccaagcttcc atgctcccga gcccaaagcc 1680
    cactacgagc ctgctgaccg agcgcgagag gtggaagata gatttaggaa aactaaccac 1740
    agctcccctg aagcccagtc taagcagaca ggcagagtat tggagccacc agtgccttcc 1800
    cgatcagagt ctttttccaa tggcaactcc gagtctgtgc atcccgccct gcagagacca 1860
    gcggagccac aggttcctgt gagaacaaca tctcgctccc ctgttctgtc ccgtcgagat 1920
    tccccactgc agggcagtgg gcagcagaat agccaggcag gacagagaaa ctccaccagt 1980
    attgagccca ggcttctgtg ggagagagtg gagaagctgg tgcccagacc tggcagtggc 2040
    agctcctcag ggtccagcaa ctcaggatcc cagcccgggt ctcaccctgg gtctcagagt 2100
    ggctccgggg aacgcttcag agtgagatca tcatccaagt ctgaaggctc tccatctcag 2160
    cgcctggaaa atgcagtgaa aaaacctgaa gataaaaagg aagttttcag acccctcaag 2220
    cctgctgatc tgaccgcact ggccaaagag cttcgagcag tggaagatgt acggccacct 2280
    cacaaagtaa cggactactc ctcatccagt gaggagtcgg ggacgacgga tgaggaggac 2340
    gacgatgtgg agcaggaagg ggctgacgag tccacctcag gaccagagga caccagagca 2400
    gcgtcatctc tgaatttgag caatggtgaa acggaatctg tgaaaaccat gattgtccat 2460
    gatgatgtag aaagtgagcc ggccatgacc ccatccaagg agggcactct aatcgtccgc 2520
    cggactcagt ccgctagtag cacactccag aaacacaaat cttcctcctc ctttacacct 2580
    tttatagacc ccagattact acagatttct ccatctagcg gaacaacagt gacatctgtg 2640
    gtgggatttt cctgtgatgg gatgagacca gaagccataa ggcaagatcc tacccggaaa 2700
    ggctcagtgg tcaatgtgaa tcctaccaac actaggccac agagtgacac cccggagatt 2760
    cgtaaataca agaagaggtt taactctgag attctgtgtg ctgccttatg gggagtgaat 2820
    ttgctagtgg gtacagagag tggcctgatg ctgctggaca gaagtggcca agggaaggtc 2880
    tatcctctta tcaaccgaag acgatttcaa caaatggacg tacttgaggg cttgaatgtc 2940
    ttggtgacaa tatctggcaa aaaggataag ttacgtgtct actatttgtc ctggttaaga 3000
    aataaaatac ttcacaatga tccagaagtt gagaagaagc agggatggac aaccgtaggg 3060
    gatttggaag gatgtgtaca ttataaagtt gtaaaatatg aaagaatcaa atttctggtg 3120
    attgctttga agagttctgt ggaagtctat gcgtgggcac caaagccata tcacaaattt 3180
    atggccttta agtcatttgg agaattggta cataagccat tactggtgga tctcactgtt 3240
    gaggaaggcc agaggttgaa agtgatctat ggatcctgtg ctggattcca tgctgttgat 3300
    gtggattcag gatcagtcta tgacatttat ctaccaacac atatccagtg tagcatcaaa 3360
    ccccatgcaa tcatcatcct ccccaataca gatggaatgg agcttctggt gtgctatgaa 3420
    gatgaggggg tttatgtaaa cacatatgga aggatcacca aggatgtagt tctacagtgg 3480
    ggagagatgc ctacatcagt agcatatatt cgatccaatc agacaatggg ctggggagag 3540
    aaggccatag agatccgatc tgtggaaact ggtcacttgg atggtgtgtt catgcacaaa 3600
    agggctcaaa gactaaaatt cttgtgtgaa cgcaatgaca aggtgttctt tgcctctgtt 3660
    cggtctggtg gcagcagtca ggtttatttc atgaccttag gcaggacttc tcttctgagc 3720
    tggtagaagc agtgtgatcc agggattact ggcctccaga gtcttcaaga tcctgagaac 3780
    ttggaattcc ttgtaact 3798
    <210> SEQ ID NO 10
    <211> LENGTH: 4055
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 10
    gctttcgggg aggtctatga gggtcgtcat gtcaaaacgg gccagcttgc agccatcaag 60
    gttatggatg tcacagggga tgaagaggaa gaaatcaaac aagaaattaa catgttgaag 120
    aaatattctc atcaccggaa tattgctaca tactatggtg cttttatcaa aaagaaccca 180
    ccaggcatgg atgaccaact ttggttggtg atggagtttt gtggtgctgg ctctgtcacc 240
    gacctgatca agaacacaaa aggtaacacg ttgaaagagg agtggattgc atacatctgc 300
    agggaaatct tacgggggct gagtcacctg caccagcata aagtgattca tcgagatatt 360
    aaagggcaaa atgtcttgct gactgaaaat gcagaagtta aactagtgga ctttggagtc 420
    agtgctcagc ttgatcgaac agtgggcagg aggaatactt tcattggaac tccctactgg 480
    atggcaccag aagttattgc ctgtgatgaa aacccagatg ccacatatga tttcaagagt 540
    gacttgtggt ctttgggtat caccgccatt gaaatggcag aaggtgctcc ccctctctgt 600
    gacatgcacc ccatgagagc tctcttcctc atcccccgga atccagcgcc tcggctgaag 660
    tctaagaagt ggtcaaaaaa attccagtca tttattgaga gctgcttggt aaagaatcac 720
    agccagcgac cagcaacaga acaattgatg aagcatccat ttatacgaga ccaacctaat 780
    gagcgacagg tccgcattca actcaaggac catattgata gaacaaagaa gaagcgagga 840
    gaaaaagatg agacagagta tgagtacagt ggaagtgagg aagaagagga ggagaatgac 900
    tcaggagagc ccagctccat cctgaatctg ccaagggagt cgacgctgcg gagggacttt 960
    ctgaggctgc agctggccaa caaggagcgt tctgaggccc tacggaggca gcagctggag 1020
    cagcagcagc gggagaatga ggagcacaag cggcagctgc tggccgagcg tcagaagcgc 1080
    atcgaggagc agaaagagca gaggcggcgg ctggaggagc aacaaaggcg agagaaggag 1140
    ctgcggaagc agcaggagag ggagcagcgc cggcactatg aggagcagat gcgccgggag 1200
    gaggagagga ggcgtgcgga gcatgaacag gaatataagc gcaaacaatt ggaagaacag 1260
    agacaagcag aaagactgca gaggcagcta aagcaagaaa gagactactt agtttccctt 1320
    cagcatcagc ggcaggagca gaggcctgtg gagaagaagc cactgtacca ttacaaagaa 1380
    ggaatgagtc ctagtgagaa gccagcatgg gccaaggagg tagaagaacg gtcaaggctc 1440
    aaccggcaaa gttcccctgc catgcctcac aaggttgcca acaggatatc tgaccccaac 1500
    ctgcccccaa ggtcggagtc cttcagcatt agtggagttc agcctgctcg aacacccccc 1560
    atgctcagac cagtcgatcc ccagatccca catctggtag ctgtaaaatc ccagggacct 1620
    gccttgaccg cctcccagtc agtgcacgag cagcccacaa agggcctctc tgggtttcag 1680
    gaggctctga acgtgacctc ccaccgcgtg gagatgccac gccagaactc agatcccacc 1740
    tcggaaaatc ctcctctccc cactcgcatt gaaaagtttg accgaagctc ttggttacga 1800
    caggaagaag acattccacc aaaggtgcct caaagaacaa cttctatatc cccagcatta 1860
    gccagaaaga attctcctgg gaatggtagt gctctgggac ccagactagg atctcaaccc 1920
    atcagagcaa gcaaccctga tctccggaga actgagccca tcttggagag ccccttgcag 1980
    aggaccagca gtggcagttc ctccagctcc agcaccccta gctcccagcc cagctcccaa 2040
    ggaggctccc agcctggatc acaagcagga tccagtgaac gcaccagagt tcgagccaac 2100
    agtaagtcag aaggatcacc tgtgctcccc catgagcctg ccaaggtgaa accagaagaa 2160
    tccagggaca ttacccggcc cagtcgacca gctagctaca aaaaagctat agatgaggat 2220
    ctgacggcat tagccaaaga actaagagaa ctccggattg aagaaacaaa ccgcccaatg 2280
    aagaaggtga ctgattactc ctcctccagt gaggagtcag aaagtagcga ggaagaggag 2340
    gaagatggag agagcgagac ccatgatggg acagtggctg tcagcgacat acccagactg 2400
    ataccaacag gagctccagg cagcaacgag cagtacaatg tgggaatggt ggggacgcat 2460
    gggctggaga cctctcatgc ggacagtttc agcggcagta tttcaagaga aggaaccttg 2520
    atgattagag agacgtctgg agagaagaag cgatctggcc acagtgacag caatggcttt 2580
    gctggccaca tcaacctccc tgacctggtg cagcagagcc attctccagc tggaaccccg 2640
    actgagggac tggggcgcgt ctcaacccat tcccaggaga tggactctgg gactgaatat 2700
    ggcatgggga gcagcaccaa agcctccttc accccctttg tggaccccag agtataccag 2760
    acgtctccca ctgatgaaga tgaagaggat gaggaatcat cagccgcagc tctgtttact 2820
    ggcgaacttc ttaggcaaga acaggccaaa ctcaatgaag caagaaagat ttcggtggta 2880
    aatgtaaacc caaccaacat tcggcctcat agcgacacac cagaaatcag aaaatacaag 2940
    aaacgattca actcagaaat actttgtgca gctctgtggg gtgtaaacct tctggtgggg 3000
    actgaaaatg gcctgatgct tttggaccga agtgggcaag gcaaagtcta taatctgatc 3060
    aaccggaggc gatttcagca gatggatgtg ctagagggac tgaatgtcct tgtgacaatt 3120
    tcaggaaaga agaataagct acgagtttac tatctttcat ggttaagaaa cagaatacta 3180
    cataatgacc cagaagtaga aaagaaacaa ggctggatca ctgttgggga cttggaaggc 3240
    tgtatacatt ataaagttgt taaatatgaa aggatcaaat ttttggtgat tgccttaaag 3300
    aatgctgtgg aaatatatgc ttgggctcct aaaccgtatc ataaattcat ggcatttaag 3360
    tcttttgcag atctccagca caagcctctg ctagttgatc tcacggtaga agaaggtcaa 3420
    agattaaagg ttatttttgg ttcacacact ggtttccatg taattgatgt tgattcagga 3480
    aactcttatg atatctacac accatctcat attcagggca atatcactcc tcatgctatt 3540
    gtcatcttgc ctaaaacaga tggaatggaa atgcttgttt gctatgagga tgagggggtg 3600
    tatgtaaaca cctatggccg gataactaag gatgtggtgc tccaatgggg agaaatgccc 3660
    acgtctgtgg cctacattca ttccaatcag ataatgggct ggggcgagaa agctattgag 3720
    atccggtcag tggaaacagg acatttggat ggagtattta tgcataagcg agctcaaagg 3780
    ttaaagtttc tatgtgaaag aaatgataag gtattttttg catccgtgcg atctggagga 3840
    agtagccaag tgtttttcat gaccctcaac agaaattcca tgatgaactg gtaacagaag 3900
    agcacttggc acttatcttc atggcgttat ttctaattta aaagaacata actcatgtgg 3960
    acttatgcca gtctagaggc agaatcagaa ggcttggttg aacatatcgc tttccctttt 4020
    tcctctccct ccgcccctcc cagtacagtc catct 4055
    <210> SEQ ID NO 11
    <211> LENGTH: 4133
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 11
    gcatttgggg aggtgtatga gggtcggcat gtcaagacgg ggcagctggc tgccatcaag 60
    gtcatggatg tcacggagga cgaggaggaa gagatcaaac aggagatcaa catgctgaaa 120
    aagtactctc accaccgcaa catcgccacc tactacggag ccttcatcaa gaagagcccc 180
    ccgggaaacg atgaccagct ctggctggtg atggagttct gtggtgctgg ttcagtgact 240
    gacctggtaa agaacacaaa aggcaacgcc ctgaaggagg actgtatcgc ctatatctgc 300
    agggagatcc tcaggggtct ggcccatctc catgcccaca aggtgatcca tcgagacatc 360
    aaggggcaga atgtgctgct gacagagaat gctgaggtca agctagtgga ttttggggtg 420
    agtgctcagc tggaccgcac cgtgggcaga cggaacactt tcattgggac tccctactgg 480
    atggctccag aggtcatcgc ctgtgatgag aaccctgatg ccacctatga ttacaggagt 540
    gatatttggt ctctaggaat cacagccatc gagatggcag agggagcccc ccctctgtgt 600
    gacatgcacc ccatgcgagc cctcttcctc attcctcgga accctccgcc caggctcaag 660
    tccaagaagt ggtctaagaa gttcattgac ttcattgaca catgtctcat caagacttac 720
    ctgagccgcc cacccacgga gcagctactg aagtttccct tcatccggga ccagcccacg 780
    gagcggcagg tccgcatcca gcttaaggac cacattgacc gatcccggaa gaagcggggt 840
    gagaaagagg agacagaata tgagtacagc ggcagcgagg aggaagatga cagccatgga 900
    gaggaaggag agccaagctc catcatgaac gtgcctggag agtcgactct acgccgggag 960
    tttctccggc tccagcagga aaataagagc aactcagagg ctttaaaaca gcagcagcag 1020
    ctgcagcagc agcagcagcg agaccccgag gcacacatca aacacctgct gcaccagcgg 1080
    cagcggcgca tagaggagca gaaggaggag cggcgccgcg tggaggagca acagcggcgg 1140
    gagcgggagc agcggaagct gcaggagaag gagcagcagc ggcggctgga ggacatgcag 1200
    gctctgcggc gggaggagga gcggcggcag gcggagcgcg agcaggaata tattcgtcac 1260
    aggctagagg aggagcagcg acagctcgag atccttcagc aacagctgct ccaggaacag 1320
    gccctgctgc tggaatacaa gcggaagcag ctggaggagc agcggcagtc agaacgtctc 1380
    cagaggcagc tgcagcagga gcatgcctac ctcaagtccc tgcagcagca gcaacagcag 1440
    cagcagcttc agaaacaaca gcagcagcag ctcctgcctg gggacaggaa gcccctgtac 1500
    cattatggtc ggggcatgaa tcccgctgac aaaccagcct gggcccgaga ggtagaagag 1560
    agaacaagga tgaacaagca gcagaactct cccttggcca agagcaagcc aggcagcacg 1620
    gggcctgagc cccccatccc ccaggcctcc ccagggcccc caggacccct ttcccagact 1680
    cctcctatgc agaggccggt ggagccccag gagggaccgc acaagagcct ggtggcacac 1740
    cgggtcccac tgaagccata tgcagcacct gtaccccgat cccagtccct gcaggaccag 1800
    cccacccgaa acctggctgc cttcccagcc tcccatgacc ccgaccctgc catccccgca 1860
    cccactgcca cgcccagtgc ccgaggagct gtcatccgcc agaattcaga ccccacctct 1920
    gaaggacctg gccccagccc gaatccccca gcctgggtcc gcccagataa cgaggcccca 1980
    cccaaggtgc ctcagaggac ctcatctatc gccactgccc ttaacaccag tggggccgga 2040
    gggtcccggc cagcccaggc agtccgtgcc agacctcgca gcaactccgc ctggcaaatc 2100
    tatctgcaaa ggcgggcaga gcggggcacc ccaaagcctc cagggccccc tgctcagccc 2160
    cctggcccgc ccaacgcctc tagtaacccc gacctcagga ggagcgaccc tggctgggaa 2220
    cgctcggaca gcgtccttcc agcctctcac gggcacctcc cccaggctgg ctcactggag 2280
    cggaaccgcg tgggagtctc ctccaaaccg gacagctccc ctgtgctctc ccctgggaat 2340
    aaagccaagc ccgacgacca ccgctcacgg ccaggccggc ccgcagactt tgtgttgctg 2400
    aaagagcgga ctctggacga ggcccctcgg cctcccaaga aggccatgga ctactcgtcg 2460
    tccagcgagg aggtggaaag cagtgaggac gacgaggagg aaggcgaagg cgggccagca 2520
    gaggggagca gagatacccc tgggggccgc gatggggata cagacagcgt cagcaccatg 2580
    gtggtccacg acgtcgagga gatcaccggg acccagcccc catacggggg cggcaccatg 2640
    gtggtccagc gcacccctga agaggagcgg aacctgctgc atgctgacag caatgggtac 2700
    acaaacctgc ctgacgtggt ccagcccagc cactcaccca ccgagaacag caaaggccaa 2760
    agcccaccct cgaaggatgg gagtggtgac taccagtctc gtgggctggt aaaggcccct 2820
    ggcaagagct cgttcacgat gtttgtggat ctagggatct accagcctgg aggcagtggg 2880
    gacagcatcc ccatcacagc cctagtgggt ggagagggca ctcggctcga ccagctgcag 2940
    tacgacgtga ggaagggttc tgtggtcaac gtgaatccca ccaacacccg ggcccacagt 3000
    gagacccctg agatccggaa gtacaagaag cgattcaact ccgagatcct ctgtgcagcc 3060
    ctttgggggg tcaacctgct ggtgggcacg gagaacgggc tgatgttgct ggaccgaagt 3120
    gggcagggca aggtgtatgg actcattggg cggcgacgct tccagcagat ggatgtgctg 3180
    gaggggctca acctgctcat caccatctca gggaaaagga acaaactgcg ggtgtattac 3240
    ttgtcctggc tccggaacaa gattctgcac aatgacccag aagtggagaa gaagcagggc 3300
    tggaccaccg tgggggacat ggagggctgc gggcactacc gtgttgtgaa atacgagcgg 3360
    attaagttcc tggtcatcgc cctcaagagc tccgtggagg tgtatgcctg ggcccccaaa 3420
    ccctaccaca aattcatggc cttcaagtcc tttgccgacc tcccccaccg ccctctgctg 3480
    gtcgacctga cagtagagga ggggcagcgg ctcaaggtca tctatggctc cagtgctggc 3540
    ttccatgctg tggatgtcga ctcggggaac agctatgaca tctacatccc tgtgcacatc 3600
    cagagccaga tcacgcccca tgccatcatc ttcctcccca acaccgacgg catggagatg 3660
    ctgctgtgct acgaggacga gggtgtctac gtcaacacgt acgggcgcat cattaaggat 3720
    gtggtgctgc agtgggggga gatgcctact tctgtggcct acatctgctc caaccagata 3780
    atgggctggg gtgagaaagc cattgagatc cgctctgtgg agacgggcca cctcgacggg 3840
    gtcttcatgc acaaacgagc tcagaggctc aagttcctgt gtgagcggaa tgacaaggtg 3900
    ttttttgcct cagtccgctc tgggggcagc agccaagttt acttcatgac tctgaaccgt 3960
    aaccgcatca tgaactggtg acggggccct gggctggggc tgtcccacac tggacccagc 4020
    tctccccctg cagccaggct tcccgggccg cccctctttc ccctccctgg gcttttgctt 4080
    ttactggttt gatttcactg gagcctgctg ggaacgtgac ctctgacccc tga 4133
    <210> SEQ ID NO 12
    <211> LENGTH: 1459
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 12
    caatgttaac ccactctatg tctctcctgc atgtaaaaaa ccactaatcc acatgtatga 60
    aaaggagttc acttctgaga tctgctgtgg ttctttgtgg ggagtcaatt tgctgttggg 120
    aacccgatct aatctatatc tgatggacag aagtggaaag gctgacatta ctaaacttat 180
    aaggcgaaga ccattccgcc agattcaagt cttagagcca ctcaatttgc tgattaccat 240
    ctcaggtcat aagaacagac ttcgggtgta tcatctgacc tggttgagga acaagatttt 300
    gaataatgat ccagaaagta aaagaaggca agaagaaatg ctgaagacag aggaagcctg 360
    caaagctatt gataagttaa caggctgtga acacttcagt gtcctccaac atgaagaaac 420
    aacatatatt gcaattgctt tgaaatcatc aattcacctt tatgcatggg caccaaagtc 480
    ctttgatgaa agcactgcta ttaaagtatt tccaacactt gatcataagc cagtgacagt 540
    tgacctggct attggttctg aaaaaagact aaagattttc ttcagctcag cagatggata 600
    tcacctcatc gatgcagaat ctgaggttat gtctgatgtg accctgccaa agaatcccct 660
    ggaaatcatt ataccacaga atatcatcat tttacctgat tgcttgggaa ttggcatgat 720
    gctcaccttc aatgctgaag ccctctctgt ggaagcaaat gaacaactct tcaagaagat 780
    ccttgaaatg tggaaagaca taccatcttc tatagctttt gaatgtacac agcgaaccac 840
    aggatggggc caaaaggcca ttgaagtgcg ctctttgcaa tccagggttc tggaaagtga 900
    gctgaagcgc aggtcaatta agaagctgag attcctgtgc acccggggtg acaagctgtt 960
    ctttacctct accctgcgca atcaccacag ccgggtttac ttcatgacac ttggaaaact 1020
    tgaagagctc caaagcaatt atgatgtcta aaagtttcca gtgatttatt accacattat 1080
    aaacatcatg tataggcagt ctgcatcttc agatttcaga gattaaatga gtattcagtt 1140
    ttatttttag taaagattaa atccaaaact ttacttttaa tgtagcacag aatagtttta 1200
    atgagaaatg cagctttatg tataaaatta actatagcaa gctctaggta ctccaatggt 1260
    gtacaatgtc ttttgcacaa actttgtaac ttttgttact gtgaattcaa acattactct 1320
    ttggacagtt tggacagtat ctgtattcag attttacaac atggagtaaa gaaacctgtt 1380
    atgaattaga ttacaagcag ccttcaaaag aattggcact gggataagat ttttcagaaa 1440
    agaaaaacat cggcaaact 1459
    <210> SEQ ID NO 13
    <211> LENGTH: 1239
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 13
    Met Ala Asn Asp Ser Pro Ala Lys Ser Leu Val Asp Ile Asp Leu Ser
    1 5 10 15
    Ser Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu Val Val Gly
    20 25 30
    Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val Lys Thr Gly
    35 40 45
    Gln Leu Ala Ala Ile Lys Val Met Asp Val Thr Glu Asp Glu Glu Glu
    50 55 60
    Glu Ile Lys Leu Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg
    65 70 75 80
    Asn Ile Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly
    85 90 95
    His Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser
    100 105 110
    Ile Thr Asp Leu Val Lys Asn Thr Lys Gly Asn Thr Leu Lys Glu Asp
    115 120 125
    Trp Ile Ala Tyr Ile Ser Arg Glu Ile Leu Arg Gly Leu Ala His Leu
    130 135 140
    His Ile His His Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu
    145 150 155 160
    Leu Thr Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala
    165 170 175
    Gln Leu Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro
    180 185 190
    Tyr Trp Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala
    195 200 205
    Thr Tyr Asp Tyr Arg Ser Asp Leu Trp Ser Cys Gly Ile Thr Ala Ile
    210 215 220
    Glu Met Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg
    225 230 235 240
    Ala Leu Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser Lys
    245 250 255
    Lys Trp Ser Lys Lys Phe Phe Ser Phe Ile Glu Gly Cys Leu Val Lys
    260 265 270
    Asn Tyr Met Gln Arg Pro Ser Thr Glu Gln Leu Leu Lys His Pro Phe
    275 280 285
    Ile Arg Asp Gln Pro Asn Glu Arg Gln Val Arg Ile Gln Leu Lys Asp
    290 295 300
    His Ile Asp Arg Thr Arg Lys Lys Arg Gly Glu Lys Asp Glu Thr Glu
    305 310 315 320
    Tyr Glu Tyr Ser Gly Ser Glu Glu Glu Glu Glu Glu Val Pro Glu Gln
    325 330 335
    Glu Gly Glu Pro Ser Ser Ile Val Asn Val Pro Gly Glu Ser Thr Leu
    340 345 350
    Arg Arg Asp Phe Leu Arg Leu Gln Gln Glu Asn Lys Glu Arg Ser Glu
    355 360 365
    Ala Leu Arg Arg Gln Gln Leu Leu Gln Glu Gln Gln Leu Arg Glu Gln
    370 375 380
    Glu Glu Tyr Lys Arg Gln Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu
    385 390 395 400
    Gln Gln Lys Glu Gln Arg Arg Arg Leu Glu Glu Gln Gln Arg Arg Glu
    405 410 415
    Arg Glu Ala Arg Arg Gln Gln Glu Arg Glu Gln Arg Arg Arg Glu Gln
    420 425 430
    Glu Glu Lys Arg Arg Leu Glu Glu Leu Glu Arg Arg Arg Lys Glu Glu
    435 440 445
    Glu Glu Arg Arg Arg Ala Glu Glu Glu Lys Arg Arg Val Glu Arg Glu
    450 455 460
    Gln Glu Tyr Ile Arg Arg Gln Leu Glu Glu Glu Gln Arg His Leu Glu
    465 470 475 480
    Val Leu Gln Gln Gln Leu Leu Gln Glu Gln Ala Met Leu Leu Glu Cys
    485 490 495
    Arg Trp Arg Glu Met Glu Glu His Arg Gln Ala Glu Arg Leu Gln Arg
    500 505 510
    Gln Leu Gln Gln Glu Gln Ala Tyr Leu Leu Ser Leu Gln His Asp His
    515 520 525
    Arg Arg Pro His Pro Gln His Ser Gln Gln Pro Pro Pro Pro Gln Gln
    530 535 540
    Glu Arg Ser Lys Pro Ser Phe His Ala Pro Glu Pro Lys Ala His Tyr
    545 550 555 560
    Glu Pro Ala Asp Arg Ala Arg Glu Val Glu Asp Arg Phe Arg Lys Thr
    565 570 575
    Asn His Ser Ser Pro Glu Ala Gln Ser Lys Gln Thr Gly Arg Val Leu
    580 585 590
    Glu Pro Pro Val Pro Ser Arg Ser Glu Ser Phe Ser Asn Gly Asn Ser
    595 600 605
    Glu Ser Val His Pro Ala Leu Gln Arg Pro Ala Glu Pro Gln Val Pro
    610 615 620
    Val Arg Thr Thr Ser Arg Ser Pro Val Leu Ser Arg Arg Asp Ser Pro
    625 630 635 640
    Leu Gln Gly Ser Gly Gln Gln Asn Ser Gln Ala Gly Gln Arg Asn Ser
    645 650 655
    Thr Ser Ile Glu Pro Arg Leu Leu Trp Glu Arg Val Glu Lys Leu Val
    660 665 670
    Pro Arg Pro Gly Ser Gly Ser Ser Ser Gly Ser Ser Asn Ser Gly Ser
    675 680 685
    Gln Pro Gly Ser His Pro Gly Ser Gln Ser Gly Ser Gly Glu Arg Phe
    690 695 700
    Arg Val Arg Ser Ser Ser Lys Ser Glu Gly Ser Pro Ser Gln Arg Leu
    705 710 715 720
    Glu Asn Ala Val Lys Lys Pro Glu Asp Lys Lys Glu Val Phe Arg Pro
    725 730 735
    Leu Lys Pro Ala Asp Leu Thr Ala Leu Ala Lys Glu Leu Arg Ala Val
    740 745 750
    Glu Asp Val Arg Pro Pro His Lys Val Thr Asp Tyr Ser Ser Ser Ser
    755 760 765
    Glu Glu Ser Gly Thr Thr Asp Glu Glu Asp Asp Asp Val Glu Gln Glu
    770 775 780
    Gly Ala Asp Glu Ser Thr Ser Gly Pro Glu Asp Thr Arg Ala Ala Ser
    785 790 795 800
    Ser Leu Asn Leu Ser Asn Gly Glu Thr Glu Ser Val Lys Thr Met Ile
    805 810 815
    Val His Asp Asp Val Glu Ser Glu Pro Ala Met Thr Pro Ser Lys Glu
    820 825 830
    Gly Thr Leu Ile Val Arg Arg Thr Gln Ser Ala Ser Ser Thr Leu Gln
    835 840 845
    Lys His Lys Ser Ser Ser Ser Phe Thr Pro Phe Ile Asp Pro Arg Leu
    850 855 860
    Leu Gln Ile Ser Pro Ser Ser Gly Thr Thr Val Thr Ser Val Val Gly
    865 870 875 880
    Phe Ser Cys Asp Gly Met Arg Pro Glu Ala Ile Arg Gln Asp Pro Thr
    885 890 895
    Arg Lys Gly Ser Val Val Asn Val Asn Pro Thr Asn Thr Arg Pro Gln
    900 905 910
    Ser Asp Thr Pro Glu Ile Arg Lys Tyr Lys Lys Arg Phe Asn Ser Glu
    915 920 925
    Ile Leu Cys Ala Ala Leu Trp Gly Val Asn Leu Leu Val Gly Thr Glu
    930 935 940
    Ser Gly Leu Met Leu Leu Asp Arg Ser Gly Gln Gly Lys Val Tyr Pro
    945 950 955 960
    Leu Ile Asn Arg Arg Arg Phe Gln Gln Met Asp Val Leu Glu Gly Leu
    965 970 975
    Asn Val Leu Val Thr Ile Ser Gly Lys Lys Asp Lys Leu Arg Val Tyr
    980 985 990
    Tyr Leu Ser Trp Leu Arg Asn Lys Ile Leu His Asn Asp Pro Glu Val
    995 1000 1005
    Glu Lys Lys Gln Gly Trp Thr Thr Val Gly Asp Leu Glu Gly Cys Val
    1010 1015 1020
    His Tyr Lys Val Val Lys Tyr Glu Arg Ile Lys Phe Leu Val Ile Ala
    1025 1030 1035 1040
    Leu Lys Ser Ser Val Glu Val Tyr Ala Trp Ala Pro Lys Pro Tyr His
    1045 1050 1055
    Lys Phe Met Ala Phe Lys Ser Phe Gly Glu Leu Val His Lys Pro Leu
    1060 1065 1070
    Leu Val Asp Leu Thr Val Glu Glu Gly Gln Arg Leu Lys Val Ile Tyr
    1075 1080 1085
    Gly Ser Cys Ala Gly Phe His Ala Val Asp Val Asp Ser Gly Ser Val
    1090 1095 1100
    Tyr Asp Ile Tyr Leu Pro Thr His Ile Gln Cys Ser Ile Lys Pro His
    1105 1110 1115 1120
    Ala Ile Ile Ile Leu Pro Asn Thr Asp Gly Met Glu Leu Leu Val Cys
    1125 1130 1135
    Tyr Glu Asp Glu Gly Val Tyr Val Asn Thr Tyr Gly Arg Ile Thr Lys
    1140 1145 1150
    Asp Val Val Leu Gln Trp Gly Glu Met Pro Thr Ser Val Ala Tyr Ile
    1155 1160 1165
    Arg Ser Asn Gln Thr Met Gly Trp Gly Glu Lys Ala Ile Glu Ile Arg
    1170 1175 1180
    Ser Val Glu Thr Gly His Leu Asp Gly Val Phe Met His Lys Arg Ala
    1185 1190 1195 1200
    Gln Arg Leu Lys Phe Leu Cys Glu Arg Asn Asp Lys Val Phe Phe Ala
    1205 1210 1215
    Ser Val Arg Ser Gly Gly Ser Ser Gln Val Tyr Phe Met Thr Leu Gly
    1220 1225 1230
    Arg Thr Ser Leu Leu Ser Trp
    1235
    <210> SEQ ID NO 14
    <211> LENGTH: 1297
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 14
    Ala Phe Gly Glu Val Tyr Glu Gly Arg His Val Lys Thr Gly Gln Leu
    1 5 10 15
    Ala Ala Ile Lys Val Met Asp Val Thr Gly Asp Glu Glu Glu Glu Ile
    20 25 30
    Lys Gln Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg Asn Ile
    35 40 45
    Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Asn Pro Pro Gly Met Asp
    50 55 60
    Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser Val Thr
    65 70 75 80
    Asp Leu Ile Lys Asn Thr Lys Gly Asn Thr Leu Lys Glu Glu Trp Ile
    85 90 95
    Ala Tyr Ile Cys Arg Glu Ile Leu Arg Gly Leu Ser His Leu His Gln
    100 105 110
    His Lys Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu Leu Thr
    115 120 125
    Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln Leu
    130 135 140
    Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp
    145 150 155 160
    Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala Thr Tyr
    165 170 175
    Asp Phe Lys Ser Asp Leu Trp Ser Leu Gly Ile Thr Ala Ile Glu Met
    180 185 190
    Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg Ala Leu
    195 200 205
    Phe Leu Ile Pro Arg Asn Pro Ala Pro Arg Leu Lys Ser Lys Lys Trp
    210 215 220
    Ser Lys Lys Phe Gln Ser Phe Ile Glu Ser Cys Leu Val Lys Asn His
    225 230 235 240
    Ser Gln Arg Pro Ala Thr Glu Gln Leu Met Lys His Pro Phe Ile Arg
    245 250 255
    Asp Gln Pro Asn Glu Arg Gln Val Arg Ile Gln Leu Lys Asp His Ile
    260 265 270
    Asp Arg Thr Lys Lys Lys Arg Gly Glu Lys Asp Glu Thr Glu Tyr Glu
    275 280 285
    Tyr Ser Gly Ser Glu Glu Glu Glu Glu Glu Asn Asp Ser Gly Glu Pro
    290 295 300
    Ser Ser Ile Leu Asn Leu Pro Arg Glu Ser Thr Leu Arg Arg Asp Phe
    305 310 315 320
    Leu Arg Leu Gln Leu Ala Asn Lys Glu Arg Ser Glu Ala Leu Arg Arg
    325 330 335
    Gln Gln Leu Glu Gln Gln Gln Arg Glu Asn Glu Glu His Lys Arg Gln
    340 345 350
    Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu Glu Gln Lys Glu Gln Arg
    355 360 365
    Arg Arg Leu Glu Glu Gln Gln Arg Arg Glu Lys Glu Leu Arg Lys Gln
    370 375 380
    Gln Glu Arg Glu Gln Arg Arg His Tyr Glu Glu Gln Met Arg Arg Glu
    385 390 395 400
    Glu Glu Arg Arg Arg Ala Glu His Glu Gln Glu Tyr Lys Arg Lys Gln
    405 410 415
    Leu Glu Glu Gln Arg Gln Ala Glu Arg Leu Gln Arg Gln Leu Lys Gln
    420 425 430
    Glu Arg Asp Tyr Leu Val Ser Leu Gln His Gln Arg Gln Glu Gln Arg
    435 440 445
    Pro Val Glu Lys Lys Pro Leu Tyr His Tyr Lys Glu Gly Met Ser Pro
    450 455 460
    Ser Glu Lys Pro Ala Trp Ala Lys Glu Val Glu Glu Arg Ser Arg Leu
    465 470 475 480
    Asn Arg Gln Ser Ser Pro Ala Met Pro His Lys Val Ala Asn Arg Ile
    485 490 495
    Ser Asp Pro Asn Leu Pro Pro Arg Ser Glu Ser Phe Ser Ile Ser Gly
    500 505 510
    Val Gln Pro Ala Arg Thr Pro Pro Met Leu Arg Pro Val Asp Pro Gln
    515 520 525
    Ile Pro His Leu Val Ala Val Lys Ser Gln Gly Pro Ala Leu Thr Ala
    530 535 540
    Ser Gln Ser Val His Glu Gln Pro Thr Lys Gly Leu Ser Gly Phe Gln
    545 550 555 560
    Glu Ala Leu Asn Val Thr Ser His Arg Val Glu Met Pro Arg Gln Asn
    565 570 575
    Ser Asp Pro Thr Ser Glu Asn Pro Pro Leu Pro Thr Arg Ile Glu Lys
    580 585 590
    Phe Asp Arg Ser Ser Trp Leu Arg Gln Glu Glu Asp Ile Pro Pro Lys
    595 600 605
    Val Pro Gln Arg Thr Thr Ser Ile Ser Pro Ala Leu Ala Arg Lys Asn
    610 615 620
    Ser Pro Gly Asn Gly Ser Ala Leu Gly Pro Arg Leu Gly Ser Gln Pro
    625 630 635 640
    Ile Arg Ala Ser Asn Pro Asp Leu Arg Arg Thr Glu Pro Ile Leu Glu
    645 650 655
    Ser Pro Leu Gln Arg Thr Ser Ser Gly Ser Ser Ser Ser Ser Ser Thr
    660 665 670
    Pro Ser Ser Gln Pro Ser Ser Gln Gly Gly Ser Gln Pro Gly Ser Gln
    675 680 685
    Ala Gly Ser Ser Glu Arg Thr Arg Val Arg Ala Asn Ser Lys Ser Glu
    690 695 700
    Gly Ser Pro Val Leu Pro His Glu Pro Ala Lys Val Lys Pro Glu Glu
    705 710 715 720
    Ser Arg Asp Ile Thr Arg Pro Ser Arg Pro Ala Ser Tyr Lys Lys Ala
    725 730 735
    Ile Asp Glu Asp Leu Thr Ala Leu Ala Lys Glu Leu Arg Glu Leu Arg
    740 745 750
    Ile Glu Glu Thr Asn Arg Pro Met Lys Lys Val Thr Asp Tyr Ser Ser
    755 760 765
    Ser Ser Glu Glu Ser Glu Ser Ser Glu Glu Glu Glu Glu Asp Gly Glu
    770 775 780
    Ser Glu Thr His Asp Gly Thr Val Ala Val Ser Asp Ile Pro Arg Leu
    785 790 795 800
    Ile Pro Thr Gly Ala Pro Gly Ser Asn Glu Gln Tyr Asn Val Gly Met
    805 810 815
    Val Gly Thr His Gly Leu Glu Thr Ser His Ala Asp Ser Phe Ser Gly
    820 825 830
    Ser Ile Ser Arg Glu Gly Thr Leu Met Ile Arg Glu Thr Ser Gly Glu
    835 840 845
    Lys Lys Arg Ser Gly His Ser Asp Ser Asn Gly Phe Ala Gly His Ile
    850 855 860
    Asn Leu Pro Asp Leu Val Gln Gln Ser His Ser Pro Ala Gly Thr Pro
    865 870 875 880
    Thr Glu Gly Leu Gly Arg Val Ser Thr His Ser Gln Glu Met Asp Ser
    885 890 895
    Gly Thr Glu Tyr Gly Met Gly Ser Ser Thr Lys Ala Ser Phe Thr Pro
    900 905 910
    Phe Val Asp Pro Arg Val Tyr Gln Thr Ser Pro Thr Asp Glu Asp Glu
    915 920 925
    Glu Asp Glu Glu Ser Ser Ala Ala Ala Leu Phe Thr Gly Glu Leu Leu
    930 935 940
    Arg Gln Glu Gln Ala Lys Leu Asn Glu Ala Arg Lys Ile Ser Val Val
    945 950 955 960
    Asn Val Asn Pro Thr Asn Ile Arg Pro His Ser Asp Thr Pro Glu Ile
    965 970 975
    Arg Lys Tyr Lys Lys Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala Leu
    980 985 990
    Trp Gly Val Asn Leu Leu Val Gly Thr Glu Asn Gly Leu Met Leu Leu
    995 1000 1005
    Asp Arg Ser Gly Gln Gly Lys Val Tyr Asn Leu Ile Asn Arg Arg Arg
    1010 1015 1020
    Phe Gln Gln Met Asp Val Leu Glu Gly Leu Asn Val Leu Val Thr Ile
    1025 1030 1035 1040
    Ser Gly Lys Lys Asn Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Arg
    1045 1050 1055
    Asn Arg Ile Leu His Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Trp
    1060 1065 1070
    Ile Thr Val Gly Asp Leu Glu Gly Cys Ile His Tyr Lys Val Val Lys
    1075 1080 1085
    Tyr Glu Arg Ile Lys Phe Leu Val Ile Ala Leu Lys Asn Ala Val Glu
    1090 1095 1100
    Ile Tyr Ala Trp Ala Pro Lys Pro Tyr His Lys Phe Met Ala Phe Lys
    1105 1110 1115 1120
    Ser Phe Ala Asp Leu Gln His Lys Pro Leu Leu Val Asp Leu Thr Val
    1125 1130 1135
    Glu Glu Gly Gln Arg Leu Lys Val Ile Phe Gly Ser His Thr Gly Phe
    1140 1145 1150
    His Val Ile Asp Val Asp Ser Gly Asn Ser Tyr Asp Ile Tyr Thr Pro
    1155 1160 1165
    Ser His Ile Gln Gly Asn Ile Thr Pro His Ala Ile Val Ile Leu Pro
    1170 1175 1180
    Lys Thr Asp Gly Met Glu Met Leu Val Cys Tyr Glu Asp Glu Gly Val
    1185 1190 1195 1200
    Tyr Val Asn Thr Tyr Gly Arg Ile Thr Lys Asp Val Val Leu Gln Trp
    1205 1210 1215
    Gly Glu Met Pro Thr Ser Val Ala Tyr Ile His Ser Asn Gln Ile Met
    1220 1225 1230
    Gly Trp Gly Glu Lys Ala Ile Glu Ile Arg Ser Val Glu Thr Gly His
    1235 1240 1245
    Leu Asp Gly Val Phe Met His Lys Arg Ala Gln Arg Leu Lys Phe Leu
    1250 1255 1260
    Cys Glu Arg Asn Asp Lys Val Phe Phe Ala Ser Val Arg Ser Gly Gly
    1265 1270 1275 1280
    Ser Ser Gln Val Phe Phe Met Thr Leu Asn Arg Asn Ser Met Met Asn
    1285 1290 1295
    Trp
    <210> SEQ ID NO 15
    <211> LENGTH: 1326
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 15
    Ala Phe Gly Glu Val Tyr Glu Gly Arg His Val Lys Thr Gly Gln Leu
    1 5 10 15
    Ala Ala Ile Lys Val Met Asp Val Thr Glu Asp Glu Glu Glu Glu Ile
    20 25 30
    Lys Gln Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg Asn Ile
    35 40 45
    Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly Asn Asp
    50 55 60
    Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser Val Thr
    65 70 75 80
    Asp Leu Val Lys Asn Thr Lys Gly Asn Ala Leu Lys Glu Asp Cys Ile
    85 90 95
    Ala Tyr Ile Cys Arg Glu Ile Leu Arg Gly Leu Ala His Leu His Ala
    100 105 110
    His Lys Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu Leu Thr
    115 120 125
    Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln Leu
    130 135 140
    Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp
    145 150 155 160
    Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala Thr Tyr
    165 170 175
    Asp Tyr Arg Ser Asp Ile Trp Ser Leu Gly Ile Thr Ala Ile Glu Met
    180 185 190
    Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg Ala Leu
    195 200 205
    Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser Lys Lys Trp
    210 215 220
    Ser Lys Lys Phe Ile Asp Phe Ile Asp Thr Cys Leu Ile Lys Thr Tyr
    225 230 235 240
    Leu Ser Arg Pro Pro Thr Glu Gln Leu Leu Lys Phe Pro Phe Ile Arg
    245 250 255
    Asp Gln Pro Thr Glu Arg Gln Val Arg Ile Gln Leu Lys Asp His Ile
    260 265 270
    Asp Arg Ser Arg Lys Lys Arg Gly Glu Lys Glu Glu Thr Glu Tyr Glu
    275 280 285
    Tyr Ser Gly Ser Glu Glu Glu Asp Asp Ser His Gly Glu Glu Gly Glu
    290 295 300
    Pro Ser Ser Ile Met Asn Val Pro Gly Glu Ser Thr Leu Arg Arg Glu
    305 310 315 320
    Phe Leu Arg Leu Gln Gln Glu Asn Lys Ser Asn Ser Glu Ala Leu Lys
    325 330 335
    Gln Gln Gln Gln Leu Gln Gln Gln Gln Gln Arg Asp Pro Glu Ala His
    340 345 350
    Ile Lys His Leu Leu His Gln Arg Gln Arg Arg Ile Glu Glu Gln Lys
    355 360 365
    Glu Glu Arg Arg Arg Val Glu Glu Gln Gln Arg Arg Glu Arg Glu Gln
    370 375 380
    Arg Lys Leu Gln Glu Lys Glu Gln Gln Arg Arg Leu Glu Asp Met Gln
    385 390 395 400
    Ala Leu Arg Arg Glu Glu Glu Arg Arg Gln Ala Glu Arg Glu Gln Glu
    405 410 415
    Tyr Ile Arg His Arg Leu Glu Glu Glu Gln Arg Gln Leu Glu Ile Leu
    420 425 430
    Gln Gln Gln Leu Leu Gln Glu Gln Ala Leu Leu Leu Glu Tyr Lys Arg
    435 440 445
    Lys Gln Leu Glu Glu Gln Arg Gln Ser Glu Arg Leu Gln Arg Gln Leu
    450 455 460
    Gln Gln Glu His Ala Tyr Leu Lys Ser Leu Gln Gln Gln Gln Gln Gln
    465 470 475 480
    Gln Gln Leu Gln Lys Gln Gln Gln Gln Gln Leu Leu Pro Gly Asp Arg
    485 490 495
    Lys Pro Leu Tyr His Tyr Gly Arg Gly Met Asn Pro Ala Asp Lys Pro
    500 505 510
    Ala Trp Ala Arg Glu Val Glu Glu Arg Thr Arg Met Asn Lys Gln Gln
    515 520 525
    Asn Ser Pro Leu Ala Lys Ser Lys Pro Gly Ser Thr Gly Pro Glu Pro
    530 535 540
    Pro Ile Pro Gln Ala Ser Pro Gly Pro Pro Gly Pro Leu Ser Gln Thr
    545 550 555 560
    Pro Pro Met Gln Arg Pro Val Glu Pro Gln Glu Gly Pro His Lys Ser
    565 570 575
    Leu Val Ala His Arg Val Pro Leu Lys Pro Tyr Ala Ala Pro Val Pro
    580 585 590
    Arg Ser Gln Ser Leu Gln Asp Gln Pro Thr Arg Asn Leu Ala Ala Phe
    595 600 605
    Pro Ala Ser His Asp Pro Asp Pro Ala Ile Pro Ala Pro Thr Ala Thr
    610 615 620
    Pro Ser Ala Arg Gly Ala Val Ile Arg Gln Asn Ser Asp Pro Thr Ser
    625 630 635 640
    Glu Gly Pro Gly Pro Ser Pro Asn Pro Pro Ala Trp Val Arg Pro Asp
    645 650 655
    Asn Glu Ala Pro Pro Lys Val Pro Gln Arg Thr Ser Ser Ile Ala Thr
    660 665 670
    Ala Leu Asn Thr Ser Gly Ala Gly Gly Ser Arg Pro Ala Gln Ala Val
    675 680 685
    Arg Ala Arg Pro Arg Ser Asn Ser Ala Trp Gln Ile Tyr Leu Gln Arg
    690 695 700
    Arg Ala Glu Arg Gly Thr Pro Lys Pro Pro Gly Pro Pro Ala Gln Pro
    705 710 715 720
    Pro Gly Pro Pro Asn Ala Ser Ser Asn Pro Asp Leu Arg Arg Ser Asp
    725 730 735
    Pro Gly Trp Glu Arg Ser Asp Ser Val Leu Pro Ala Ser His Gly His
    740 745 750
    Leu Pro Gln Ala Gly Ser Leu Glu Arg Asn Arg Val Gly Val Ser Ser
    755 760 765
    Lys Pro Asp Ser Ser Pro Val Leu Ser Pro Gly Asn Lys Ala Lys Pro
    770 775 780
    Asp Asp His Arg Ser Arg Pro Gly Arg Pro Ala Asp Phe Val Leu Leu
    785 790 795 800
    Lys Glu Arg Thr Leu Asp Glu Ala Pro Arg Pro Pro Lys Lys Ala Met
    805 810 815
    Asp Tyr Ser Ser Ser Ser Glu Glu Val Glu Ser Ser Glu Asp Asp Glu
    820 825 830
    Glu Glu Gly Glu Gly Gly Pro Ala Glu Gly Ser Arg Asp Thr Pro Gly
    835 840 845
    Gly Arg Asp Gly Asp Thr Asp Ser Val Ser Thr Met Val Val His Asp
    850 855 860
    Val Glu Glu Ile Thr Gly Thr Gln Pro Pro Tyr Gly Gly Gly Thr Met
    865 870 875 880
    Val Val Gln Arg Thr Pro Glu Glu Glu Arg Asn Leu Leu His Ala Asp
    885 890 895
    Ser Asn Gly Tyr Thr Asn Leu Pro Asp Val Val Gln Pro Ser His Ser
    900 905 910
    Pro Thr Glu Asn Ser Lys Gly Gln Ser Pro Pro Ser Lys Asp Gly Ser
    915 920 925
    Gly Asp Tyr Gln Ser Arg Gly Leu Val Lys Ala Pro Gly Lys Ser Ser
    930 935 940
    Phe Thr Met Phe Val Asp Leu Gly Ile Tyr Gln Pro Gly Gly Ser Gly
    945 950 955 960
    Asp Ser Ile Pro Ile Thr Ala Leu Val Gly Gly Glu Gly Thr Arg Leu
    965 970 975
    Asp Gln Leu Gln Tyr Asp Val Arg Lys Gly Ser Val Val Asn Val Asn
    980 985 990
    Pro Thr Asn Thr Arg Ala His Ser Glu Thr Pro Glu Ile Arg Lys Tyr
    995 1000 1005
    Lys Lys Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala Leu Trp Gly Val
    1010 1015 1020
    Asn Leu Leu Val Gly Thr Glu Asn Gly Leu Met Leu Leu Asp Arg Ser
    1025 1030 1035 1040
    Gly Gln Gly Lys Val Tyr Gly Leu Ile Gly Arg Arg Arg Phe Gln Gln
    1045 1050 1055
    Met Asp Val Leu Glu Gly Leu Asn Leu Leu Ile Thr Ile Ser Gly Lys
    1060 1065 1070
    Arg Asn Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Arg Asn Lys Ile
    1075 1080 1085
    Leu His Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Trp Thr Thr Val
    1090 1095 1100
    Gly Asp Met Glu Gly Cys Gly His Tyr Arg Val Val Lys Tyr Glu Arg
    1105 1110 1115 1120
    Ile Lys Phe Leu Val Ile Ala Leu Lys Ser Ser Val Glu Val Tyr Ala
    1125 1130 1135
    Trp Ala Pro Lys Pro Tyr His Lys Phe Met Ala Phe Lys Ser Phe Ala
    1140 1145 1150
    Asp Leu Pro His Arg Pro Leu Leu Val Asp Leu Thr Val Glu Glu Gly
    1155 1160 1165
    Gln Arg Leu Lys Val Ile Tyr Gly Ser Ser Ala Gly Phe His Ala Val
    1170 1175 1180
    Asp Val Asp Ser Gly Asn Ser Tyr Asp Ile Tyr Ile Pro Val His Ile
    1185 1190 1195 1200
    Gln Ser Gln Ile Thr Pro His Ala Ile Ile Phe Leu Pro Asn Thr Asp
    1205 1210 1215
    Gly Met Glu Met Leu Leu Cys Tyr Glu Asp Glu Gly Val Tyr Val Asn
    1220 1225 1230
    Thr Tyr Gly Arg Ile Ile Lys Asp Val Val Leu Gln Trp Gly Glu Met
    1235 1240 1245
    Pro Thr Ser Val Ala Tyr Ile Cys Ser Asn Gln Ile Met Gly Trp Gly
    1250 1255 1260
    Glu Lys Ala Ile Glu Ile Arg Ser Val Glu Thr Gly His Leu Asp Gly
    1265 1270 1275 1280
    Val Phe Met His Lys Arg Ala Gln Arg Leu Lys Phe Leu Cys Glu Arg
    1285 1290 1295
    Asn Asp Lys Val Phe Phe Ala Ser Val Arg Ser Gly Gly Ser Ser Gln
    1300 1305 1310
    Val Tyr Phe Met Thr Leu Asn Arg Asn Arg Ile Met Asn Trp
    1315 1320 1325
    <210> SEQ ID NO 16
    <211> LENGTH: 349
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 16
    Asn Val Asn Pro Leu Tyr Val Ser Pro Ala Cys Lys Lys Pro Leu Ile
    1 5 10 15
    His Met Tyr Glu Lys Glu Phe Thr Ser Glu Ile Cys Cys Gly Ser Leu
    20 25 30
    Trp Gly Val Asn Leu Leu Leu Gly Thr Arg Ser Asn Leu Tyr Leu Met
    35 40 45
    Asp Arg Ser Gly Lys Ala Asp Ile Thr Lys Leu Ile Arg Arg Arg Pro
    50 55 60
    Phe Arg Gln Ile Gln Val Leu Glu Pro Leu Asn Leu Leu Ile Thr Ile
    65 70 75 80
    Ser Gly His Lys Asn Arg Leu Arg Val Tyr His Leu Thr Trp Leu Arg
    85 90 95
    Asn Lys Ile Leu Asn Asn Asp Pro Glu Ser Lys Arg Arg Gln Glu Glu
    100 105 110
    Met Leu Lys Thr Glu Glu Ala Cys Lys Ala Ile Asp Lys Leu Thr Gly
    115 120 125
    Cys Glu His Phe Ser Val Leu Gln His Glu Glu Thr Thr Tyr Ile Ala
    130 135 140
    Ile Ala Leu Lys Ser Ser Ile His Leu Tyr Ala Trp Ala Pro Lys Ser
    145 150 155 160
    Phe Asp Glu Ser Thr Ala Ile Lys Val Phe Pro Thr Leu Asp His Lys
    165 170 175
    Pro Val Thr Val Asp Leu Ala Ile Gly Ser Glu Lys Arg Leu Lys Ile
    180 185 190
    Phe Phe Ser Ser Ala Asp Gly Tyr His Leu Ile Asp Ala Glu Ser Glu
    195 200 205
    Val Met Ser Asp Val Thr Leu Pro Lys Asn Pro Leu Glu Ile Ile Ile
    210 215 220
    Pro Gln Asn Ile Ile Ile Leu Pro Asp Cys Leu Gly Ile Gly Met Met
    225 230 235 240
    Leu Thr Phe Asn Ala Glu Ala Leu Ser Val Glu Ala Asn Glu Gln Leu
    245 250 255
    Phe Lys Lys Ile Leu Glu Met Trp Lys Asp Ile Pro Ser Ser Ile Ala
    260 265 270
    Phe Glu Cys Thr Gln Arg Thr Thr Gly Trp Gly Gln Lys Ala Ile Glu
    275 280 285
    Val Arg Ser Leu Gln Ser Arg Val Leu Glu Ser Glu Leu Lys Arg Arg
    290 295 300
    Ser Ile Lys Lys Leu Arg Phe Leu Cys Thr Arg Gly Asp Lys Leu Phe
    305 310 315 320
    Phe Thr Ser Thr Leu Arg Asn His His Ser Arg Val Tyr Phe Met Thr
    325 330 335
    Leu Gly Lys Leu Glu Glu Leu Gln Ser Asn Tyr Asp Val
    340 345
    SEQ ID NO 17
    LENGTH: 4023
    TYPE: DNA
    ORGANISM: Homo sapiens
    SEQUENCE: 17
    ccgccatgaa ccccggcttc gatttgtccc gccggaaccc gcaggaggac ttcgagctga 60
    ttcagcgcat cggcagcggc acctacggcg acgtctacaa ggcacggaat gttaacactg 120
    gtgaattagc agcaattaaa gtaataaaat tggaaccagg agaagacttt gcagttgtgc 180
    agcaagaaat tattatgatg aaagactgta aacacccaaa tattgttgct tattttggaa 240
    gctatctcag gcgagataag ctttggattt gcatggagtt ttgtggaggt ggttctttac 300
    aggatattta tcacgtaact ggacctctgt cagaactgca aattgcatat gttagcagag 360
    aaacactgca gggattatat tatcttcaca gtaaaggaaa aatgcacaga gatataaagg 420
    gagctaacat tctattaacg gataatggtc atgtgaaatt ggctgatttt ggagtatctg 480
    cacagataac agctacaatt gccaaacgga agtctttcat tggcacacca tattggatgg 540
    ctccagaagt tgcagctgtt gagaggaagg ggggttacaa tcaactctgt gatctctggg 600
    cagtgggaat cactgccata gaacttgcag agcttcagcc tcctatgttt gacttacacc 660
    caatgagagc attatttcta atgacaaaaa gcaattttca gcctcctaaa ctaaaggata 720
    aaatgaaatg gtcaaatagt tttcatcact ttgtgaaaat ggcacttacc aaaaatccga 780
    aaaaaagacc tactgctgaa aaattattac agcatccttt tgtaacacaa catttgacac 840
    ggtctttggc aatcgagctg ttggataaag taaataatcc agatcattcc acttaccatg 900
    atttcgatga tgatgatcct gagcctcttg ttgctgtacc acatagaatt cactcaacaa 960
    gtagaaacgt gagagaagaa aaaacacgct cagagataac ctttggccaa gtgaaatttg 1020
    atccaccctt aagaaaggag acagaaccac atcatgaact tcccgacagt gatggttttt 1080
    tggacagttc agaagaaata tactacactg caagatctaa tctggatctg caactggaat 1140
    atggacaagg acaccaaggt ggttactttt taggtgcaaa caagagtctt ctcaagtctg 1200
    ttgaagaaga attgcatcag cgaggacacg tcgcacattt agaagatgat gaaggagatg 1260
    atgatgaatc taaacactca actctgaaag caaaaattcc acctcctttg ccaccaaagc 1320
    ctaagtctat cttcatacca caggaaatgc attctactga ggatgaaaat caaggaacaa 1380
    tcaagagatg tcccatgtca gggagcccag caaagccatc ccaagttcca cctagaccac 1440
    cacctcccag attaccccca cacaaacctg ttgccttagg aaatggaatg agctccttcc 1500
    agttaaatgg tgaacgagat ggctcattat gtcaacaaca gaatgaacat agaggcacaa 1560
    acctttcaag aaaagaaaag aaagatgtac caaagcctat tagtaatggt cttcctccaa 1620
    cacctaaagt gcatatgggt gcatgttttt caaaagtttt taatgggtgt cccttgaaaa 1680
    ttcactgtgc atcatcatgg ataaacccag atacaagaga tcagtacttg atatttggtg 1740
    ccgaagaagg gatttatacc ctcaatctta atgaacttca tgaaacatca atggaacagc 1800
    tattccctcg aaggtgtaca tggttgtatg taatgaacaa ttgcttgcta tcaatatctg 1860
    gtaaagcttc tcagctttat tcccataatt taccagggct ttttgattat gcaagacaaa 1920
    tgcaaaagtt acctgttgct attccagcac acaaactccc tgacagaata ctgccaagga 1980
    aattttctgt atcagcaaaa atccctgaaa ccaaatggtg ccagaagtgt tgtgttgtaa 2040
    gaaatcctta cacgggccat aaatacctat gtggagcact tcagactagc attgttctat 2100
    tagaatgggt tgaaccaatg cagaaattta tgttaattaa gcacatagat tttcctatac 2160
    catgtccact tagaatgttt gaaatgctgg tagttcctga acaggagtac cctttagttt 2220
    gtgttggtgt cagtagaggt agagacttca accaagtggt tcgatttgag acggtcaatc 2280
    caaattctac ctcttcatgg tttacagaat cagatacccc acagacaaat gttactcatg 2340
    taacccaact ggagagagat accatccttg tatgcttgga ctgttgtata aaaatagtaa 2400
    atctccaagg aagattaaaa tctagcagga aattgtcatc agaactcacc tttgatttcc 2460
    agattgaatc aatagtgtgc ctacaagaca gtgtgctagc tttctggaaa catggaatgc 2520
    aaggtagaag ttttagatct aatgaggtaa cacaagaaat ttcagatagc acaagaattt 2580
    tcaggctgct tggatctgac agggtcgtgg ttttggaaag taggccaact gataacccca 2640
    cagcaaatag caatttgtac atcctggcgg gtcatgaaaa cagttactga gaattgttgt 2700
    gctttgacag ttaactctag aaagaaagaa cactaccact gcaacattaa tggatgcttg 2760
    aagctgtaca aaagctgcag taacctgtct tcagttactt tgtaatttat tgtggcatga 2820
    gataagatgg ggaaaatttt gttttaagtg gtatggatat atttagcata ttgaaccaca 2880
    caagtgctta attcattgtt atgtaatctt tgtacatata ggcagtattt tttctgtgaa 2940
    acttcatatt gctgaagaca tacactaaga atttatgtag ataatgtact tttatgagat 3000
    gtacaagtaa gtgtcttatc tgtacagatg taaatgttga tgaaaatgca attggggtta 3060
    atattttaag aattctttag tatattcttg ggtgtggcta tattacaaaa tgggatgctg 3120
    gcaatgaaac aatacattta acactattgt atttttatta tatgtaattt agtaatatga 3180
    atataaatct tgtaactttt aaaattgtaa tggaggctgt aatcatttta taatcttttt 3240
    aattttaatg caagtacact ggtgtttata tttgcacaaa gtattgatat gtgatgtatt 3300
    aagtcacaaa agtaagctgt gacattgtct ataagcattt ggctccacaa atgtatttgg 3360
    attgttttct atgtgaagca aaccaattat aattaaccac atgttgtagt aactggtctt 3420
    tttatattta agcagaatcc tgtaagattg cttgtctttg cttaaaaaca atacctttga 3480
    acatttttga atcacagaat agcggtacca tgatagaata ctgcaattgt ggtcagaatt 3540
    acagtatgca caaagaatta attagcatta ttaaagagtc ctcactaaac atttcatatg 3600
    atcacactga agaactgtaa cattccatag agtgaagtgg ttcaaatttc tcttggaatt 3660
    tttacttttg ttggccttat tttatgatcc ttttcatatt tcttttgact tagagtatta 3720
    atacatggcc aaaataattt agttactacc tcatacaaac aatataatgg ttactacaca 3780
    tcacaggaac ttagttttgg tttaagtcat ttttgattgc ttttttccaa tggaatatgt 3840
    atataccagg ttttagcaaa atgcacactt ttggctcttt ttggtatatg ttctttatat 3900
    tttaatgtga gtatatacac taagaacaaa ctaaattgtg atttatgatc ttcatttatt 3960
    ttaatgataa tggttttaaa atatgttcct gattgtacat attgtaaaat aaacatgttt 4020
    ttt 4023
    <210> SEQ ID NO 18
    <211> LENGTH: 894
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 18
    Met Asn Pro Gly Phe Asp Leu Ser Arg Arg Asn Pro Gln Glu Asp Phe
    1 5 10 15
    Glu Leu Ile Gln Arg Ile Gly Ser Gly Thr Tyr Gly Asp Val Tyr Lys
    20 25 30
    Ala Arg Asn Val Asn Thr Gly Glu Leu Ala Ala Ile Lys Val Ile Lys
    35 40 45
    Leu Glu Pro Gly Glu Asp Phe Ala Val Val Gln Gln Glu Ile Ile Met
    50 55 60
    Met Lys Asp Cys Lys His Pro Asn Ile Val Ala Tyr Phe Gly Ser Tyr
    65 70 75 80
    Leu Arg Arg Asp Lys Leu Trp Ile Cys Met Glu Phe Cys Gly Gly Gly
    85 90 95
    Ser Leu Gln Asp Ile Tyr His Val Thr Gly Pro Leu Ser Glu Leu Gln
    100 105 110
    Ile Ala Tyr Val Ser Arg Glu Thr Leu Gln Gly Leu Tyr Tyr Leu His
    115 120 125
    Ser Lys Gly Lys Met His Arg Asp Ile Lys Gly Ala Asn Ile Leu Leu
    130 135 140
    Thr Asp Asn Gly His Val Lys Leu Ala Asp Phe Gly Val Ser Ala Gln
    145 150 155 160
    Ile Thr Ala Thr Ile Ala Lys Arg Lys Ser Phe Ile Gly Thr Pro Tyr
    165 170 175
    Trp Met Ala Pro Glu Val Ala Ala Val Glu Arg Lys Gly Gly Tyr Asn
    180 185 190
    Gln Leu Cys Asp Leu Trp Ala Val Gly Ile Thr Ala Ile Glu Leu Ala
    195 200 205
    Glu Leu Gln Pro Pro Met Phe Asp Leu His Pro Met Arg Ala Leu Phe
    210 215 220
    Leu Met Thr Lys Ser Asn Phe Gln Pro Pro Lys Leu Lys Asp Lys Met
    225 230 235 240
    Lys Trp Ser Asn Ser Phe His His Phe Val Lys Met Ala Leu Thr Lys
    245 250 255
    Asn Pro Lys Lys Arg Pro Thr Ala Glu Lys Leu Leu Gln His Pro Phe
    260 265 270
    Val Thr Gln His Leu Thr Arg Ser Leu Ala Ile Glu Leu Leu Asp Lys
    275 280 285
    Val Asn Asn Pro Asp His Ser Thr Tyr His Asp Phe Asp Asp Asp Asp
    290 295 300
    Pro Glu Pro Leu Val Ala Val Pro His Arg Ile His Ser Thr Ser Arg
    305 310 315 320
    Asn Val Arg Glu Glu Lys Thr Arg Ser Glu Ile Thr Phe Gly Gln Val
    325 330 335
    Lys Phe Asp Pro Pro Leu Arg Lys Glu Thr Glu Pro His His Glu Leu
    340 345 350
    Pro Asp Ser Asp Gly Phe Leu Asp Ser Ser Glu Glu Ile Tyr Tyr Thr
    355 360 365
    Ala Arg Ser Asn Leu Asp Leu Gln Leu Glu Tyr Gly Gln Gly His Gln
    370 375 380
    Gly Gly Tyr Phe Leu Gly Ala Asn Lys Ser Leu Leu Lys Ser Val Glu
    385 390 395 400
    Glu Glu Leu His Gln Arg Gly His Val Ala His Leu Glu Asp Asp Glu
    405 410 415
    Gly Asp Asp Asp Glu Ser Lys His Ser Thr Leu Lys Ala Lys Ile Pro
    420 425 430
    Pro Pro Leu Pro Pro Lys Pro Lys Ser Ile Phe Ile Pro Gln Glu Met
    435 440 445
    His Ser Thr Glu Asp Glu Asn Gln Gly Thr Ile Lys Arg Cys Pro Met
    450 455 460
    Ser Gly Ser Pro Ala Lys Pro Ser Gln Val Pro Pro Arg Pro Pro Pro
    465 470 475 480
    Pro Arg Leu Pro Pro His Lys Pro Val Ala Leu Gly Asn Gly Met Ser
    485 490 495
    Ser Phe Gln Leu Asn Gly Glu Arg Asp Gly Ser Leu Cys Gln Gln Gln
    500 505 510
    Asn Glu His Arg Gly Thr Asn Leu Ser Arg Lys Glu Lys Lys Asp Val
    515 520 525
    Pro Lys Pro Ile Ser Asn Gly Leu Pro Pro Thr Pro Lys Val His Met
    530 535 540
    Gly Ala Cys Phe Ser Lys Val Phe Asn Gly Cys Pro Leu Lys Ile His
    545 550 555 560
    Cys Ala Ser Ser Trp Ile Asn Pro Asp Thr Arg Asp Gln Tyr Leu Ile
    565 570 575
    Phe Gly Ala Glu Glu Gly Ile Tyr Thr Leu Asn Leu Asn Glu Leu His
    580 585 590
    Glu Thr Ser Met Glu Gln Leu Phe Pro Arg Arg Cys Thr Trp Leu Tyr
    595 600 605
    Val Met Asn Asn Cys Leu Leu Ser Ile Ser Gly Lys Ala Ser Gln Leu
    610 615 620
    Tyr Ser His Asn Leu Pro Gly Leu Phe Asp Tyr Ala Arg Gln Met Gln
    625 630 635 640
    Lys Leu Pro Val Ala Ile Pro Ala His Lys Leu Pro Asp Arg Ile Leu
    645 650 655
    Pro Arg Lys Phe Ser Val Ser Ala Lys Ile Pro Glu Thr Lys Trp Cys
    660 665 670
    Gln Lys Cys Cys Val Val Arg Asn Pro Tyr Thr Gly His Lys Tyr Leu
    675 680 685
    Cys Gly Ala Leu Gln Thr Ser Ile Val Leu Leu Glu Trp Val Glu Pro
    690 695 700
    Met Gln Lys Phe Met Leu Ile Lys His Ile Asp Phe Pro Ile Pro Cys
    705 710 715 720
    Pro Leu Arg Met Phe Glu Met Leu Val Val Pro Glu Gln Glu Tyr Pro
    725 730 735
    Leu Val Cys Val Gly Val Ser Arg Gly Arg Asp Phe Asn Gln Val Val
    740 745 750
    Arg Phe Glu Thr Val Asn Pro Asn Ser Thr Ser Ser Trp Phe Thr Glu
    755 760 765
    Ser Asp Thr Pro Gln Thr Asn Val Thr His Val Thr Gln Leu Glu Arg
    770 775 780
    Asp Thr Ile Leu Val Cys Leu Asp Cys Cys Ile Lys Ile Val Asn Leu
    785 790 795 800
    Gln Gly Arg Leu Lys Ser Ser Arg Lys Leu Ser Ser Glu Leu Thr Phe
    805 810 815
    Asp Phe Gln Ile Glu Ser Ile Val Cys Leu Gln Asp Ser Val Leu Ala
    820 825 830
    Phe Trp Lys His Gly Met Gln Gly Arg Ser Phe Arg Ser Asn Glu Val
    835 840 845
    Thr Gln Glu Ile Ser Asp Ser Thr Arg Ile Phe Arg Leu Leu Gly Ser
    850 855 860
    Asp Arg Val Val Val Leu Glu Ser Arg Pro Thr Asp Asn Pro Thr Ala
    865 870 875 880
    Asn Ser Asn Leu Tyr Ile Leu Ala Gly His Glu Asn Ser Tyr
    885 890
    <210> SEQ ID NO 19
    <211> LENGTH: 4196
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 19
    gggagggtcc ttgtggcgcc gggcggcggg gtcctgcgtg gagagtggga cgcaacgccg 60
    agaccgcgag cagaggctgc gcacagccgg atccggcact cagcgaccgg acccaaggat 120
    ccgccgggga acaagccaca ggagagcgac tcaggaacaa gtgtgggaga ggaagcggcg 180
    gcggcggcgc cgggcccggg ggtggtgaca gcaggtctga ggttgcatca taaatacaaa 240
    ggactgaagt tataaaagag aaaagagaag tttgctgcta aaatgaatct gagcaatatg 300
    gaatattttg tgccacacac aaaaaggtac tgaagattta ccccccaaaa aaaattgtca 360
    atgagaaata aagctaactg atatcaaaaa gcagagcctg ctctactggc catcatgcgt 420
    aaaggggtgc tgaaggaccc agagattgac gatctattct acaaagatga tcctgaggaa 480
    ctttttattg gtttgcatga aattggacat ggaagttttg gagcagttta ttttgctaca 540
    aatgctcaca ccaatgaggt ggtggcaatt aagaagatgt cctatagtgg gaagcagacc 600
    catgagaaat ggcaagatat tcttaaggaa gttaaatttt tacgacaatt gaagcatcct 660
    aatactattg agtacaaagg ctgttacttg aaagaacaca ctgcttggtt ggtgatggaa 720
    tattgcttag gctcagcctc tgatttatta gaagttcata aaaaaccact tcaggaagtg 780
    gagatcgctg ccattactca tggagccttg catggactag cctacctaca ttctcatgca 840
    ttgattcata gggatattaa agcaggaaat attcttctaa cagagccagg tcaggtaaaa 900
    ctagctgatt ttggatctgc ttcaatggct tctcctgcca actccttcgt gggcacacct 960
    tactggatgg ctccagaggt gatcttagct atggatgaag gacagtatga tgggaaagtt 1020
    gatatttggt cacttggcat cacttgtatt gaattggcgg aacggaagcc gccccttttc 1080
    aacatgaatg caatgagtgc cttatatcac attgcccaga atgactcccc aacgttacag 1140
    tctaatgaat ggacagactc ctttaggaga tttgttgatt actgcttgca gaaaatacct 1200
    caggaaaggc caacatcagc agaactatta aggcatgact ttgttcgacg agaccggcca 1260
    ctacgtgtcc tcattgacct catacagagg acaaaagatg cagttcgtga gctagataac 1320
    ctacagtacc gaaaaatgaa aaaaatactt ttccaagaga cacggaatgg acccttgaat 1380
    gagtcacagg aggatgagga agacagtgaa catggaacca gcctgaacag ggaaatggac 1440
    agcctgggca gcaaccattc cattccaagc atgtccgtga gcacaggcag ccagagcagc 1500
    agtgtgaaca gcatgcagga agtcatggac gagagcagtt ccgaacttgt catgatgcac 1560
    gatgacgaaa gcacaatcaa ttccagctcc tccgtcgtgc ataagaaaga tcatgtattc 1620
    acaagggatg aggcgggcca cggcgatccc aggcctgagc cgcggcctac ccagtcagtt 1680
    cagagccagg ccctccacta ccggaacaga gagcgctttg ccacgatcaa atcagcatct 1740
    ttggttacac gacagatcca tgagcatgag caggagaacg agttgcggga acagatgtca 1800
    ggttataagc ggatgcggcg ccagcaccag aagcagctga tcgccctgga gaacaagctg 1860
    aaggctgaga tggacgagca ccgcctcaag ctacagaagg aggtggagac gcatgccaac 1920
    aactcgtcca tcgagctgga gaagctggcc aagaagcaag tggctatcat agaaaaggag 1980
    gcaaaggtag ctgcagcaga tgagaagaag ttccagcaac agatcttggc ccagcagaag 2040
    aaagatttga caactttctt agaaagtcag aagaagcagt ataagatttg taaggaaaaa 2100
    ataaaagagg aaatgaatga ggaccatagc acacccaaga aagagaagca agagcggatc 2160
    tccaaacata aagagaactt gcagcacaca caggctgaag aggaagccca ccttctcact 2220
    caacagagac tgtactacga caaaaattgt cgtttcttca agcggaaaat aatgatcaag 2280
    cggcacgagg tggagcagca gaacattcgg gaggaactaa ataaaaagag gacccagaag 2340
    gagatggagc atgccatgct aatccggcac gacgagtcca cccgagagct agagtacagg 2400
    cagctgcaca cgttacagaa gctacgcatg gatctgatcc gtttacagca ccagacggaa 2460
    ctggaaaacc agctggagta caataagagg cgagaaagag aactgcacag aaagcatgtc 2520
    atgggacttc ggcaacagcc aaaaaactta aaggccatgg aaatgcaaat taaaaaacag 2580
    tttcaggaca cttgcaaagt acagaccaaa cagtataaag cactcaagaa tcaccagttg 2640
    gaagttactc caaagaatga gcacaaaaca atcttaaaga cactgaaaga tgagcagaca 2700
    agaaaacttg ccattttggc agagcagtat gaacagagta taaatgaaat gatggcctct 2760
    caagcgttac ggctagatga ggctcaagaa gcagaatgcc aggccttgag gctacagctc 2820
    cagcaggaaa tggagctgct caacgcctac cagagcaaaa tcaagatgca aacagaggca 2880
    caacatgaac gtgagctcca gaagctagag cagagagtgt ctctgcgcag agcacacctt 2940
    gagcagaaga ttgaagagga gctggctgcc cttcagaagg aacgcagcga gagaataaag 3000
    aacctattgg aaaggcaaga gcgagagatt gaaacttttg acatggagag cctcagaatg 3060
    ggatttggga atttggttac attagatttt cctaaggagg actacagatg agattaaatt 3120
    ttttgccatt tacaaaaaaa aaaaaaaaaa agaaaacaga aaaaaattca gaccctgcaa 3180
    aaccacattc cccattttaa cgggcgttgc tctcactctc tctctctctt actcttactg 3240
    acatcgtgtc ggactagtgc ctgtttattc ttactccatc aggggccccc ttcctccccc 3300
    cgtgtcaact ttcagtgctg gccaaaacct ggccgtctct tctattcaca gtacacgtca 3360
    cagtattgat gtgattcaaa atgtttcagt gaaaactttg gagacagttt taacaaaacc 3420
    aataaaccaa caacaaaaaa agtggatgta tattgcttta agcaatcact cattaccacc 3480
    aatctgtgaa agtaaagcaa aaaataataa taataaatgc caagggggag agagacacaa 3540
    tatccgcagc cttacacctt aactagctgc tgcattattt tattttattt tatttttttg 3600
    gtatttattc atcaggaata aaaaaaacaa agttttatta aagattgaaa atttgataca 3660
    ttttacagaa actaattgtg atgtacatat cagtggtgac atattattac ttttttgggg 3720
    acggggggtg ggtggggtga agagatcttg tgatttttaa gaacctgctg gcaagagttt 3780
    aacttgtctt cagcatattc tgattgtatc ataatcattt tctgctgttg cagaggatgt 3840
    gaatacactt aaggagctca cagaatccca gtagcacaaa ttgggctttg gcaaatcgtg 3900
    tattttgtgt atagaaggaa tttaaggaga ggtattactt attttcatat tgtattttaa 3960
    ctgtttctct gatcaaattt ttttacttcc tcctcctgtt cctccccacc tccctccttt 4020
    tccagttcag tatttggagt tcaacactgt ctctcaatca gatcatcttg atctttttct 4080
    ttatctccct tccccttcct aagtcccatt tcttggtcat aaatattgca ttattcacac 4140
    tttcaaactg tgtattttct tacaataaaa aatgatgaaa aaaaaaaaaa aaaaaa 4196
    <210> SEQ ID NO 20
    <211> LENGTH: 3824
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 20
    tattgaattg gcggaacgga agcctccttt atttaatatg aatgcaatga gtgccttata 60
    tcacatagcc caaaatgaat cccctacact acagtctaat gaatggtctg attattttcg 120
    caactttgta gattcttgcc tccagaaaat ccctcaagat cgacctacat cagaggaact 180
    tttaaagcac atatttgttc ttcgggagcg ccctgaaacc gtgttaatag atctcattca 240
    gaggacaaag gatgcagtaa gagagctgga caatctgcag tatcgaaaga tgaagaaact 300
    ccttttccag gaggcacata atggaccagc agtagaagca caggaagaag aagaggaaca 360
    agatcatggt gttggccgga caggaacagt taatagtgtt ggaagtaatc aatccattcc 420
    cagcatgtcc atcagtgcca gcagccaaag cagtagtgtt aacagtcttc cagatgtctc 480
    agatgacaag agtgagctag acatgatgga gggagaccac acagtgatgt ctaacagttc 540
    tgttatccat ttaaaaccag aggaagaaaa ttacagagaa gagggagatc ctagaacaag 600
    agcatcagat ccacaatctc caccccaagt atctcgtcac aaatcacact atcgtaatcg 660
    agaacacttt gctactatac ggacagcatc actggttacg aggcaaatgc aagaacatga 720
    gcaggactct gagcttagag aacaaatgtc tggctataag cgaatgaggc gacaacatca 780
    aaagcaactg atgactctgg aaaacaagct aaaggctgag atggatgaac atcgcctcag 840
    attagacaaa gatcttgaaa ctcagcgtaa caattttgct gcagaaatgg agaaacttat 900
    caagaaacac caggctgcca tggagaaaga ggctaaagtg atgtccaatg aagagaaaaa 960
    atttcagcaa catattcagg cccaacagaa gaaagaactg aatagttttc tcgagtccca 1020
    gaaaagagag tataaacttc gaaaagagca gcttaaagag gagctaaatg aaaaccagag 1080
    tacccccaaa aaagaaaaac aggagtggct ttcaaagcag aaggagaata tacagcattt 1140
    ccaagcagaa gaagaagcta accttcttcg acgtcaaaga caatacctag agctggaatg 1200
    ccgtcgcttc aagagaagaa tgttacttgg gcgtcataac ttagagcagg accttgtcag 1260
    ggaggagtta aacaaaagac agactcagaa ggacttagag catgccatgc tactccgaca 1320
    gcatgaatct atgcaagaac tggagttccg ccacctcaac acaattcaga agatgcgctg 1380
    tgagttgatc agattacagc atcaaactga gctcactaac cagctggaat ataataagcg 1440
    aagagaacga gaactaagac gaaagcatgt catggaagtt cgacaacagc ctaagagttt 1500
    gaagtctaaa gaactccaaa taaaaaagca gtttcaggat acctgcaaaa tccaaaccag 1560
    acagtacaaa gcattaagaa atcacctgct ggagactaca ccaaagagtg agcacaaagc 1620
    tgttctgaaa cggctcaagg aggaacagac ccggaaatta gctatcttgg ctgagcagta 1680
    tgatcacagc attaatgaaa tgctctccac acaagccctg cgtttggatg aagcacagga 1740
    agcagagtgc caggttttga agatgcagct gcagcaggaa ctggagctgt tgaatgcgta 1800
    tcagagcaaa atcaagatgc aagctgaggc acaacatgat cgagagcttc gcgagcttga 1860
    acagagggtc tccctccgga gggcactctt agaacaaaag attgaagaag agatgttggc 1920
    tttgcagaat gagcgcacag aacgaatacg aagcctgttg gaacgtcaag ccagagagat 1980
    tgaagctttt gactctgaaa gcatgagact aggttttagt aatatggtcc tttctaatct 2040
    ctcccctgag gcattcagcc acagctaccc gggagcttct ggttggtcac acaaccctac 2100
    tgggggtcca ggacctcact ggggtcatcc catgggtggc ccaccacaag cttggggcca 2160
    tccaatgcaa ggtggacccc agccatgggg tcacccttca gggccaatgc aaggggtacc 2220
    tcgaggtagc agtatgggag tccgcaatag cccccaggct ctgaggcgga cagcttctgg 2280
    gggacggacg gagcagggca tgagcagaag cacgagtgtc acttcacaaa tatccaatgg 2340
    gtcacacatg tcttatacat aacttaataa ttgagagtgg caattccgct ggagctgtct 2400
    gccaaaagaa actgcctaca gacatcatca cagcagcctc ctcacttggg tactacagtg 2460
    tggaagctga gtgcatatgg tatattttat tcatttttgt aaagcgttct gttttgtgtt 2520
    tactaattgg gatgtcatag tacttggctg ccgggtttgt ttgtttttgg ggaaattttg 2580
    aaaagtggag ttgatattaa aaataaatgt gtatgtgtgt acatatatat acacacacat 2640
    acacatatat tatgcatgtg gtgaaaagaa ttggctagat aggggatttt tctgaacact 2700
    gcaaaaatag aacgtagcaa aatggcttca gttatcactt ttgggtgtct gtatcctaag 2760
    aagtttctga aaagatctaa agccttttta tcccatatcc caaattctta tgagccactc 2820
    acagcaggca gcatatgttg aaataagtta ttactggtac acacctgcat tgcctcacca 2880
    gtgtatttat ttgttattaa attgatctga cttctcagcc tcatttggac taaaaaaaga 2940
    aagcagaaat ccatgaacac attgcttctc ggccttttgg ctaagatcaa gtgtagaaat 3000
    ccatgaacac taaaggactt cattgatttt ttcagagagt agaaaacaac ttagtttttc 3060
    ttttttcctg aatgcgtcat aggcttgtga gtgatttttg tccattcaat tgtgccttct 3120
    ttgtattatg ataagatggg ggtacttaag gagatcacaa gttgtgtgag gattgcatta 3180
    acaaacctat gagccttcaa tggggaagac cagaagggtg agaggggccc tgaaagttca 3240
    tatggtgggt atgtcccgca gcagagtgag gagatgaagc ttacgtgtcc tgacgttttg 3300
    ttgcttatac tgtgatatct catcctagct aagctctata atgcccaaga ccccaaacag 3360
    tacttttact ttgtttgtac aaaaacaaag acatatagcc aatacaaatc aaatgccgga 3420
    ggtgtttgat gccatatttg caaattgcca tctattgaaa ttctcgtcac actacataga 3480
    cataattgtt atctcctttt ggcttatgtg attttctgtt tacaagtaga atagccaatt 3540
    atttaaatgt ttagttgcca cagtgaacca ggagtcactg agccaatgac tttaccagct 3600
    gctgactaat cttcatcacc actgtagatt ttgctgcatg tgcaggtcct ctatttttaa 3660
    ttgctgtttt cgttgctgca gtactttaca aacttctagt tcgttgagac ttagtgacca 3720
    tttggcatca agttaacatc acacaatagg aaacaccact tccacaagtc tcaagcctca 3780
    gtgctaaagt actactgaaa aggaactagg aagtttggcc aatt 3824
    <210> SEQ ID NO 21
    <211> LENGTH: 2249
    <212> TYPE: DNA
    <213> ORGANISM: Murine sp.
    <400> SEQUENCE: 21
    gcaggatgcc atcaactaac agagcaggca gtctaaagga ccctgaaatt gcagagctct 60
    tcttcaaaga agatccggaa aagctcttca cagatctcag agaaatcggc catgggagct 120
    ttggagcagt atattttgca cgagatgtgc gtactaatga agtggtggcc atcaagaaaa 180
    tgtcttatag tggaaagcag tctactgaga aatggcagga tattattaag gaagtcaagt 240
    ttctacaaag aataaaacat cccaacagta tagaatacaa aggctgctat ttacgtgaac 300
    acacagcatg gcttgtaatg gaatattgtt taggatctgc ttcagattta ttagaagttc 360
    ataaaaagcc attacaagaa gtggaaatag cagcaattac acatggtgct ctccagggac 420
    tagcttattt acattctcat accatgatcc atagagatat caaagcagga aatatccttc 480
    tgacagaacc aggccaagtg aaacttgctg actttggatc tgcttccatg gcttcccctg 540
    ccaattcttt tgtgggaaca ccatattgga tggccccaga agtaatttta gccatggatg 600
    aaggacagta tgatggcaaa gttgatgtat ggtctcttgg aataacgtgt attgaattag 660
    ccgagaggaa gcctccttta tttaatatga atgcaatgag tgccttatat cacatagccc 720
    aaaatgaatc ccctacacta caatctaata tgaatgattc ttgcctccag aaaatccctc 780
    aagatcgccc tacatcagag gaacttttaa agcacatgtt tgttcttcga gagcgccctg 840
    aaacagtgtt aatagatctt attcaaagga caaaggatgc agtaagagag ctggacaatc 900
    tgcagtatcg aaagatgaag aaactccttt tccaggaggc acataatggg ccagcggtag 960
    aagcacagga agaagaggag gagcaagatc atggtgttgg ccgaacagga acagtgaata 1020
    gtgttggaag caatcagtct atccctagta tgtctatcag tgccagcagt caaagcagca 1080
    gtgttaatag tcttccagat gcatcagatg acaagagtga gctagacatg atggagggag 1140
    accatacagt gatgtctaac agttctgtca tccacttaaa acctgaggag gaaaattacc 1200
    aggaagaagg agatcctaga acaagagcat cagacccaca gtctccccct caggtgtctc 1260
    gtcacaagtc acattatcgt aatagagaac actttgcaac catacgaaca gcatcactgg 1320
    ttacaagaca gatgcaagaa catgagcagg actctgaact tagagaacag atgtctggtt 1380
    ataagcggat gaggcgacag catcaaaagc agctgatgac gctggaaaat aaactgaagg 1440
    cagagatgga cgaacatcgg ctcagattag acaaagatct tgaaactcag cgtaacaatt 1500
    tcgctgcaga aatggagaaa cttattaaga aacaccaagc tgctatggaa aaagaggcta 1560
    aagtgatggc caatgaggag aaaaaattcc agcaacacat tcaggctcaa cagaaaaaag 1620
    aactgaatag ctttttggag tctcaaaaaa gagaatataa acttcgcaaa gagcagctta 1680
    aggaggagct gaatgaaaac cagagcacac ctaaaaaaga aaagcaggaa tggctttcaa 1740
    agcagaagga gaatatacag cattttcagg cagaagaaga agctaatctt cttcgacgtc 1800
    aaaggcagta tctagagcta gaatgtcgtc gcttcaaaag aagaatgtta cttgggcgac 1860
    ataacttgga acaggacctt gtcagggagg agttaaacaa aaggcagact caaaaggact 1920
    tggaacatgc aatgctattg cgacagcatg aatcaatgca agaactggag tttcgccatc 1980
    tcaacactat tcagaagatg cgctgtgagt tgatcagact gcagcatcaa actgagctca 2040
    ctaaccagct agagtacaat aagagaaggg aacgggaact gaggcgaaaa catgtcatgg 2100
    aagttcgaca acaacctaag agtctgaagt ctaaagaact ccaaataaaa aagcagtttc 2160
    aggatacctg caaaattcaa accagacagt acaaagcatt aaggaatcac ctactggaga 2220
    ctacaccaaa gaatgagcac aaagcaatc 2249
    <210> SEQ ID NO 22
    <211> LENGTH: 898
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 22
    Met Arg Lys Gly Val Leu Lys Asp Pro Glu Ile Asp Asp Leu Phe Tyr
    1 5 10 15
    Lys Asp Asp Pro Glu Glu Leu Phe Ile Gly Leu His Glu Ile Gly His
    20 25 30
    Gly Ser Phe Gly Ala Val Tyr Phe Ala Thr Asn Ala His Thr Asn Glu
    35 40 45
    Val Val Ala Ile Lys Lys Met Ser Tyr Ser Gly Lys Gln Thr His Glu
    50 55 60
    Lys Trp Gln Asp Ile Leu Lys Glu Val Lys Phe Leu Arg Gln Leu Lys
    65 70 75 80
    His Pro Asn Thr Ile Glu Tyr Lys Gly Cys Tyr Leu Lys Glu His Thr
    85 90 95
    Ala Trp Leu Val Met Glu Tyr Cys Leu Gly Ser Ala Ser Asp Leu Leu
    100 105 110
    Glu Val His Lys Lys Pro Leu Gln Glu Val Glu Ile Ala Ala Ile Thr
    115 120 125
    His Gly Ala Leu His Gly Leu Ala Tyr Leu His Ser His Ala Leu Ile
    130 135 140
    His Arg Asp Ile Lys Ala Gly Asn Ile Leu Leu Thr Glu Pro Gly Gln
    145 150 155 160
    Val Lys Leu Ala Asp Phe Gly Ser Ala Ser Met Ala Ser Pro Ala Asn
    165 170 175
    Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Leu Ala
    180 185 190
    Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Ile Trp Ser Leu Gly
    195 200 205
    Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro Leu Phe Asn Met
    210 215 220
    Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn Asp Ser Pro Thr
    225 230 235 240
    Leu Gln Ser Asn Glu Trp Thr Asp Ser Phe Arg Arg Phe Val Asp Tyr
    245 250 255
    Cys Leu Gln Lys Ile Pro Gln Glu Arg Pro Thr Ser Ala Glu Leu Leu
    260 265 270
    Arg His Asp Phe Val Arg Arg Asp Arg Pro Leu Arg Val Leu Ile Asp
    275 280 285
    Leu Ile Gln Arg Thr Lys Asp Ala Val Arg Glu Leu Asp Asn Leu Gln
    290 295 300
    Tyr Arg Lys Met Lys Lys Ile Leu Phe Gln Glu Thr Arg Asn Gly Pro
    305 310 315 320
    Leu Asn Glu Ser Gln Glu Asp Glu Glu Asp Ser Glu His Gly Thr Ser
    325 330 335
    Leu Asn Arg Glu Met Asp Ser Leu Gly Ser Asn His Ser Ile Pro Ser
    340 345 350
    Met Ser Val Ser Thr Gly Ser Gln Ser Ser Ser Val Asn Ser Met Gln
    355 360 365
    Glu Val Met Asp Glu Ser Ser Ser Glu Leu Val Met Met His Asp Asp
    370 375 380
    Glu Ser Thr Ile Asn Ser Ser Ser Ser Val Val His Lys Lys Asp His
    385 390 395 400
    Val Phe Thr Arg Asp Glu Ala Gly His Gly Asp Pro Arg Pro Glu Pro
    405 410 415
    Arg Pro Thr Gln Ser Val Gln Ser Gln Ala Leu His Tyr Arg Asn Arg
    420 425 430
    Glu Arg Phe Ala Thr Ile Lys Ser Ala Ser Leu Val Thr Arg Gln Ile
    435 440 445
    His Glu His Glu Gln Glu Asn Glu Leu Arg Glu Gln Met Ser Gly Tyr
    450 455 460
    Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu Ile Ala Leu Glu Asn
    465 470 475 480
    Lys Leu Lys Ala Glu Met Asp Glu His Arg Leu Lys Leu Gln Lys Glu
    485 490 495
    Val Glu Thr His Ala Asn Asn Ser Ser Ile Glu Leu Glu Lys Leu Ala
    500 505 510
    Lys Lys Gln Val Ala Ile Ile Glu Lys Glu Ala Lys Val Ala Ala Ala
    515 520 525
    Asp Glu Lys Lys Phe Gln Gln Gln Ile Leu Ala Gln Gln Lys Lys Asp
    530 535 540
    Leu Thr Thr Phe Leu Glu Ser Gln Lys Lys Gln Tyr Lys Ile Cys Lys
    545 550 555 560
    Glu Lys Ile Lys Glu Glu Met Asn Glu Asp His Ser Thr Pro Lys Lys
    565 570 575
    Glu Lys Gln Glu Arg Ile Ser Lys His Lys Glu Asn Leu Gln His Thr
    580 585 590
    Gln Ala Glu Glu Glu Ala His Leu Leu Thr Gln Gln Arg Leu Tyr Tyr
    595 600 605
    Asp Lys Asn Cys Arg Phe Phe Lys Arg Lys Ile Met Ile Lys Arg His
    610 615 620
    Glu Val Glu Gln Gln Asn Ile Arg Glu Glu Leu Asn Lys Lys Arg Thr
    625 630 635 640
    Gln Lys Glu Met Glu His Ala Met Leu Ile Arg His Asp Glu Ser Thr
    645 650 655
    Arg Glu Leu Glu Tyr Arg Gln Leu His Thr Leu Gln Lys Leu Arg Met
    660 665 670
    Asp Leu Ile Arg Leu Gln His Gln Thr Glu Leu Glu Asn Gln Leu Glu
    675 680 685
    Tyr Asn Lys Arg Arg Glu Arg Glu Leu His Arg Lys His Val Met Gly
    690 695 700
    Leu Arg Gln Gln Pro Lys Asn Leu Lys Ala Met Glu Met Gln Ile Lys
    705 710 715 720
    Lys Gln Phe Gln Asp Thr Cys Lys Val Gln Thr Lys Gln Tyr Lys Ala
    725 730 735
    Leu Lys Asn His Gln Leu Glu Val Thr Pro Lys Asn Glu His Lys Thr
    740 745 750
    Ile Leu Lys Thr Leu Lys Asp Glu Gln Thr Arg Lys Leu Ala Ile Leu
    755 760 765
    Ala Glu Gln Tyr Glu Gln Ser Ile Asn Glu Met Met Ala Ser Gln Ala
    770 775 780
    Leu Arg Leu Asp Glu Ala Gln Glu Ala Glu Cys Gln Ala Leu Arg Leu
    785 790 795 800
    Gln Leu Gln Gln Glu Met Glu Leu Leu Asn Ala Tyr Gln Ser Lys Ile
    805 810 815
    Lys Met Gln Thr Glu Ala Gln His Glu Arg Glu Leu Gln Lys Leu Glu
    820 825 830
    Gln Arg Val Ser Leu Arg Arg Ala His Leu Glu Gln Lys Ile Glu Glu
    835 840 845
    Glu Leu Ala Ala Leu Gln Lys Glu Arg Ser Glu Arg Ile Lys Asn Leu
    850 855 860
    Leu Glu Arg Gln Glu Arg Glu Ile Glu Thr Phe Asp Met Glu Ser Leu
    865 870 875 880
    Arg Met Gly Phe Gly Asn Leu Val Thr Leu Asp Phe Pro Lys Glu Asp
    885 890 895
    Tyr Arg
    <210> SEQ ID NO 23
    <211> LENGTH: 786
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 23
    Ile Glu Leu Ala Glu Arg Lys Pro Pro Leu Phe Asn Met Asn Ala Met
    1 5 10 15
    Ser Ala Leu Tyr His Ile Ala Gln Asn Glu Ser Pro Thr Leu Gln Ser
    20 25 30
    Asn Glu Trp Ser Asp Tyr Phe Arg Asn Phe Val Asp Ser Cys Leu Gln
    35 40 45
    Lys Ile Pro Gln Asp Arg Pro Thr Ser Glu Glu Leu Leu Lys His Ile
    50 55 60
    Phe Val Leu Arg Glu Arg Pro Glu Thr Val Leu Ile Asp Leu Ile Gln
    65 70 75 80
    Arg Thr Lys Asp Ala Val Arg Glu Leu Asp Asn Leu Gln Tyr Arg Lys
    85 90 95
    Met Lys Lys Leu Leu Phe Gln Glu Ala His Asn Gly Pro Ala Val Glu
    100 105 110
    Ala Gln Glu Glu Glu Glu Glu Gln Asp His Gly Val Gly Arg Thr Gly
    115 120 125
    Thr Val Asn Ser Val Gly Ser Asn Gln Ser Ile Pro Ser Met Ser Ile
    130 135 140
    Ser Ala Ser Ser Gln Ser Ser Ser Val Asn Ser Leu Pro Asp Val Ser
    145 150 155 160
    Asp Asp Lys Ser Glu Leu Asp Met Met Glu Gly Asp His Thr Val Met
    165 170 175
    Ser Asn Ser Ser Val Ile His Leu Lys Pro Glu Glu Glu Asn Tyr Arg
    180 185 190
    Glu Glu Gly Asp Pro Arg Thr Arg Ala Ser Asp Pro Gln Ser Pro Pro
    195 200 205
    Gln Val Ser Arg His Lys Ser His Tyr Arg Asn Arg Glu His Phe Ala
    210 215 220
    Thr Ile Arg Thr Ala Ser Leu Val Thr Arg Gln Met Gln Glu His Glu
    225 230 235 240
    Gln Asp Ser Glu Leu Arg Glu Gln Met Ser Gly Tyr Lys Arg Met Arg
    245 250 255
    Arg Gln His Gln Lys Gln Leu Met Thr Leu Glu Asn Lys Leu Lys Ala
    260 265 270
    Glu Met Asp Glu His Arg Leu Arg Leu Asp Lys Asp Leu Glu Thr Gln
    275 280 285
    Arg Asn Asn Phe Ala Ala Glu Met Glu Lys Leu Ile Lys Lys His Gln
    290 295 300
    Ala Ala Met Glu Lys Glu Ala Lys Val Met Ser Asn Glu Glu Lys Lys
    305 310 315 320
    Phe Gln Gln His Ile Gln Ala Gln Gln Lys Lys Glu Leu Asn Ser Phe
    325 330 335
    Leu Glu Ser Gln Lys Arg Glu Tyr Lys Leu Arg Lys Glu Gln Leu Lys
    340 345 350
    Glu Glu Leu Asn Glu Asn Gln Ser Thr Pro Lys Lys Glu Lys Gln Glu
    355 360 365
    Trp Leu Ser Lys Gln Lys Glu Asn Ile Gln His Phe Gln Ala Glu Glu
    370 375 380
    Glu Ala Asn Leu Leu Arg Arg Gln Arg Gln Tyr Leu Glu Leu Glu Cys
    385 390 395 400
    Arg Arg Phe Lys Arg Arg Met Leu Leu Gly Arg His Asn Leu Glu Gln
    405 410 415
    Asp Leu Val Arg Glu Glu Leu Asn Lys Arg Gln Thr Gln Lys Asp Leu
    420 425 430
    Glu His Ala Met Leu Leu Arg Gln His Glu Ser Met Gln Glu Leu Glu
    435 440 445
    Phe Arg His Leu Asn Thr Ile Gln Lys Met Arg Cys Glu Leu Ile Arg
    450 455 460
    Leu Gln His Gln Thr Glu Leu Thr Asn Gln Leu Glu Tyr Asn Lys Arg
    465 470 475 480
    Arg Glu Arg Glu Leu Arg Arg Lys His Val Met Glu Val Arg Gln Gln
    485 490 495
    Pro Lys Ser Leu Lys Ser Lys Glu Leu Gln Ile Lys Lys Gln Phe Gln
    500 505 510
    Asp Thr Cys Lys Ile Gln Thr Arg Gln Tyr Lys Ala Leu Arg Asn His
    515 520 525
    Leu Leu Glu Thr Thr Pro Lys Ser Glu His Lys Ala Val Leu Lys Arg
    530 535 540
    Leu Lys Glu Glu Gln Thr Arg Lys Leu Ala Ile Leu Ala Glu Gln Tyr
    545 550 555 560
    Asp His Ser Ile Asn Glu Met Leu Ser Thr Gln Ala Leu Arg Leu Asp
    565 570 575
    Glu Ala Gln Glu Ala Glu Cys Gln Val Leu Lys Met Gln Leu Gln Gln
    580 585 590
    Glu Leu Glu Leu Leu Asn Ala Tyr Gln Ser Lys Ile Lys Met Gln Ala
    595 600 605
    Glu Ala Gln His Asp Arg Glu Leu Arg Glu Leu Glu Gln Arg Val Ser
    610 615 620
    Leu Arg Arg Ala Leu Leu Glu Gln Lys Ile Glu Glu Glu Met Leu Ala
    625 630 635 640
    Leu Gln Asn Glu Arg Thr Glu Arg Ile Arg Ser Leu Leu Glu Arg Gln
    645 650 655
    Ala Arg Glu Ile Glu Ala Phe Asp Ser Glu Ser Met Arg Leu Gly Phe
    660 665 670
    Ser Asn Met Val Leu Ser Asn Leu Ser Pro Glu Ala Phe Ser His Ser
    675 680 685
    Tyr Pro Gly Ala Ser Gly Trp Ser His Asn Pro Thr Gly Gly Pro Gly
    690 695 700
    Pro His Trp Gly His Pro Met Gly Gly Pro Pro Gln Ala Trp Gly His
    705 710 715 720
    Pro Met Gln Gly Gly Pro Gln Pro Trp Gly His Pro Ser Gly Pro Met
    725 730 735
    Gln Gly Val Pro Arg Gly Ser Ser Met Gly Val Arg Asn Ser Pro Gln
    740 745 750
    Ala Leu Arg Arg Thr Ala Ser Gly Gly Arg Thr Glu Gln Gly Met Ser
    755 760 765
    Arg Ser Thr Ser Val Thr Ser Gln Ile Ser Asn Gly Ser His Met Ser
    770 775 780
    Tyr Thr
    785
    SEQ ID NO 24
    LENGTH: 748
    <212> TYPE: PRT
    <213> ORGANISM: Murine sp.
    <400> SEQUENCE: 24
    Met Pro Ser Thr Asn Arg Ala Gly Ser Leu Lys Asp Pro Glu Ile Ala
    1 5 10 15
    Glu Leu Phe Phe Lys Glu Asp Pro Glu Lys Leu Phe Thr Asp Leu Arg
    20 25 30
    Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Arg Asp Val
    35 40 45
    Arg Thr Asn Glu Val Val Ala Ile Lys Lys Met Ser Tyr Ser Gly Lys
    50 55 60
    Gln Ser Thr Glu Lys Trp Gln Asp Ile Ile Lys Glu Val Lys Phe Leu
    65 70 75 80
    Gln Arg Ile Lys His Pro Asn Ser Ile Glu Tyr Lys Gly Cys Tyr Leu
    85 90 95
    Arg Glu His Thr Ala Trp Leu Val Met Glu Tyr Cys Leu Gly Ser Ala
    100 105 110
    Ser Asp Leu Leu Glu Val His Lys Lys Pro Leu Gln Glu Val Glu Ile
    115 120 125
    Ala Ala Ile Thr His Gly Ala Leu Gln Gly Leu Ala Tyr Leu His Ser
    130 135 140
    His Thr Met Ile His Arg Asp Ile Lys Ala Gly Asn Ile Leu Leu Thr
    145 150 155 160
    Glu Pro Gly Gln Val Lys Leu Ala Asp Phe Gly Ser Ala Ser Met Ala
    165 170 175
    Ser Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu
    180 185 190
    Val Ile Leu Ala Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Val
    195 200 205
    Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro
    210 215 220
    Leu Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn
    225 230 235 240
    Glu Ser Pro Thr Leu Gln Ser Asn Met Asn Asp Ser Cys Leu Gln Lys
    245 250 255
    Ile Pro Gln Asp Arg Pro Thr Ser Glu Glu Leu Leu Lys His Met Phe
    260 265 270
    Val Leu Arg Glu Arg Pro Glu Thr Val Leu Ile Asp Leu Ile Gln Arg
    275 280 285
    Thr Lys Asp Ala Val Arg Glu Leu Asp Asn Leu Gln Tyr Arg Lys Met
    290 295 300
    Lys Lys Leu Leu Phe Gln Glu Ala His Asn Gly Pro Ala Val Glu Ala
    305 310 315 320
    Gln Glu Glu Glu Glu Glu Gln Asp His Gly Val Gly Arg Thr Gly Thr
    325 330 335
    Val Asn Ser Val Gly Ser Asn Gln Ser Ile Pro Ser Met Ser Ile Ser
    340 345 350
    Ala Ser Ser Gln Ser Ser Ser Val Asn Ser Leu Pro Asp Ala Ser Asp
    355 360 365
    Asp Lys Ser Glu Leu Asp Met Met Glu Gly Asp His Thr Val Met Ser
    370 375 380
    Asn Ser Ser Val Ile His Leu Lys Pro Glu Glu Glu Asn Tyr Gln Glu
    385 390 395 400
    Glu Gly Asp Pro Arg Thr Arg Ala Ser Asp Pro Gln Ser Pro Pro Gln
    405 410 415
    Val Ser Arg His Lys Ser His Tyr Arg Asn Arg Glu His Phe Ala Thr
    420 425 430
    Ile Arg Thr Ala Ser Leu Val Thr Arg Gln Met Gln Glu His Glu Gln
    435 440 445
    Asp Ser Glu Leu Arg Glu Gln Met Ser Gly Tyr Lys Arg Met Arg Arg
    450 455 460
    Gln His Gln Lys Gln Leu Met Thr Leu Glu Asn Lys Leu Lys Ala Glu
    465 470 475 480
    Met Asp Glu His Arg Leu Arg Leu Asp Lys Asp Leu Glu Thr Gln Arg
    485 490 495
    Asn Asn Phe Ala Ala Glu Met Glu Lys Leu Ile Lys Lys His Gln Ala
    500 505 510
    Ala Met Glu Lys Glu Ala Lys Val Met Ala Asn Glu Glu Lys Lys Phe
    515 520 525
    Gln Gln His Ile Gln Ala Gln Gln Lys Lys Glu Leu Asn Ser Phe Leu
    530 535 540
    Glu Ser Gln Lys Arg Glu Tyr Lys Leu Arg Lys Glu Gln Leu Lys Glu
    545 550 555 560
    Glu Leu Asn Glu Asn Gln Ser Thr Pro Lys Lys Glu Lys Gln Glu Trp
    565 570 575
    Leu Ser Lys Gln Lys Glu Asn Ile Gln His Phe Gln Ala Glu Glu Glu
    580 585 590
    Ala Asn Leu Leu Arg Arg Gln Arg Gln Tyr Leu Glu Leu Glu Cys Arg
    595 600 605
    Arg Phe Lys Arg Arg Met Leu Leu Gly Arg His Asn Leu Glu Gln Asp
    610 615 620
    Leu Val Arg Glu Glu Leu Asn Lys Arg Gln Thr Gln Lys Asp Leu Glu
    625 630 635 640
    His Ala Met Leu Leu Arg Gln His Glu Ser Met Gln Glu Leu Glu Phe
    645 650 655
    Arg His Leu Asn Thr Ile Gln Lys Met Arg Cys Glu Leu Ile Arg Leu
    660 665 670
    Gln His Gln Thr Glu Leu Thr Asn Gln Leu Glu Tyr Asn Lys Arg Arg
    675 680 685
    Glu Arg Glu Leu Arg Arg Lys His Val Met Glu Val Arg Gln Gln Pro
    690 695 700
    Lys Ser Leu Lys Ser Lys Glu Leu Gln Ile Lys Lys Gln Phe Gln Asp
    705 710 715 720
    Thr Cys Lys Ile Gln Thr Arg Gln Tyr Lys Ala Leu Arg Asn His Leu
    725 730 735
    Leu Glu Thr Thr Pro Lys Asn Glu His Lys Ala Ile
    740 745
    <210> SEQ ID NO 25
    <211> LENGTH: 2795
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 25
    cgaagccaca gcccgagccc gagcccgagc ccgagccggc gccaccgcgc ccccggccat 60
    ggcttttgcc aatttccgcc gcatcctgcg cctgtctacc ttcgagaaga gaaagtcccg 120
    cgaatatgag cacgtccgcc gcgacctgga ccccaacgag gtgtgggaga tcgtgggcga 180
    gctgggcgac ggcgccttcg gcaaggttta caaggccaag aataaggaga cgggtgcttt 240
    ggctgcggcc aaagtcattg aaaccaagag tgaggaggag ctggaggact acatcgtgga 300
    gattgagatc ctggccacct gcgaccaccc ctacattgtg aagctcctgg gagcctacta 360
    tcacgacggg aagctgtgga tcatgattga gttctgtcca gggggagccg tggacgccat 420
    catgctggag ctggacagag gcctcacgga gccccagata caggtggttt gccgccagat 480
    gctagaagcc ctcaacttcc tgcacagcaa gaggatcatc caccgagatc tgaaagctgg 540
    caacgtgctg atgaccctcg agggagacat caggctggct gactttggtg tgtctgccaa 600
    gaatctgaag actctacaga aacgagattc cttcatcggc acgccttact ggatggcccc 660
    cgaggtggtc atgtgtgaga ccatgaaaga cacgccctac gactacaaag ccgacatctg 720
    gtccctgggc atcacgctga ttgagatggc ccagatcgag ccgccacacc acgagctcaa 780
    ccccatgcgg gtcctgctaa agatcgccaa gtcggaccct cccacgctgc tcacgccctc 840
    caagtggtct gtagagttcc gtgacttcct gaagatagcc ctggataaga acccagaaac 900
    ccgacccagt gccgcgcagc tgctggagca tcccttcgtc agcagcatca ccagtaacaa 960
    ggctctgcgg gagctggtgg ctgaggccaa ggccgaggtg atggaagaga tcgaagacgg 1020
    ccgggatgag ggggaagagg aggacgccgt ggatgccgcc tccaccctgg agaaccatac 1080
    tcagaactcc tctgaggtga gtccgccaag cctcaatgct gacaagcctc tcgaggagtc 1140
    accttccacc ccgctggcac ccagccagtc tcaggacagt gtgaatgagc cctgcagcca 1200
    gccctctggg gacagatccc tccaaaccac cagtccccca gtcgtggccc ctggaaatga 1260
    gaacggcctg gcagtgcctg tgcccctgcg gaagtcccga cccgtgtcaa tggatgccag 1320
    aattcaggta gcccaggaga agcaagttgc tgagcagggt ggggacctca gcccagcagc 1380
    caacagatct caaaaggcca gccagagccg gcccaacagc agcgccctgg agaccttggg 1440
    tggggagaag ctggccaatg gcagcctgga gccacctgcc caggcagctc cagggccttc 1500
    caagagggac tcggactgca gcagcctctg cacctctgag agcatggact atggtaccaa 1560
    tctctccact gacctgtcgc tgaacaaaga gatgggctct ctgtccatca aggacccgaa 1620
    actgtacaaa aaaaccctca agcggacacg caaatttgtg gtggatggtg tggaggtgag 1680
    catcaccacc tccaagatca tcagcgaaga tgagaagaag gatgaggaga tgagatttct 1740
    caggcgccag gaactccgag agcttcggct gctccagaaa gaagagcatc ggaaccagac 1800
    ccagctgagt aacaagcatg agctgcagct ggagcaaatg cataaacgtt ttgaacagga 1860
    aatcaacgcc aagaagaagt tctttgacac ggaattagag aacctggagc gtcagcaaaa 1920
    gcagcaagtg gagaagatgg agcaagacca tgccgtgcgc cgccgggagg aggccaggcg 1980
    gatccgcctg gagcaggatc gggactacac caggttccaa gagcagctca aactgatgaa 2040
    gaaagaggtg aagaacgagg tggagaagct cccccgacag cagcggaagg aaagcatgaa 2100
    gcagaagatg gaggagcaca cgcagaaaaa gcagcttctt gaccgggact ttgtagccaa 2160
    gcagaaggag gacctggagc tggccatgaa gaggctcacc accgacaaca ggcgggagat 2220
    ctgtgacaag gagcgcgagt gcctcatgaa gaagcaggag ctccttcgag accgggaagc 2280
    agccctgtgg gagatggaag agcaccagct gcaggagagg caccagctgg tgaagcagca 2340
    gctcaaagac cagtacttcc tccagcggca cgagctgctg cgcaagcatg agaaggagcg 2400
    ggagcagatg cagcgctaca accagcgcat gatagagcag ctgaaggtgc ggcagcaaca 2460
    ggaaaaggcg cggctgccca agatccagag gagtgagggc aagacgcgca tggccatgta 2520
    caagaagagc ctccacatca acggcggggg cagcgcagct gagcagcgtg agaagatcaa 2580
    gcagttctcc cagcaggagg agaagaggca gaagtcggag cggctgcagc aacagcagaa 2640
    acacgagaac cagatgcggg acatgctggc gcagtgcgag agcaacatga gcgagctgca 2700
    gcagctgcag aatgaaaagt gccacctcct ggtagagcac gaaacccaga aactgaaggc 2760
    cctggatgag agccataacc agaacctgaa ggaat 2795
    <210> SEQ ID NO 26
    <211> LENGTH: 912
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 26
    Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
    1 5 10 15
    Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp Leu Asp Pro
    20 25 30
    Asn Glu Val Trp Glu Ile Val Gly Glu Leu Gly Asp Gly Ala Phe Gly
    35 40 45
    Lys Val Tyr Lys Ala Lys Asn Lys Glu Thr Gly Ala Leu Ala Ala Ala
    50 55 60
    Lys Val Ile Glu Thr Lys Ser Glu Glu Glu Leu Glu Asp Tyr Ile Val
    65 70 75 80
    Glu Ile Glu Ile Leu Ala Thr Cys Asp His Pro Tyr Ile Val Lys Leu
    85 90 95
    Leu Gly Ala Tyr Tyr His Asp Gly Lys Leu Trp Ile Met Ile Glu Phe
    100 105 110
    Cys Pro Gly Gly Ala Val Asp Ala Ile Met Leu Glu Leu Asp Arg Gly
    115 120 125
    Leu Thr Glu Pro Gln Ile Gln Val Val Cys Arg Gln Met Leu Glu Ala
    130 135 140
    Leu Asn Phe Leu His Ser Lys Arg Ile Ile His Arg Asp Leu Lys Ala
    145 150 155 160
    Gly Asn Val Leu Met Thr Leu Glu Gly Asp Ile Arg Leu Ala Asp Phe
    165 170 175
    Gly Val Ser Ala Lys Asn Leu Lys Thr Leu Gln Lys Arg Asp Ser Phe
    180 185 190
    Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Met Cys Glu Thr
    195 200 205
    Met Lys Asp Thr Pro Tyr Asp Tyr Lys Ala Asp Ile Trp Ser Leu Gly
    210 215 220
    Ile Thr Leu Ile Glu Met Ala Gln Ile Glu Pro Pro His His Glu Leu
    225 230 235 240
    Asn Pro Met Arg Val Leu Leu Lys Ile Ala Lys Ser Asp Pro Pro Thr
    245 250 255
    Leu Leu Thr Pro Ser Lys Trp Ser Val Glu Phe Arg Asp Phe Leu Lys
    260 265 270
    Ile Ala Leu Asp Lys Asn Pro Glu Thr Arg Pro Ser Ala Ala Gln Leu
    275 280 285
    Leu Glu His Pro Phe Val Ser Ser Ile Thr Ser Asn Lys Ala Leu Arg
    290 295 300
    Glu Leu Val Ala Glu Ala Lys Ala Glu Val Met Glu Glu Ile Glu Asp
    305 310 315 320
    Gly Arg Asp Glu Gly Glu Glu Glu Asp Ala Val Asp Ala Ala Ser Thr
    325 330 335
    Leu Glu Asn His Thr Gln Asn Ser Ser Glu Val Ser Pro Pro Ser Leu
    340 345 350
    Asn Ala Asp Lys Pro Leu Glu Glu Ser Pro Ser Thr Pro Leu Ala Pro
    355 360 365
    Ser Gln Ser Gln Asp Ser Val Asn Glu Pro Cys Ser Gln Pro Ser Gly
    370 375 380
    Asp Arg Ser Leu Gln Thr Thr Ser Pro Pro Val Val Ala Pro Gly Asn
    385 390 395 400
    Glu Asn Gly Leu Ala Val Pro Val Pro Leu Arg Lys Ser Arg Pro Val
    405 410 415
    Ser Met Asp Ala Arg Ile Gln Val Ala Gln Glu Lys Gln Val Ala Glu
    420 425 430
    Gln Gly Gly Asp Leu Ser Pro Ala Ala Asn Arg Ser Gln Lys Ala Ser
    435 440 445
    Gln Ser Arg Pro Asn Ser Ser Ala Leu Glu Thr Leu Gly Gly Glu Lys
    450 455 460
    Leu Ala Asn Gly Ser Leu Glu Pro Pro Ala Gln Ala Ala Pro Gly Pro
    465 470 475 480
    Ser Lys Arg Asp Ser Asp Cys Ser Ser Leu Cys Thr Ser Glu Ser Met
    485 490 495
    Asp Tyr Gly Thr Asn Leu Ser Thr Asp Leu Ser Leu Asn Lys Glu Met
    500 505 510
    Gly Ser Leu Ser Ile Lys Asp Pro Lys Leu Tyr Lys Lys Thr Leu Lys
    515 520 525
    Arg Thr Arg Lys Phe Val Val Asp Gly Val Glu Val Ser Ile Thr Thr
    530 535 540
    Ser Lys Ile Ile Ser Glu Asp Glu Lys Lys Asp Glu Glu Met Arg Phe
    545 550 555 560
    Leu Arg Arg Gln Glu Leu Arg Glu Leu Arg Leu Leu Gln Lys Glu Glu
    565 570 575
    His Arg Asn Gln Thr Gln Leu Ser Asn Lys His Glu Leu Gln Leu Glu
    580 585 590
    Gln Met His Lys Arg Phe Glu Gln Glu Ile Asn Ala Lys Lys Lys Phe
    595 600 605
    Phe Asp Thr Glu Leu Glu Asn Leu Glu Arg Gln Gln Lys Gln Gln Val
    610 615 620
    Glu Lys Met Glu Gln Asp His Ala Val Arg Arg Arg Glu Glu Ala Arg
    625 630 635 640
    Arg Ile Arg Leu Glu Gln Asp Arg Asp Tyr Thr Arg Phe Gln Glu Gln
    645 650 655
    Leu Lys Leu Met Lys Lys Glu Val Lys Asn Glu Val Glu Lys Leu Pro
    660 665 670
    Arg Gln Gln Arg Lys Glu Ser Met Lys Gln Lys Met Glu Glu His Thr
    675 680 685
    Gln Lys Lys Gln Leu Leu Asp Arg Asp Phe Val Ala Lys Gln Lys Glu
    690 695 700
    Asp Leu Glu Leu Ala Met Lys Arg Leu Thr Thr Asp Asn Arg Arg Glu
    705 710 715 720
    Ile Cys Asp Lys Glu Arg Glu Cys Leu Met Lys Lys Gln Glu Leu Leu
    725 730 735
    Arg Asp Arg Glu Ala Ala Leu Trp Glu Met Glu Glu His Gln Leu Gln
    740 745 750
    Glu Arg His Gln Leu Val Lys Gln Gln Leu Lys Asp Gln Tyr Phe Leu
    755 760 765
    Gln Arg His Glu Leu Leu Arg Lys His Glu Lys Glu Arg Glu Gln Met
    770 775 780
    Gln Arg Tyr Asn Gln Arg Met Ile Glu Gln Leu Lys Val Arg Gln Gln
    785 790 795 800
    Gln Glu Lys Ala Arg Leu Pro Lys Ile Gln Arg Ser Glu Gly Lys Thr
    805 810 815
    Arg Met Ala Met Tyr Lys Lys Ser Leu His Ile Asn Gly Gly Gly Ser
    820 825 830
    Ala Ala Glu Gln Arg Glu Lys Ile Lys Gln Phe Ser Gln Gln Glu Glu
    835 840 845
    Lys Arg Gln Lys Ser Glu Arg Leu Gln Gln Gln Gln Lys His Glu Asn
    850 855 860
    Gln Met Arg Asp Met Leu Ala Gln Cys Glu Ser Asn Met Ser Glu Leu
    865 870 875 880
    Gln Gln Leu Gln Asn Glu Lys Cys His Leu Leu Val Glu His Glu Thr
    885 890 895
    Gln Lys Leu Lys Ala Leu Asp Glu Ser His Asn Gln Asn Leu Lys Glu
    900 905 910
    <210> SEQ ID NO 27
    <211> LENGTH: 3604
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 27
    cgttcctggg cttcccgctc cgcaggcctg cggaggactg gcccagcaag gtcccaggtc 60
    ttccctctcc ttagcgccta agagagaggc ccagtgcggg tgaggagtcg cgaggaagag 120
    gcggaaggcg ccggaaggca ccatgttccg caagaaaaag aagaaacgcc ctgagatctc 180
    agcgccacag aacttccagc accgtgtcca cacctccttc gaccccaaag aaggcaagtt 240
    tgtgggcctc cccccacaat ggcagaacat cctggacaca ctgcggcgcc ccaagcccgt 300
    ggtggaccct tcgcgaatca cacgggtgca gctccagccc atgaagacag tggtgcgggg 360
    cagcgcgatg cctgtggatg gctacatctc ggggctgctc aacgacatcc agaagttgtc 420
    agtcatcagc tccaacaccc tgcgtggccg cagccccacc agccggcggc gggcacagtc 480
    cctggggctg ctgggggatg agcactgggc caccgaccca gacatgtacc tccagagccc 540
    ccagtctgag cgcactgacc cccacggcct ctacctcagc tgcaacgggg gcacaccagc 600
    aggccacaag cagatgccgt ggcccgagcc acagagccca cgggtcctgc ccaatgggct 660
    ggctgcaaag gcacagtccc tgggccccgc cgagtttcag ggtgcctcgc agcgctgtct 720
    gcagctgggt gcctgcctgc agagctcccc accaggagcc tcgcccccca cgggcaccaa 780
    taggcatgga atgaaggctg ccaagcatgg ctctgaggag gcccggccac agtcctgcct 840
    ggtgggctca gccacaggca ggccaggtgg ggaaggcagc cctagcccta agacccggga 900
    gagcagcctg aagcgcaggc tattccgaag catgttcctg tccactgctg ccacagcccc 960
    tccaagcagc agcaagccag gccctccacc acagagcaag cccaactcct ctttccgacc 1020
    gccgcagaaa gacaaccccc caagcctggt ggccaaggcc cagtccttgc cctcggacca 1080
    gccggtgggg accttcagcc ctctgaccac ttcggatacc agcagccccc agaagtccct 1140
    ccgcacagcc ccggccacag gccagcttcc aggccggtct tccccagcgg gatccccccg 1200
    cacctggcac gcccagatca gcaccagcaa cctgtacctg ccccaggacc ccacggttgc 1260
    caagggtgcc ctggctggtg aggacacagg tgttgtgaca catgagcagt tcaaggctgc 1320
    gctcaggatg gtggtggacc agggtgaccc ccggctgctg ctggacagct acgtgaagat 1380
    tggcgagggc tccaccggca tcgtctgctt ggcccgggag aagcactcgg gccgccaggt 1440
    ggccgtcaag atgatggacc tcaggaagca gcagcgcagg gagctgctct tcaacgaggt 1500
    ggtgatcatg cgggactacc agcacttcaa cgtggtggag atgtacaaga gctacctggt 1560
    gggcgaggag ctgtgggtgc tcatggagtt cctgcaggga ggagccctca cagacatcgt 1620
    ctcccaagtc aggctgaatg aggagcagat tgccactgtg tgtgaggctg tgctgcaggc 1680
    cctggcctac ctgcatgctc agggtgtcat ccaccgggac atcaagagtg actccatcct 1740
    gctgaccctc gatggcaggg tgaagctctc ggacttcgga ttctgtgctc agatcagcaa 1800
    agacgtccct aagaggaagt ccctggtggg aaccccctac tggatggctc ctgaagtgat 1860
    ctccaggtct ttgtatgcca ctgaggtgga tatctggtct ctgggcatca tggtgattga 1920
    gatggtagat ggggagccac cgtacttcag tgactcccca gtgcaagcca tgaagaggct 1980
    ccgggacagc cccccaccca agctgaaaaa ctctcacaag gtctccccag tgctgcgaga 2040
    cttcctggag cggatgctgg tgcgggaccc ccaagagaga gccacagccc aggagctcct 2100
    agaccacccc ttcctgctgc agacagggct acctgagtgc ctggtgcccc tgatccagct 2160
    ctaccgaaag cagacctcca cctgctgagc ccaccccaag tatgcctgcc acctacgccc 2220
    acaggcaggg cacactgggc agccagcctg ccggcaggac ttgcctgcct cctcctctca 2280
    gtattctctc caaagattga aatgtgaagc cccagcccca ccctctgccc ttcagcctac 2340
    tgggccaggc cggacctgcc ccctcagtgt ctctccctcc cgagtcccca gatggagacc 2400
    cctttctaca ggatgacccc ttgatatttg cacagggata tttctaagaa acgcagaggc 2460
    cagcgttcct ggcctctgca gccaacacag tagaaaaggc tgctgtggtt ttttaaaggc 2520
    agttgtccac tagtgtccta ggccactgca gagggcagac tgctggtctc cacagatacc 2580
    tgctgttctc agctccagct tcaaacctcg agtctcgaga gggccacggg gtggttttta 2640
    tgaccggaat cccgcttcct ccctcacgtc tgatgtcctg aaggtgcagt cccacctgta 2700
    cagcccctcc ccgccaagaa ctgtgaatgg cctgctccag gccatggctg ggggcaggga 2760
    gtgaggggac aatttctgag tgaaagagaa agaatggggt cggtggtgaa ggtgctctca 2820
    ctttacagaa tggagagaac atcgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 2880
    tgtgtgtgtg tgtgtgtgtg tgtgtgtaag gggaggaaag ccaccttgac agcccaggtc 2940
    cctccaggtc acccacagcc agtttcagga aggctgcccc tctctcccac taagttctgg 3000
    cctgaaggga cctgctttct tggcctggct tccacctctc cactcctgtg tctacctggc 3060
    cagtggagtg gtccatgcta agtctaacac tcctgggagc tcaggaggct tctgagcttc 3120
    tcctgtactg tgcatcgtga gggccagaga caggaatgta aggattggca actgtgttac 3180
    ctttcaagtt tatctcaata accaggtcat cagggaccca ttgttctctt cagaacccta 3240
    tctgggagag aaggcgaacc acctccgggt ttccatcatg tcaaggtcac aggcatccat 3300
    gtgtgcaaac catctgcccc agctgcctcc acagactgct gtctccttgt cctcctcggc 3360
    cctgccccac ttcagggctg ctgtgagatg gaattccagg aaagaacttc aggtgtctgg 3420
    accctttcta tctagataat atttttagat tcttctgctc cctagtgacc tacctggggg 3480
    caaagaaatt gcaaggactt ttttttaagg gtcagagttt tcaaaacaaa agcatcttcc 3540
    ctagaaattt ttgtgaattg tttgcacttg tgcctgtttt aaattaaatt gagtgttcaa 3600
    agcc 3604
    <210> SEQ ID NO 28
    <211> LENGTH: 2050
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 28
    ggccagtggg gcgaaactgg cagctggccg gccctttaac acctacccga gggctgacac 60
    ggaccaccca tcccggggtg cccaggggga gcctcatgac gtggccccta acgggccatc 120
    agcggggggc ctggccatcc cccagtcctc ctcctcctcc tcccggcctc ccacccgagc 180
    ccgaggtgcc cccagccctg gagtgctggg accccacgcc tcagagcccc agctggcccc 240
    tccagcctgc acccccgccg cccctgctgt tcctgggccc cctggccccc gctcaccaca 300
    gcgggagcca cagcgagtat cccatgagca gttccgggct gccctgcagc tggtggtgga 360
    cccaggcgac ccccgctcct acctggacaa cttcatcaag attggcgagg gctccacggg 420
    catcgtgtgc atcgccaccg tgcgcagctc gggcaagctg gtggccgtca agaagatgga 480
    cctgcgcaag cagcagaggc gcgagctgct cttcaacgag gtggtaatca tgagggacta 540
    ccagcacgag aatgtggtgg agatgtacaa cagctacctg gtgggggacg agctctgggt 600
    ggtcatggag ttcctggaag gaggcgccct caccgacatc gtcacccaca ccaggatgaa 660
    cgaggagcag atcgcggccg tgtgccttgc agtgctgcag gccctgtcgg tgctccacgc 720
    ccagggcgtc atccaccggg acatcaagag cgactcgatc ctgctgaccc atgatggcag 780
    ggtgaagctg tcagactttg ggttctgcgc ccaggtgagc aaggaagtgc cccgaaggaa 840
    gtcgctggtc ggcacgccct actggatggc cccagagctc atctcccgcc ttccctacgg 900
    gccagaggta gacatctggt cgctggggat aatggtgatt gagatggtgg acggagagcc 960
    cccctacttc aacgagccac ccctcaaagc catgaagatg attcgggaca acctgccacc 1020
    ccgactgaag aacctgcaca aggtgtcgcc atccctgaag ggcttcctgg accgcctgct 1080
    ggtgcgagac cctgcccagc gggccacggc agccgagctg ctgaagcacc cattcctggc 1140
    caaggcaggg ccgcctgcca gcatcgtgcc cctcatgcgc cagaaccgca ccagatgagg 1200
    cccagcgccc ttcccctcaa ccaaagagcc cccccgggtc acccccgccc cactgaggcc 1260
    agtagggggc caggcctccc actcctccca gcccgggaga tgctccgcgt ggcaccaccc 1320
    tccttgctgg gggtagatga gaccctacta ctgaactcca gttttgatct cgtgactttt 1380
    agaaaaacac agggactcgt gggagcaagc gaggctccca ggacccccac cctctgggac 1440
    aggccctccc ccatgttctt ctgtctccag gaagggcagc ggccctccca tcactggaag 1500
    tctgcagtgg gggtcgctgg gggtggagag aacactaaga ggtgaacatg tatgagtgtg 1560
    tgcacgcgtg tgagtgtgca tgtgtgtgtg tgtgcaaagg tccagccacc ccgtcctcca 1620
    gcccgcaagg ggtgtctggc gccttgcctg acacccagcc ccctctcccc ctgagccatt 1680
    gtgggggtcg atcatgaatg tccgaagagt ggccttttcc cgtagccctg cgcccccttt 1740
    ctgtggctgg atggggagac aggtcagggc cccccaccct ctccagcccc tgcagcaaat 1800
    gactactgca cctggacagc ctcctctttt ctagaagtct atttatattg tcattttata 1860
    acactctagc ccctgccctt attgggggac agatggtccc tgtcctgcgg ggtggccctg 1920
    gcagaaccac tgcctgaaga accaggttcc tgcccggtca gcgcagcccc agcccgccca 1980
    cccctgcctc gagttagttt tacaattaaa acattgtctt gttttgtgaa aaaaaaaaaa 2040
    aaaaaaaaaa 2050
    <210> SEQ ID NO 29
    <211> LENGTH: 681
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 29
    Met Phe Arg Lys Lys Lys Lys Lys Arg Pro Glu Ile Ser Ala Pro Gln
    1 5 10 15
    Asn Phe Gln His Arg Val His Thr Ser Phe Asp Pro Lys Glu Gly Lys
    20 25 30
    Phe Val Gly Leu Pro Pro Gln Trp Gln Asn Ile Leu Asp Thr Leu Arg
    35 40 45
    Arg Pro Lys Pro Val Val Asp Pro Ser Arg Ile Thr Arg Val Gln Leu
    50 55 60
    Gln Pro Met Lys Thr Val Val Arg Gly Ser Ala Met Pro Val Asp Gly
    65 70 75 80
    Tyr Ile Ser Gly Leu Leu Asn Asp Ile Gln Lys Leu Ser Val Ile Ser
    85 90 95
    Ser Asn Thr Leu Arg Gly Arg Ser Pro Thr Ser Arg Arg Arg Ala Gln
    100 105 110
    Ser Leu Gly Leu Leu Gly Asp Glu His Trp Ala Thr Asp Pro Asp Met
    115 120 125
    Tyr Leu Gln Ser Pro Gln Ser Glu Arg Thr Asp Pro His Gly Leu Tyr
    130 135 140
    Leu Ser Cys Asn Gly Gly Thr Pro Ala Gly His Lys Gln Met Pro Trp
    145 150 155 160
    Pro Glu Pro Gln Ser Pro Arg Val Leu Pro Asn Gly Leu Ala Ala Lys
    165 170 175
    Ala Gln Ser Leu Gly Pro Ala Glu Phe Gln Gly Ala Ser Gln Arg Cys
    180 185 190
    Leu Gln Leu Gly Ala Cys Leu Gln Ser Ser Pro Pro Gly Ala Ser Pro
    195 200 205
    Pro Thr Gly Thr Asn Arg His Gly Met Lys Ala Ala Lys His Gly Ser
    210 215 220
    Glu Glu Ala Arg Pro Gln Ser Cys Leu Val Gly Ser Ala Thr Gly Arg
    225 230 235 240
    Pro Gly Gly Glu Gly Ser Pro Ser Pro Lys Thr Arg Glu Ser Ser Leu
    245 250 255
    Lys Arg Arg Leu Phe Arg Ser Met Phe Leu Ser Thr Ala Ala Thr Ala
    260 265 270
    Pro Pro Ser Ser Ser Lys Pro Gly Pro Pro Pro Gln Ser Lys Pro Asn
    275 280 285
    Ser Ser Phe Arg Pro Pro Gln Lys Asp Asn Pro Pro Ser Leu Val Ala
    290 295 300
    Lys Ala Gln Ser Leu Pro Ser Asp Gln Pro Val Gly Thr Phe Ser Pro
    305 310 315 320
    Leu Thr Thr Ser Asp Thr Ser Ser Pro Gln Lys Ser Leu Arg Thr Ala
    325 330 335
    Pro Ala Thr Gly Gln Leu Pro Gly Arg Ser Ser Pro Ala Gly Ser Pro
    340 345 350
    Arg Thr Trp His Ala Gln Ile Ser Thr Ser Asn Leu Tyr Leu Pro Gln
    355 360 365
    Asp Pro Thr Val Ala Lys Gly Ala Leu Ala Gly Glu Asp Thr Gly Val
    370 375 380
    Val Thr His Glu Gln Phe Lys Ala Ala Leu Arg Met Val Val Asp Gln
    385 390 395 400
    Gly Asp Pro Arg Leu Leu Leu Asp Ser Tyr Val Lys Ile Gly Glu Gly
    405 410 415
    Ser Thr Gly Ile Val Cys Leu Ala Arg Glu Lys His Ser Gly Arg Gln
    420 425 430
    Val Ala Val Lys Met Met Asp Leu Arg Lys Gln Gln Arg Arg Glu Leu
    435 440 445
    Leu Phe Asn Glu Val Val Ile Met Arg Asp Tyr Gln His Phe Asn Val
    450 455 460
    Val Glu Met Tyr Lys Ser Tyr Leu Val Gly Glu Glu Leu Trp Val Leu
    465 470 475 480
    Met Glu Phe Leu Gln Gly Gly Ala Leu Thr Asp Ile Val Ser Gln Val
    485 490 495
    Arg Leu Asn Glu Glu Gln Ile Ala Thr Val Cys Glu Ala Val Leu Gln
    500 505 510
    Ala Leu Ala Tyr Leu His Ala Gln Gly Val Ile His Arg Asp Ile Lys
    515 520 525
    Ser Asp Ser Ile Leu Leu Thr Leu Asp Gly Arg Val Lys Leu Ser Asp
    530 535 540
    Phe Gly Phe Cys Ala Gln Ile Ser Lys Asp Val Pro Lys Arg Lys Ser
    545 550 555 560
    Leu Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Ser Arg Ser
    565 570 575
    Leu Tyr Ala Thr Glu Val Asp Ile Trp Ser Leu Gly Ile Met Val Ile
    580 585 590
    Glu Met Val Asp Gly Glu Pro Pro Tyr Phe Ser Asp Ser Pro Val Gln
    595 600 605
    Ala Met Lys Arg Leu Arg Asp Ser Pro Pro Pro Lys Leu Lys Asn Ser
    610 615 620
    His Lys Val Ser Pro Val Leu Arg Asp Phe Leu Glu Arg Met Leu Val
    625 630 635 640
    Arg Asp Pro Gln Glu Arg Ala Thr Ala Gln Glu Leu Leu Asp His Pro
    645 650 655
    Phe Leu Leu Gln Thr Gly Leu Pro Glu Cys Leu Val Pro Leu Ile Gln
    660 665 670
    Leu Tyr Arg Lys Gln Thr Ser Thr Cys
    675 680
    <210> SEQ ID NO 30
    <211> LENGTH: 398
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 30
    Ala Ser Gly Ala Lys Leu Ala Ala Gly Arg Pro Phe Asn Thr Tyr Pro
    1 5 10 15
    Arg Ala Asp Thr Asp His Pro Ser Arg Gly Ala Gln Gly Glu Pro His
    20 25 30
    Asp Val Ala Pro Asn Gly Pro Ser Ala Gly Gly Leu Ala Ile Pro Gln
    35 40 45
    Ser Ser Ser Ser Ser Ser Arg Pro Pro Thr Arg Ala Arg Gly Ala Pro
    50 55 60
    Ser Pro Gly Val Leu Gly Pro His Ala Ser Glu Pro Gln Leu Ala Pro
    65 70 75 80
    Pro Ala Cys Thr Pro Ala Ala Pro Ala Val Pro Gly Pro Pro Gly Pro
    85 90 95
    Arg Ser Pro Gln Arg Glu Pro Gln Arg Val Ser His Glu Gln Phe Arg
    100 105 110
    Ala Ala Leu Gln Leu Val Val Asp Pro Gly Asp Pro Arg Ser Tyr Leu
    115 120 125
    Asp Asn Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val Cys Ile
    130 135 140
    Ala Thr Val Arg Ser Ser Gly Lys Leu Val Ala Val Lys Lys Met Asp
    145 150 155 160
    Leu Arg Lys Gln Gln Arg Arg Glu Leu Leu Phe Asn Glu Val Val Ile
    165 170 175
    Met Arg Asp Tyr Gln His Glu Asn Val Val Glu Met Tyr Asn Ser Tyr
    180 185 190
    Leu Val Gly Asp Glu Leu Trp Val Val Met Glu Phe Leu Glu Gly Gly
    195 200 205
    Ala Leu Thr Asp Ile Val Thr His Thr Arg Met Asn Glu Glu Gln Ile
    210 215 220
    Ala Ala Val Cys Leu Ala Val Leu Gln Ala Leu Ser Val Leu His Ala
    225 230 235 240
    Gln Gly Val Ile His Arg Asp Ile Lys Ser Asp Ser Ile Leu Leu Thr
    245 250 255
    His Asp Gly Arg Val Lys Leu Ser Asp Phe Gly Phe Cys Ala Gln Val
    260 265 270
    Ser Lys Glu Val Pro Arg Arg Lys Ser Leu Val Gly Thr Pro Tyr Trp
    275 280 285
    Met Ala Pro Glu Leu Ile Ser Arg Leu Pro Tyr Gly Pro Glu Val Asp
    290 295 300
    Ile Trp Ser Leu Gly Ile Met Val Ile Glu Met Val Asp Gly Glu Pro
    305 310 315 320
    Pro Tyr Phe Asn Glu Pro Pro Leu Lys Ala Met Lys Met Ile Arg Asp
    325 330 335
    Asn Leu Pro Pro Arg Leu Lys Asn Leu His Lys Val Ser Pro Ser Leu
    340 345 350
    Lys Gly Phe Leu Asp Arg Leu Leu Val Arg Asp Pro Ala Gln Arg Ala
    355 360 365
    Thr Ala Ala Glu Leu Leu Lys His Pro Phe Leu Ala Lys Ala Gly Pro
    370 375 380
    Pro Ala Ser Ile Val Pro Leu Met Arg Gln Asn Arg Thr Arg
    385 390 395
    <210> SEQ ID NO 31
    <211> LENGTH: 1001
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    murine/human SULU3
    <400> SEQUENCE: 31
    Met Pro Ser Thr Asn Arg Ala Gly Ser Leu Lys Asp Pro Glu Ile Ala
    1 5 10 15
    Glu Leu Phe Phe Lys Glu Asp Pro Glu Lys Leu Phe Thr Asp Leu Arg
    20 25 30
    Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Arg Asp Val
    35 40 45
    Arg Thr Asn Glu Val Val Ala Ile Lys Lys Met Ser Tyr Ser Gly Lys
    50 55 60
    Gln Ser Thr Glu Lys Trp Gln Asp Ile Ile Lys Glu Val Lys Phe Leu
    65 70 75 80
    Gln Arg Ile Lys His Pro Asn Ser Ile Glu Tyr Lys Gly Cys Tyr Leu
    85 90 95
    Arg Glu His Thr Ala Trp Leu Val Met Glu Tyr Cys Leu Gly Ser Ala
    100 105 110
    Ser Asp Leu Leu Glu Val His Lys Lys Pro Leu Gln Glu Val Glu Ile
    115 120 125
    Ala Ala Ile Thr His Gly Ala Leu Gln Gly Leu Ala Tyr Leu His Ser
    130 135 140
    His Thr Met Ile His Arg Asp Ile Lys Ala Gly Asn Ile Leu Leu Thr
    145 150 155 160
    Glu Pro Gly Gln Val Lys Leu Ala Asp Phe Gly Ser Ala Ser Met Ala
    165 170 175
    Ser Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu
    180 185 190
    Val Ile Leu Ala Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Val
    195 200 205
    Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro
    210 215 220
    Leu Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn
    225 230 235 240
    Glu Ser Pro Thr Leu Gln Ser Asn Glu Trp Ser Asp Tyr Phe Arg Asn
    245 250 255
    Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gln Asp Arg Pro Thr Ser
    260 265 270
    Glu Glu Leu Leu Lys His Ile Phe Val Leu Arg Glu Arg Pro Glu Thr
    275 280 285
    Val Leu Ile Asp Leu Ile Gln Arg Thr Lys Asp Ala Val Arg Glu Leu
    290 295 300
    Asp Asn Leu Gln Tyr Arg Lys Met Lys Lys Leu Leu Phe Gln Glu Ala
    305 310 315 320
    His Asn Gly Pro Ala Val Glu Ala Gln Glu Glu Glu Glu Glu Gln Asp
    325 330 335
    His Gly Val Gly Arg Thr Gly Thr Val Asn Ser Val Gly Ser Asn Gln
    340 345 350
    Ser Ile Pro Ser Met Ser Ile Ser Ala Ser Ser Gln Ser Ser Ser Val
    355 360 365
    Asn Ser Leu Pro Asp Val Ser Asp Asp Lys Ser Glu Leu Asp Met Met
    370 375 380
    Glu Gly Asp His Thr Val Met Ser Asn Ser Ser Val Ile His Leu Lys
    385 390 395 400
    Pro Glu Glu Glu Asn Tyr Arg Glu Glu Gly Asp Pro Arg Thr Arg Ala
    405 410 415
    Ser Asp Pro Gln Ser Pro Pro Gln Val Ser Arg His Lys Ser His Tyr
    420 425 430
    Arg Asn Arg Glu His Phe Ala Thr Ile Arg Thr Ala Ser Leu Val Thr
    435 440 445
    Arg Gln Met Gln Glu His Glu Gln Asp Ser Glu Leu Arg Glu Gln Met
    450 455 460
    Ser Gly Tyr Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu Met Thr
    465 470 475 480
    Leu Glu Asn Lys Leu Lys Ala Glu Met Asp Glu His Arg Leu Arg Leu
    485 490 495
    Asp Lys Asp Leu Glu Thr Gln Arg Asn Asn Phe Ala Ala Glu Met Glu
    500 505 510
    Lys Leu Ile Lys Lys His Gln Ala Ala Met Glu Lys Glu Ala Lys Val
    515 520 525
    Met Ser Asn Glu Glu Lys Lys Phe Gln Gln His Ile Gln Ala Gln Gln
    530 535 540
    Lys Lys Glu Leu Asn Ser Phe Leu Glu Ser Gln Lys Arg Glu Tyr Lys
    545 550 555 560
    Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu Asn Glu Asn Gln Ser Thr
    565 570 575
    Pro Lys Lys Glu Lys Gln Glu Trp Leu Ser Lys Gln Lys Glu Asn Ile
    580 585 590
    Gln His Phe Gln Ala Glu Glu Glu Ala Asn Leu Leu Arg Arg Gln Arg
    595 600 605
    Gln Tyr Leu Glu Leu Glu Cys Arg Arg Phe Lys Arg Arg Met Leu Leu
    610 615 620
    Gly Arg His Asn Leu Glu Gln Asp Leu Val Arg Glu Glu Leu Asn Lys
    625 630 635 640
    Arg Gln Thr Gln Lys Asp Leu Glu His Ala Met Leu Leu Arg Gln His
    645 650 655
    Glu Ser Met Gln Glu Leu Glu Phe Arg His Leu Asn Thr Ile Gln Lys
    660 665 670
    Met Arg Cys Glu Leu Ile Arg Leu Gln His Gln Thr Glu Leu Thr Asn
    675 680 685
    Gln Leu Glu Tyr Asn Lys Arg Arg Glu Arg Glu Leu Arg Arg Lys His
    690 695 700
    Val Met Glu Val Arg Gln Gln Pro Lys Ser Leu Lys Ser Lys Glu Leu
    705 710 715 720
    Gln Ile Lys Lys Gln Phe Gln Asp Thr Cys Lys Ile Gln Thr Arg Gln
    725 730 735
    Tyr Lys Ala Leu Arg Asn His Leu Leu Glu Thr Thr Pro Lys Ser Glu
    740 745 750
    His Lys Ala Val Leu Lys Arg Leu Lys Glu Glu Gln Thr Arg Lys Leu
    755 760 765
    Ala Ile Leu Ala Glu Gln Tyr Asp His Ser Ile Asn Glu Met Leu Ser
    770 775 780
    Thr Gln Ala Leu Arg Leu Asp Glu Ala Gln Glu Ala Glu Cys Gln Val
    785 790 795 800
    Leu Lys Met Gln Leu Gln Gln Glu Leu Glu Leu Leu Asn Ala Tyr Gln
    805 810 815
    Ser Lys Ile Lys Met Gln Ala Glu Ala Gln His Asp Arg Glu Leu Arg
    820 825 830
    Glu Leu Glu Gln Arg Val Ser Leu Arg Arg Ala Leu Leu Glu Gln Lys
    835 840 845
    Ile Glu Glu Glu Met Leu Ala Leu Gln Asn Glu Arg Thr Glu Arg Ile
    850 855 860
    Arg Ser Leu Leu Glu Arg Gln Ala Arg Glu Ile Glu Ala Phe Asp Ser
    865 870 875 880
    Glu Ser Met Arg Leu Gly Phe Ser Asn Met Val Leu Ser Asn Leu Ser
    885 890 895
    Pro Glu Ala Phe Ser His Ser Tyr Pro Gly Ala Ser Gly Trp Ser His
    900 905 910
    Asn Pro Thr Gly Gly Pro Gly Pro His Trp Gly His Pro Met Gly Gly
    915 920 925
    Pro Pro Gln Ala Trp Gly His Pro Met Gln Gly Gly Pro Gln Pro Trp
    930 935 940
    Gly His Pro Ser Gly Pro Met Gln Gly Val Pro Arg Gly Ser Ser Met
    945 950 955 960
    Gly Val Arg Asn Ser Pro Gln Ala Leu Arg Arg Thr Ala Ser Gly Gly
    965 970 975
    Arg Thr Glu Gln Gly Met Ser Arg Ser Thr Ser Val Thr Ser Gln Ile
    980 985 990
    Ser Asn Gly Ser His Met Ser Tyr Thr
    995 1000
    SEQ ID NO 32
    LENGTH: 25
    TYPE: DNA
    ORGANISM: Artificial Sequence
    FEATURE:
    OTHER INFORMATION: Description of Artificial Sequence: Primer
    FEATURE:
    NAME/KEY: modified_base
    LOCATION: (11)
    OTHER INFORMATION: a, t, c, g, other or unknown
    FEATURE:
    NAME/KEY: modified_base
    LOCATION: (14)
    OTHER INFORMATION: a, t, c, g, other or unknown
    FEATURE:
    NAME/KEY: modified_base
    LOCATION: (20)
    OTHER INFORMATION: a, t, c, g, other or unknown
    SEQUENCE: 32
    ctgaattcgg ngcnttyggn aargt 25
    SEQ ID NO 33
    LENGTH: 24
    TYPE: DNA
    ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (13)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (16)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <400> SEQUENCE: 33
    gctggatccy tcnggnggca tcca 24
    <210> SEQ ID NO 34
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (3)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (9)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (15)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <400> SEQUENCE: 34
    gcnttyggng argtntayga rgg 23
    <210> SEQ ID NO 35
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (13)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (16)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <400> SEQUENCE: 35
    gctggatccy tcnggnswca tcca 24
    <210> SEQ ID NO 36
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (9)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (15)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <220> FEATURE:
    <221> NAME/KEY: modified_base
    <222> LOCATION: (21)
    <223> OTHER INFORMATION: a, t, c, g, other or unknown
    <400> SEQUENCE: 36
    gagttyggng argtnttyyt ngc 23
    <210> SEQ ID NO 37
    <211> LENGTH: 6
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    motif
    <400> SEQUENCE: 37
    Gly Ala Phe Gly Lys Val
    1 5
    <210> SEQ ID NO 38
    <211> LENGTH: 5
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    motif
    <400> SEQUENCE: 38
    Trp Met Pro Pro Glu
    1 5
    <210> SEQ ID NO 39
    <211> LENGTH: 8
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    motif
    <400> SEQUENCE: 39
    Ala Phe Gly Glu Val Tyr Glu Gly
    1 5
    <210> SEQ ID NO 40
    <211> LENGTH: 5
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    motif
    <400> SEQUENCE: 40
    Trp Met Ser Pro Glu
    1 5
    <210> SEQ ID NO 41
    <211> LENGTH: 8
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    motif
    <400> SEQUENCE: 41
    Glu Phe Gly Glu Val Tyr Glu Gly
    1 5
    <210> SEQ ID NO 42
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 42
    cacagaaacg gtcagattca c 21
    <210> SEQ ID NO 43
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 43
    gatcagggtg acatcaaggg ac 22
    <210> SEQ ID NO 44
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 44
    ctcatctgta cacacttcat gg 22
    <210> SEQ ID NO 45
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 45
    gattcccaca ctgtagatgt c 21
    <210> SEQ ID NO 46
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 46
    ggccctcgac tacatccacc acat 24
    <210> SEQ ID NO 47
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 47
    caacgaaact aacacagcat aagg 24
    <210> SEQ ID NO 48
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 48
    atggcgaacg actctcccgc gaa 23
    <210> SEQ ID NO 49
    <211> LENGTH: 26
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 49
    acaccaaaat caacaagttt cacctc 26
    <210> SEQ ID NO 50
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 50
    agttacaagg aattccaagt tct 23
    <210> SEQ ID NO 51
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 51
    atgaagagga agaaatcaaa ctg 23
    <210> SEQ ID NO 52
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 52
    agatggactg tactgggagg 20
    <210> SEQ ID NO 53
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 53
    actttgtgca gctctgtggg 20
    <210> SEQ ID NO 54
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 54
    aaggttatgg atgtcacagg g 21
    <210> SEQ ID NO 55
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 55
    ctcacaaggt tgccaacagg 20
    <210> SEQ ID NO 56
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 56
    agtccccacc agaaggttta c 21
    <210> SEQ ID NO 57
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 57
    tcaggggtca gaggtcacg 19
    <210> SEQ ID NO 58
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 58
    cccaaaccct accacaaatt c 21
    <210> SEQ ID NO 59
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 59
    cccccgggaa acgatgacca 20
    <210> SEQ ID NO 60
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 60
    agccgctgcc cctcctctac tgt 23
    <210> SEQ ID NO 61
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 61
    accgcaacat cgccacctac tac 23
    <210> SEQ ID NO 62
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 62
    ctcgacgtcg tggaccacc 19
    <210> SEQ ID NO 63
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 63
    caatgttaac ccactctatg tctc 24
    <210> SEQ ID NO 64
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 64
    agtttgccga tgtttttctt ttc 23
    <210> SEQ ID NO 65
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 65
    ccgccatgaa ccccggctt 19
    <210> SEQ ID NO 66
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 66
    cgattgccaa agaccgtgtc a 21
    <210> SEQ ID NO 67
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 67
    agaagttgca gctgttgaga gga 23
    <210> SEQ ID NO 68
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 68
    tatggcccgt gtaaggattt c 21
    <210> SEQ ID NO 69
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 69
    gtgccagaag tgttgtgttg taa 23
    <210> SEQ ID NO 70
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 70
    tattgaattg gcggaacgga ag 22
    <210> SEQ ID NO 71
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 71
    ttgttttgtg ctcattcttt ggag 24
    <210> SEQ ID NO 72
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 72
    gattgctttg tgctcattct ttgg 24
    <210> SEQ ID NO 73
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 73
    ttgttctaag agtgccctcc g 21
    <210> SEQ ID NO 74
    <211> LENGTH: 19
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 74
    aagaccatgc cgtgcgccg 19
    <210> SEQ ID NO 75
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 75
    attccttcag gttctggtta tgg 23
    <210> SEQ ID NO 76
    <400> SEQUENCE: 76
    000
    <210> SEQ ID NO 77
    <400> SEQUENCE: 77
    000
    <210> SEQ ID NO 78
    <211> LENGTH: 12
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    peptide 540A
    <400> SEQUENCE: 78
    His Gly Asp Pro Arg Pro Glu Pro Arg Pro Thr Gln
    1 5 10
    <210> SEQ ID NO 79
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Syntbetic
    peptide 539A
    <400> SEQUENCE: 79
    Cys Leu Asp Phe Pro Lys Glu Asp Tyr Arg
    1 5 10
    <210> SEQ ID NO 80
    <400> SEQUENCE: 80
    000
    <210> SEQ ID NO 81
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    peptide 542A
    <400> SEQUENCE: 81
    Pro Ser Thr Asn Arg Ala Gly Ser Leu Lys Asp Pro Glu Cys
    1 5 10
    <210> SEQ ID NO 82
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    peptide 554A
    <400> SEQUENCE: 82
    Cys Leu Val Pro Leu Ile Gln Leu Tyr Arg Lys Gln Thr Ser Thr
    1 5 10 15
    <210> SEQ ID NO 83
    <400> SEQUENCE: 83
    000
    <210> SEQ ID NO 84
    <211> LENGTH: 426
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 84
    Met Ala His Leu Arg Gly Phe Ala Asn Gln His Ser Arg Val Asp Pro
    1 5 10 15
    Glu Glu Leu Phe Thr Lys Leu Asp Arg Ile Gly Lys Gly Ser Phe Gly
    20 25 30
    Glu Val Tyr Lys Gly Ile Asp Asn His Thr Lys Glu Val Val Ala Ile
    35 40 45
    Lys Ile Ile Asp Leu Glu Glu Ala Glu Asp Glu Ile Glu Asp Ile Gln
    50 55 60
    Gln Glu Ile Thr Val Leu Ser Gln Cys Asp Ser Pro Tyr Ile Thr Arg
    65 70 75 80
    Tyr Phe Gly Ser Tyr Leu Lys Ser Thr Lys Leu Trp Ile Ile Met Glu
    85 90 95
    Tyr Leu Gly Gly Gly Ser Ala Leu Asp Leu Leu Lys Pro Gly Pro Leu
    100 105 110
    Glu Glu Thr Tyr Ile Ala Thr Ile Leu Arg Glu Ile Leu Lys Gly Leu
    115 120 125
    Asp Tyr Leu His Ser Glu Arg Lys Ile His Arg Asp Ile Lys Ala Ala
    130 135 140
    Asn Val Leu Leu Ser Glu Gln Gly Asp Val Lys Leu Ala Asp Phe Gly
    145 150 155 160
    Val Ala Gly Gln Leu Thr Asp Thr Gln Ile Lys Arg Asn Thr Phe Val
    165 170 175
    Gly Thr Pro Phe Trp Met Ala Pro Glu Val Ile Lys Gln Ser Ala Tyr
    180 185 190
    Asp Phe Lys Ala Asp Ile Trp Ser Leu Gly Ile Thr Ala Ile Glu Leu
    195 200 205
    Ala Lys Gly Glu Pro Pro Asn Ser Asp Leu His Pro Met Arg Val Leu
    210 215 220
    Phe Leu Ile Pro Lys Asn Ser Pro Pro Thr Leu Glu Gly Gln His Ser
    225 230 235 240
    Lys Pro Phe Lys Glu Phe Val Glu Ala Cys Leu Asn Lys Asp Pro Arg
    245 250 255
    Phe Arg Pro Thr Ala Lys Glu Leu Leu Lys His Lys Phe Ile Thr Arg
    260 265 270
    Tyr Thr Lys Lys Thr Ser Phe Leu Thr Glu Leu Ile Asp Arg Tyr Lys
    275 280 285
    Arg Trp Lys Ser Glu Gly His Gly Glu Glu Ser Ser Ser Glu Asp Ser
    290 295 300
    Asp Ile Asp Gly Glu Ala Glu Asp Gly Glu Gln Gly Pro Ile Trp Thr
    305 310 315 320
    Phe Pro Pro Thr Ile Arg Pro Ser Pro His Ser Lys Leu His Lys Gly
    325 330 335
    Thr Ala Leu His Ser Ser Gln Lys Pro Ala Glu Pro Val Lys Arg Gln
    340 345 350
    Pro Arg Ser Gln Cys Leu Ser Thr Leu Val Arg Pro Val Phe Gly Glu
    355 360 365
    Leu Lys Glu Lys His Lys Gln Ser Gly Gly Ser Val Gly Ala Leu Glu
    370 375 380
    Glu Leu Glu Asn Ala Phe Ser Leu Ala Glu Glu Ser Cys Pro Gly Ile
    385 390 395 400
    Ser Asp Lys Leu Met Val His Leu Val Glu Arg Val Gln Arg Phe Ser
    405 410 415
    His Asn Arg Asn His Leu Thr Ser Thr Arg
    420 425
    <210> SEQ ID NO 85
    <211> LENGTH: 431
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 85
    Met Ala His Ser Pro Val Gln Ser Gly Leu Pro Gly Met Gln Asn Leu
    1 5 10 15
    Lys Ala Asp Pro Glu Glu Leu Phe Thr Lys Leu Glu Lys Ile Gly Lys
    20 25 30
    Gly Ser Phe Gly Glu Val Phe Lys Gly Ile Asp Asn Arg Thr Gln Lys
    35 40 45
    Val Val Ala Ile Lys Ile Ile Asp Leu Glu Glu Ala Glu Asp Glu Ile
    50 55 60
    Glu Asp Ile Gln Gln Glu Ile Thr Val Leu Ser Gln Cys Asp Ser Pro
    65 70 75 80
    Tyr Val Thr Lys Tyr Tyr Gly Ser Tyr Leu Lys Asp Thr Lys Leu Trp
    85 90 95
    Ile Ile Met Glu Tyr Leu Gly Gly Gly Ser Ala Leu Asp Leu Leu Glu
    100 105 110
    Pro Gly Pro Leu Asp Glu Thr Gln Ile Ala Thr Ile Leu Arg Glu Ile
    115 120 125
    Leu Lys Gly Leu Asp Tyr Leu His Ser Glu Lys Lys Ile His Arg Asp
    130 135 140
    Ile Lys Ala Ala Asn Val Leu Leu Ser Glu His Gly Glu Val Lys Leu
    145 150 155 160
    Ala Asp Phe Gly Val Ala Gly Gln Leu Thr Asp Thr Gln Ile Lys Arg
    165 170 175
    Asn Thr Phe Val Gly Thr Pro Phe Trp Met Ala Pro Glu Val Ile Lys
    180 185 190
    Gln Ser Ala Tyr Asp Ser Lys Ala Asp Ile Trp Ser Leu Gly Ile Thr
    195 200 205
    Ala Ile Glu Leu Ala Arg Gly Glu Pro Pro His Ser Glu Leu His Pro
    210 215 220
    Met Lys Val Leu Phe Leu Ile Pro Lys Asn Asn Pro Pro Thr Leu Glu
    225 230 235 240
    Gly Asn Tyr Ser Lys Pro Leu Lys Glu Phe Val Glu Ala Cys Leu Asn
    245 250 255
    Lys Glu Pro Ser Phe Arg Pro Thr Ala Lys Glu Leu Leu Lys His Lys
    260 265 270
    Phe Ile Leu Arg Asn Ala Lys Lys Thr Ser Tyr Leu Thr Glu Leu Ile
    275 280 285
    Asp Arg Tyr Lys Arg Trp Lys Ala Glu Gln Ser His Asp Asp Ser Ser
    290 295 300
    Ser Glu Asp Ser Asp Ala Glu Thr Asp Gly Gln Ala Ser Gly Gly Ser
    305 310 315 320
    Asp Ser Gly Asp Trp Ile Phe Thr Ile Arg Glu Lys Asp Pro Lys Asn
    325 330 335
    Leu Glu Asn Gly Ala Leu Gln Pro Ser Asp Leu Asp Arg Asn Lys Met
    340 345 350
    Lys Asp Ile Pro Lys Arg Pro Phe Ser Gln Cys Leu Ser Thr Ile Ile
    355 360 365
    Ser Pro Leu Phe Ala Glu Leu Lys Glu Lys Ser Gln Ala Cys Gly Gly
    370 375 380
    Asn Leu Gly Ser Ile Glu Glu Leu Arg Gly Ala Ile Tyr Leu Ala Glu
    385 390 395 400
    Glu Ala Cys Pro Gly Ile Ser Asp Thr Met Val Ala Gln Leu Val Gln
    405 410 415
    Arg Leu Gln Arg Tyr Ser Leu Ser Gly Gly Gly Thr Ser Ser His
    420 425 430
    <210> SEQ ID NO 86
    <211> LENGTH: 443
    <212> TYPE: PRT
    <213> ORGANISM: Caenorhabditis elegans
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (389)..(390)
    <223> OTHER INFORMATION: Any amino acid
    <400> SEQUENCE: 86
    Met Thr Thr Thr Ser Ser Asp Glu Leu Pro Arg Gln Ala Asp Asp Asp
    1 5 10 15
    Ser Met Lys Trp Asp Arg Ile Tyr Ile Gln Lys Leu Asp Pro Glu Val
    20 25 30
    Ile Phe Thr Lys Gln Glu Arg Ile Gly Arg Gly Ser Phe Gly Glu Val
    35 40 45
    Tyr Lys Gly Ile Asp Asn Arg Thr Gly Arg Val Val Ala Ile Lys Ile
    50 55 60
    Ile Asp Leu Glu Gln Ala Glu Asp Glu Ile Glu Asp Ile Gln Gln Glu
    65 70 75 80
    Ile Gln Val Leu Ser Gln Cys Asp Ser Gln Tyr Val Thr Lys Tyr Phe
    85 90 95
    Gly Ser Phe Leu Lys Gly Ser Lys Leu Trp Ile Ile Met Glu Tyr Leu
    100 105 110
    Gly Gly Gly Ser Ala Leu Asp Leu Thr Lys Ser Gly Lys Leu Asp Glu
    115 120 125
    Ser His Ile Ala Val Ile Leu Arg Glu Ile Leu Lys Gly Leu Glu Tyr
    130 135 140
    Leu His Ser Glu Arg Lys Ile His Arg Asp Ile Lys Ala Ala Asn Val
    145 150 155 160
    Leu Val Ser Glu His Gly Asp Val Lys Val Ala Asp Phe Gly Val Ala
    165 170 175
    Gly Gln Leu Thr Glu Thr Val Lys Lys Arg Ile Thr Phe Val Gly Ser
    180 185 190
    Pro Phe Trp Met Ala Pro Glu Leu Ile Lys Gln Ser Ser Tyr Asp Tyr
    195 200 205
    Lys Ala Asp Ile Trp Ser Leu Gly Ile Thr Ala Ile Glu Leu Ala Asn
    210 215 220
    Gly Glu Pro Pro His Ser Asp Leu His Pro Met Arg Val Leu Phe Leu
    225 230 235 240
    Ile Pro Lys Asn Pro Pro Pro Val Leu Gln Gly Ser Gln Trp Ser Lys
    245 250 255
    Pro Phe Lys Glu Phe Val Glu Met Cys Leu Asn Lys Asp Pro Glu Asn
    260 265 270
    Arg Pro Ser Ala Ser Thr Leu Leu Lys His Gln Phe Ile Lys Arg Ala
    275 280 285
    Lys Lys Asn Ser Ile Leu Val Asp Leu Ile Glu Arg Ala Ala Glu Tyr
    290 295 300
    Arg Leu Arg Thr Gly Val Ser Ser Asp Ser Asp Leu Asp Glu Asp Ser
    305 310 315 320
    Asp Gly Gly Gly Gly Thr Ser Lys Trp Asp Tyr Pro Thr Val Arg Gly
    325 330 335
    Pro Arg Val Ser Ala Asp Asp Asp Gly Thr Val Arg Gln Arg Thr Asp
    340 345 350
    Arg Pro Arg Ala Gln Val Asp Arg Arg Ser Pro Ser Gly Ser Pro Gly
    355 360 365
    Gly Thr Ile Val Arg Gly Ser Pro Gln Val Ala Ala Val Ala Glu Gln
    370 375 380
    Leu Arg Asn Ser Xaa Xaa Ala Leu Asp Gln Leu Arg His Val Phe Arg
    385 390 395 400
    Asp Val Glu Asp Ser Cys Pro Gly Ile Cys Asn Glu Leu Ile Glu Glu
    405 410 415
    Leu Met Gln Arg Ile Ala Val Pro Gln Val Ser Gln Ser Asp Leu Asp
    420 425 430
    Ala Ala Ile Arg Arg Leu Thr Thr Pro Pro Ser
    435 440
    <210> SEQ ID NO 87
    <211> LENGTH: 275
    <212> TYPE: PRT
    <213> ORGANISM: Saccharomyces pombe
    <400> SEQUENCE: 87
    Leu Leu Tyr Arg Asn Phe Val Lys Ile Gly Gln Gly Ala Ser Gly Asp
    1 5 10 15
    Val Tyr Ser Ala Arg Gln Val Gly Thr Asn Leu Ser Val Ala Ile Lys
    20 25 30
    Lys Met Asn Ile Asn Gln Gln Pro Lys Lys Glu Phe Ile Val Asn Glu
    35 40 45
    Ile Leu Val Met Lys Ser His His His Lys Asn Ile Val Asn Phe Ile
    50 55 60
    Asp Thr Phe Phe Tyr Lys Ser Glu Leu Trp Met Val Met Glu Tyr Met
    65 70 75 80
    Arg Gly Gly Ser Leu Thr Glu Val Val Thr Asn Asn Thr Leu Ser Glu
    85 90 95
    Gly Gln Ile Ala Ala Ile Cys Lys Glu Thr Leu Glu Gly Leu Gln His
    100 105 110
    Leu His Glu Asn Gly Ile Val His Arg Asp Ile Lys Ser Asp Asn Ile
    115 120 125
    Leu Leu Ser Leu Gln Gly Asp Ile Lys Leu Thr Asp Phe Gly Phe Cys
    130 135 140
    Ala Gln Ile Asp Ser Asn Met Thr Lys Arg Thr Thr Met Val Gly Thr
    145 150 155 160
    Pro Tyr Trp Met Ala Pro Glu Val Val Thr Arg Lys Glu Tyr Gly Phe
    165 170 175
    Lys Val Asp Val Trp Ser Leu Gly Ile Met Ala Ile Glu Met Val Glu
    180 185 190
    Gly Glu Pro Pro Tyr Leu Asn Glu Asn Pro Leu Arg Ala Leu Tyr Leu
    195 200 205
    Ile Ala Thr Ile Gly Thr Pro Lys Ile Ser Arg Pro Glu Leu Leu Ser
    210 215 220
    Ser Val Phe His Asp Phe Leu Ser Lys Ser Leu Thr Val Asn Pro Lys
    225 230 235 240
    Gln Arg Pro Ser Ser Gly Glu Leu Leu Arg His Pro Phe Leu Lys Gln
    245 250 255
    Ala Val Pro Val Ser Ser Leu Ile Pro Leu Ile Lys Ser Ile His His
    260 265 270
    Ser Gly Lys
    275
    <210> SEQ ID NO 88
    <211> LENGTH: 1109
    <212> TYPE: PRT
    <213> ORGANISM: Caenorhabditis elegans
    <400> SEQUENCE: 88
    Met Ser Ser Ser Gly Leu Asp Glu Ile Asp Leu Asn Ser Leu Arg Asp
    1 5 10 15
    Pro Ala Gly Ile Phe Glu Leu Ile Glu Val Val Gly Asn Gly Thr Tyr
    20 25 30
    Gly Gln Val Tyr Lys Gly Arg His Val Lys Thr Ala Gln Leu Ala Ala
    35 40 45
    Ile Lys Ile Met Asn Ile Asn Glu Asp Glu Glu Asp Glu Ile Lys Leu
    50 55 60
    Glu Ile Asn Met Leu Lys Lys His Ser His His Arg Asn Val Ala Thr
    65 70 75 80
    Tyr Tyr Gly Ala Phe Ile Lys Lys Leu Pro Ser Ser Thr Gly Lys His
    85 90 95
    Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ser Gly Ser Ile Thr
    100 105 110
    Asp Leu Val Lys Asn Thr Lys Gly Gly Ser Leu Lys Glu Glu Trp Ile
    115 120 125
    Ala Tyr Ile Cys Arg Glu Ile Leu Arg Gly Leu Tyr His Leu His Gln
    130 135 140
    Ser Lys Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu Leu Thr
    145 150 155 160
    Asp Ser Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln Leu
    165 170 175
    Asp Lys Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp
    180 185 190
    Met Ala Pro Glu Val Ile Ala Cys Asp Glu Ser Pro Glu Ala Thr Tyr
    195 200 205
    Asp Ser Arg Ser Asp Leu Trp Ser Leu Gly Ile Thr Ala Leu Glu Met
    210 215 220
    Ala Glu Gly His Pro Pro Leu Cys Asp Met His Pro Met Arg Ala Leu
    225 230 235 240
    Phe Leu Ile Pro Arg Asn Pro Pro Pro Lys Leu Lys Arg Asn Lys Lys
    245 250 255
    Trp Thr Lys Lys Phe Glu Thr Phe Ile Glu Thr Val Leu Val Lys Asp
    260 265 270
    Tyr His Gln Arg Pro Tyr Thr Gly Ala Leu Leu Arg His Pro Phe Ile
    275 280 285
    Lys Glu Gln Pro His Glu Gln Thr Ile Arg His Ser Ile Lys Glu His
    290 295 300
    Ile Asp Arg Asn Arg Arg Val Lys Lys Asp Asp Ala Asp Tyr Glu Tyr
    305 310 315 320
    Ser Gly Ser Glu Asp Asp Glu Pro Ser Pro Asn Asn Arg Asp Asp Ser
    325 330 335
    Glu Ser Ser Ser Met Ile Pro Met Asp Asn Thr Leu Arg Lys Gly Phe
    340 345 350
    Gln Lys Leu Gln Glu Ser Ser Arg Gly Phe Ala Glu Pro Gly Ala Gln
    355 360 365
    Gln Leu Arg Arg Leu Pro Gln Gln Pro Ala Pro Ala Pro Phe Gln Tyr
    370 375 380
    Gln Gln Ser Arg Tyr Val Glu Pro Arg Arg Glu Ser Ser Glu Val Lys
    385 390 395 400
    Leu Arg Ala Val Ser Ser Arg Gly Ala Ala Asp Gly Pro Arg His Ser
    405 410 415
    Pro Ala Ser Arg Pro Arg Pro Arg Ser Pro Gln Gln Ser His Pro Ala
    420 425 430
    Ala Pro His Leu Ala Asp Leu Ala Asn Tyr Glu Lys Arg Arg Arg Ser
    435 440 445
    Glu Arg Glu Glu Arg Arg Glu Arg Glu Arg Gln Ala His His Ala Met
    450 455 460
    Pro Ile Ala Arg Val Ser Ala Ser Val Pro Ala Pro Gln Gln Ser Arg
    465 470 475 480
    Lys Met Ser Glu Pro Leu Leu Ile Thr His Val Lys Pro Glu Asp Leu
    485 490 495
    Asp Val Leu Ala Ser Glu Leu Ser Lys Met Gly Gly His His Asn Gly
    500 505 510
    Arg Ser Arg Glu Glu Ser Met Ser Pro Pro Pro Pro Ala Pro Pro Pro
    515 520 525
    Arg Glu Ala Ser Ile Ser Ser Ile Thr Asp Thr Ile Asp Val Gly Glu
    530 535 540
    Leu Asp Asn Gly Ala Asp Ala Glu Trp Asp Asp Leu Lys Asp Ile Met
    545 550 555 560
    Met Asn Gly Glu Gly Thr Leu Arg Gly Pro Asn Lys Pro Leu Pro Pro
    565 570 575
    Thr Pro Thr Asp Gly Glu Asn Thr Leu Val Ser Asp Val Arg Arg Asn
    580 585 590
    Gly Asn Gly Asn Ser Gly His Gly Ala Tyr Lys Gly Lys Lys Ile Pro
    595 600 605
    Glu Ile Arg Pro Gly Ile Ile Ser Leu Asp Asp Asp Asp Ser Asp Ser
    610 615 620
    Asp Asn Glu Glu Gly Asn Glu Pro Leu Met Phe Lys Pro Ile Val Arg
    625 630 635 640
    Cys Pro Phe Ser Ile Phe Phe Trp Phe Leu Ser Ala Asn Val Ile His
    645 650 655
    Ser Val Asp Gly Ser Ile Pro Leu Val Lys His Leu Ile Trp Phe Gln
    660 665 670
    Asn Ala Ser Ser Ser Arg Gly Ala Leu Pro Asp Leu Leu Pro Lys Ser
    675 680 685
    Pro Asp Leu Arg Arg Gln Ile Asn Asp Gln Thr Arg Gln Met Ser Asp
    690 695 700
    Asp Arg Ala Asp Glu Gln Pro Asn Gly Phe Gln Asn Ser Asp Ser Arg
    705 710 715 720
    Ser Ser Ile Gln His Ser Phe Ser Asn Arg Asp Arg Glu Lys Ser Phe
    725 730 735
    Val Gly Tyr Phe Gly Gly Gly Ala Gly Ala Gly Gly Gly Thr Val Asn
    740 745 750
    Arg Pro Gly Arg Pro Gln Asp Ile Asn Gln Val Gln Val Asn Val Thr
    755 760 765
    Pro Asn Ser Asn Gly Thr Pro Ala Glu Asn Asp Ala Pro Glu Ile Arg
    770 775 780
    Lys Tyr Lys Lys Lys Phe Ser Gly Glu Ile Leu Cys Ala Ala Leu Trp
    785 790 795 800
    Gly Val Asn Leu Leu Ile Gly Thr Asp Ser Gly Leu Met Leu Leu Asp
    805 810 815
    Arg Ser Gly Gln Gly Lys Val Tyr Pro Leu Ile Ser Arg Arg Arg Phe
    820 825 830
    Asp Gln Met Thr Val Leu Glu Gly Gln Asn Ile Leu Ala Thr Ile Ser
    835 840 845
    Gly Arg Lys Arg Arg Ile Arg Val Tyr Tyr Leu Ser Trp Leu Arg Gln
    850 855 860
    Lys Ile Leu Arg Thr Glu Gly Ala Gly Ser Ala Asn Thr Thr Glu Lys
    865 870 875 880
    Arg Asn Gly Trp Val Asn Val Gly Asp Leu Gln Gly Ala Ile His Phe
    885 890 895
    Lys Ile Val Arg Tyr Glu Arg Ile Lys Phe Leu Val Val Gly Leu Glu
    900 905 910
    Ser Ser Ile Glu Ile Tyr Ala Trp Ala Pro Lys Pro Tyr His Lys Phe
    915 920 925
    Met Ser Phe Lys Ser Phe Gly Ser Leu Ser His Val Pro Leu Ile Val
    930 935 940
    Asp Leu Thr Val Glu Asp Asn Ala Arg Leu Lys Val Leu Tyr Gly Ser
    945 950 955 960
    Thr Gly Gly Phe His Ala Ile Asp Leu Asp Ser Ala Ala Val Tyr Asp
    965 970 975
    Ile Tyr Thr Pro Ala Gln Ser Gly Gln Thr Thr Thr Pro His Cys Ile
    980 985 990
    Val Val Leu Pro Asn Ser Asn Gly Met Gln Leu Leu Leu Cys Tyr Asp
    995 1000 1005
    Asn Glu Gly Val Tyr Val Asn Thr Tyr Gly Arg Met Thr Lys Asn Val
    1010 1015 1020
    Val Leu Gln Trp Gly Glu Met Pro Ser Ser Val Ala Tyr Ile Ser Thr
    1025 1030 1035 1040
    Gly Gln Ile Met Gly Trp Gly Asn Lys Ala Ile Glu Ile Arg Ser Val
    1045 1050 1055
    Asp Thr Gly His Leu Asp Gly Val Phe Met His Lys Lys Ala Gln Lys
    1060 1065 1070
    Leu Lys Phe Leu Cys Glu Arg Asn Asp Lys Val Phe Phe Ser Ser Ala
    1075 1080 1085
    Lys Gly Gly Gly Ser Cys Gln Ile Tyr Phe Met Thr Leu Asn Lys Pro
    1090 1095 1100
    Gly Leu Thr Asn Trp
    1105
    SEQ ID NO 89
    LENGTH: 1233
    TYPE: PRT
    ORGANISM: Murine sp.
    SEQUENCE: 89
    Met Ala Asn Asp Ser Pro Ala Lys Ser Leu Val Asp Ile Asp Leu Ser
    1 5 10 15
    Ser Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu Val Val Gly
    20 25 30
    Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val Lys Thr Val
    35 40 45
    Thr Ala Ala Ile Lys Val Met Asp Val Thr Glu Asp Glu Glu Glu Glu
    50 55 60
    Ile Thr Leu Glu Ile Asn Met Leu Lys Lys Tyr Ser His His Arg Asn
    65 70 75 80
    Ile Ala Thr Tyr Tyr Gly Ala Phe Ile Lys Lys Ser Pro Pro Gly His
    85 90 95
    Asp Asp Gln Leu Trp Leu Val Met Glu Phe Cys Gly Ala Gly Ser Ile
    100 105 110
    Thr Asp Leu Val Lys Asn Thr Lys Gly Asn Thr Leu Lys Glu Asp Trp
    115 120 125
    Ile Ala Tyr Ile Ser Arg Glu Ile Leu Arg Gly Leu Ala His Leu His
    130 135 140
    Ile His His Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu Leu
    145 150 155 160
    Thr Glu Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln
    165 170 175
    Leu Asp Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr
    180 185 190
    Trp Met Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala Thr
    195 200 205
    Tyr Asp Tyr Arg Ser Asp Leu Trp Ser Cys Gly Ile Thr Ala Ile Glu
    210 215 220
    Met Ala Glu Gly Gly Pro Pro Leu Cys Asp Met His Pro Met Arg Ala
    225 230 235 240
    Leu Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser Lys Lys
    245 250 255
    Trp Ser Lys Lys Phe Phe Ser Phe Ile Glu Gly Cys Leu Val Lys Asn
    260 265 270
    Tyr Met Gln Arg Pro Ser Thr Glu Gln Leu Leu Lys His Pro Phe Ile
    275 280 285
    Arg Asp Gln Pro Asn Glu Arg Gln Val Arg Ile Gln Leu Lys Asp His
    290 295 300
    Ile Asp Arg Thr Arg Lys Lys Arg Gly Glu Lys Asp Glu Thr Glu Tyr
    305 310 315 320
    Glu Tyr Ser Gly Ser Glu Glu Glu Glu Glu Glu Val Pro Glu Gln Glu
    325 330 335
    Gly Glu Pro Ser Ser Ile Val Asn Val Pro Gly Glu Ser Thr Leu Arg
    340 345 350
    Arg Asp Phe Leu Arg Leu Gln Gln Glu Asn Lys Glu Arg Ser Glu Ala
    355 360 365
    Leu Arg Arg Gln Gln Leu Leu Gln Glu Gln Gln Leu Arg Glu Gln Glu
    370 375 380
    Glu Tyr Lys Arg Gln Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu Gln
    385 390 395 400
    Gln Lys Glu Gln Arg Arg Arg Leu Glu Glu Gln Gln Arg Arg Glu Arg
    405 410 415
    Glu Ala Arg Arg Gln Gln Glu Arg Glu Gln Arg Arg Arg Glu Gln Glu
    420 425 430
    Glu Lys Arg Arg Leu Glu Glu Leu Glu Arg Arg Arg Lys Glu Glu Glu
    435 440 445
    Glu Arg Arg Arg Ala Glu Glu Glu Lys Arg Arg Val Glu Arg Glu Gln
    450 455 460
    Glu Tyr Ile Arg Arg Gln Leu Glu Glu Glu Gln Arg His Leu Glu Ile
    465 470 475 480
    Leu Gln Gln Gln Leu Leu Gln Glu Gln Ala Met Leu Leu His Asp His
    485 490 495
    Arg Arg Pro His Ala Gln Gln Gln Pro Pro Pro Pro Gln Gln Gln Asp
    500 505 510
    Arg Ser Lys Pro Ser Phe His Ala Pro Glu Pro Lys Pro His Tyr Asp
    515 520 525
    Pro Ala Asp Arg Ala Arg Glu Val Gln Trp Ser His Leu Ala Ser Leu
    530 535 540
    Lys Asn Asn Val Ser Pro Val Ser Arg Ser His Ser Phe Ser Asp Pro
    545 550 555 560
    Ser Pro Lys Phe Ala His His His Leu Arg Ser Gln Asp Pro Cys Pro
    565 570 575
    Pro Ser Arg Ser Glu Gly Leu Ser Gln Ser Ser Asp Ser Lys Ser Glu
    580 585 590
    Val Pro Glu Pro Thr Gln Lys Ala Trp Ser Arg Ser Asp Ser Asp Glu
    595 600 605
    Val Pro Pro Arg Val Pro Val Arg Thr Thr Ser Arg Ser Pro Val Leu
    610 615 620
    Ser Arg Arg Asp Ser Pro Leu Gln Gly Gly Gly Gln Gln Asn Ser Gln
    625 630 635 640
    Ala Gly Gln Arg Asn Ser Thr Ser Ser Ile Glu Pro Arg Leu Leu Trp
    645 650 655
    Glu Arg Val Glu Lys Leu Val Pro Arg Pro Gly Ser Gly Ser Ser Ser
    660 665 670
    Gly Ser Ser Asn Ser Gly Ser Gln Pro Gly Ser His Pro Gly Ser Gln
    675 680 685
    Ser Gly Ser Gly Glu Arg Phe Arg Val Arg Ser Ser Ser Lys Ser Glu
    690 695 700
    Gly Ser Pro Ser Pro Arg Gln Glu Ser Ala Ala Lys Lys Pro Asp Asp
    705 710 715 720
    Lys Lys Glu Val Phe Arg Ser Leu Lys Pro Ala Gly Glu Val Asp Leu
    725 730 735
    Thr Ala Leu Ala Lys Glu Leu Arg Ala Val Glu Asp Val Arg Pro Pro
    740 745 750
    His Lys Val Thr Asp Tyr Ser Ser Ser Ser Glu Glu Ser Gly Thr Thr
    755 760 765
    Asp Glu Glu Glu Glu Asp Val Glu Gln Glu Gly Ala Asp Asp Ser Thr
    770 775 780
    Ser Gly Pro Glu Asp Thr Arg Ala Ala Ser Ser Pro Asn Leu Ser Asn
    785 790 795 800
    Gly Glu Thr Glu Ser Val Lys Thr Met Ile Val His Asp Asp Val Glu
    805 810 815
    Ser Glu Pro Ala Met Thr Pro Ser Lys Glu Gly Thr Leu Ile Val Arg
    820 825 830
    Gln Thr Gln Ser Ala Ser Ser Thr Leu Gln Lys His Lys Ser Ser Ser
    835 840 845
    Ser Phe Thr Pro Phe Ile Asp Pro Arg Leu Leu Gln Ile Ser Pro Ser
    850 855 860
    Ser Gly Thr Thr Val Thr Ser Val Val Gly Phe Ser Cys Asp Gly Leu
    865 870 875 880
    Arg Pro Glu Ala Ile Arg Gln Asp Pro Thr Arg Lys Gly Ser Val Val
    885 890 895
    Asn Val Asn Pro Thr Asn Thr Arg Pro Gln Ser Asp Thr Pro Glu Ile
    900 905 910
    Arg Lys Tyr Lys Lys Arg Phe Asn Ser Glu Ile Leu Cys Ala Ala Leu
    915 920 925
    Trp Gly Val Asn Leu Leu Val Gly Thr Glu Ser Gly Leu Met Leu Leu
    930 935 940
    Asp Arg Ser Gly Gln Gly Lys Val Tyr Pro Leu Ile Ser Arg Arg Arg
    945 950 955 960
    Phe Gln Gln Met Asp Val Leu Glu Gly Leu Asn Val Leu Val Thr Ile
    965 970 975
    Ser Gly Lys Lys Asp Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Arg
    980 985 990
    Asn Lys Ile Leu His Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Trp
    995 1000 1005
    Thr Thr Val Gly Asp Leu Glu Gly Cys Val His Tyr Lys Val Val Lys
    1010 1015 1020
    Tyr Glu Arg Ile Lys Phe Leu Val Ile Ala Leu Lys Ser Ser Val Glu
    1025 1030 1035 1040
    Val Tyr Ala Trp Ala Pro Lys Pro Tyr His Lys Phe Met Ala Phe Lys
    1045 1050 1055
    Ser Phe Gly Glu Leu Leu His Lys Pro Leu Leu Val Asp Leu Thr Val
    1060 1065 1070
    Glu Glu Gly Gln Arg Leu Lys Val Ile Tyr Gly Ser Cys Ala Gly Phe
    1075 1080 1085
    His Ala Val Asp Val Asp Ser Gly Ser Val Tyr Asp Ile Tyr Leu Pro
    1090 1095 1100
    Thr His Ile Gln Cys Ser Ile Lys Pro His Ala Ile Ile Ile Leu Pro
    1105 1110 1115 1120
    Asn Thr Asp Gly Met Glu Leu Leu Val Cys Tyr Glu Asp Glu Gly Val
    1125 1130 1135
    Tyr Val Asn Thr Tyr Gly Arg Ile Thr Lys Asp Val Val Leu Gln Trp
    1140 1145 1150
    Gly Glu Met Pro Thr Ser Val Ala Tyr Ile Arg Ser Asn Gln Thr Met
    1155 1160 1165
    Gly Trp Gly Glu Lys Ala Ile Glu Ile Arg Ser Val Glu Thr Gly His
    1170 1175 1180
    Leu Asp Gly Val Phe Met His Lys Arg Ala Gln Arg Leu Lys Phe Leu
    1185 1190 1195 1200
    Cys Gly Arg Asn Asp Lys Val Phe Phe Ser Ser Val Arg Ser Gly Gly
    1205 1210 1215
    Ser Ser Gln Val Tyr Phe Met Thr Leu Gly Arg Thr Ser Leu Leu Ser
    1220 1225 1230
    Trp
    <210> SEQ ID NO 90
    <211> LENGTH: 982
    <212> TYPE: PRT
    <213> ORGANISM: Caenorhabditis elegans
    <400> SEQUENCE: 90
    Met Ala Pro Ala Val Leu Asp Lys Pro Gly Val Ile Lys Asp Pro Ser
    1 5 10 15
    Ile Ala Ala Leu Phe Ser Asn Lys Asp Pro Glu Gln Arg Tyr Gln Asp
    20 25 30
    Leu Arg Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala Tyr
    35 40 45
    Asp Lys Lys Asn Glu Gln Thr Val Ala Ile Lys Lys Met Asn Phe Ser
    50 55 60
    Gly Lys Gln Ala Val Glu Lys Trp Asn Asp Ile Leu Lys Glu Val Ser
    65 70 75 80
    Phe Leu Asn Thr Val Val His Pro His Ile Val Asp Tyr Lys Ala Cys
    85 90 95
    Phe Leu Lys Asp Thr Thr Cys Trp Leu Val Met Glu Tyr Cys Ile Gly
    100 105 110
    Ser Ala Ala Asp Ile Val Asp Val Leu Arg Lys Gly Met Arg Glu Val
    115 120 125
    Glu Ile Ala Ala Ile Cys Ser Gln Thr Leu Asp Ala Leu Arg Tyr Leu
    130 135 140
    His Ser Leu Lys Arg Ile His Arg Asp Ile Lys Ala Gly Asn Ile Leu
    145 150 155 160
    Leu Ser Asp His Ala Ile Val Lys Leu Ala Asp Phe Gly Ser Ala Ser
    165 170 175
    Leu Val Asp Pro Ala Gln Thr Phe Ile Gly Thr Pro Phe Phe Met Ala
    180 185 190
    Pro Glu Val Ile Leu Ala Met Asp Glu Gly His Tyr Thr Asp Arg Ala
    195 200 205
    Asp Ile Trp Ser Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Arg
    210 215 220
    Pro Pro Leu Phe Ser Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala
    225 230 235 240
    Gln Asn Asp Pro Pro Thr Leu Ser Pro Ile Asp Thr Ser Glu Gln Pro
    245 250 255
    Glu Trp Ser Leu Glu Phe Val Gln Phe Ile Asp Lys Cys Leu Arg Lys
    260 265 270
    Pro Ala Glu Glu Arg Met Ser Ala Glu Glu Cys Phe Arg His Pro Phe
    275 280 285
    Ile Gln Arg Ser Arg Pro Ser Asp Thr Ile Gln Glu Leu Ile Gln Arg
    290 295 300
    Thr Lys Asn Met Val Leu Glu Leu Asp Asn Phe Gln Tyr Lys Lys Met
    305 310 315 320
    Arg Lys Leu Met Tyr Leu Asp Glu Thr Glu Gly Lys Glu Gly Ser Glu
    325 330 335
    Gly Asn Gly Ala Ser Asp Asp Leu Asp Phe His Gly Asn Glu Ala Asn
    340 345 350
    Ser Ile Gly Arg Ala Gly Asp Ser Ala Ser Ser Arg Ser Ala Ser Leu
    355 360 365
    Thr Ser Phe Arg Ser Met Gln Ser Ser Gly Gly Ala Gly Leu Leu Val
    370 375 380
    Ser Thr Asn Thr Thr Gly Ala Met Asp Asn Val His Gly Ser Ser Gly
    385 390 395 400
    Tyr Gly Asn Gly Ser Ser Ser Thr Thr Ser Ser Ala Arg Arg Arg Pro
    405 410 415
    Pro Ile Pro Ser Gln Met Leu Ser Ser Thr Ser Thr Ser Gly Val Gly
    420 425 430
    Thr Met Pro Ser His Gly Ser Val Gly Ala Ser Ile Thr Ala Ile Ala
    435 440 445
    Val Asn Pro Thr Pro Ser Pro Ser Glu Pro Ile Pro Thr Ser Gln Pro
    450 455 460
    Thr Ser Lys Ser Glu Ser Ser Ser Ile Leu Glu Thr Ala His Asp Asp
    465 470 475 480
    Pro Leu Asp Thr Ser Ile Arg Ala Pro Val Lys Asp Leu His Met Pro
    485 490 495
    His Arg Ala Val Lys Glu Arg Ile Ala Thr Leu Gln Asn His Lys Phe
    500 505 510
    Ala Thr Leu Arg Ser Gln Arg Ile Ile Asn Gln Glu Gln Glu Glu Tyr
    515 520 525
    Thr Lys Glu Asn Asn Met Tyr Glu Gln Met Ser Lys Tyr Lys His Leu
    530 535 540
    Arg Gln Ala His His Lys Glu Leu Gln Gln Phe Glu Glu Arg Cys Ala
    545 550 555 560
    Leu Asp Arg Glu Gln Leu Arg Val Lys Met Asp Arg Glu Leu Glu Gln
    565 570 575
    Leu Thr Thr Thr Tyr Ser Lys Glu Lys Met Arg Val Arg Cys Ser Gln
    580 585 590
    Asn Asn Glu Leu Asp Lys Arg Lys Lys Asp Ile Glu Asp Gly Glu Lys
    595 600 605
    Lys Met Lys Lys Thr Lys Asn Ser Gln Asn Gln Gln Gln Met Lys Leu
    610 615 620
    Tyr Ser Ala Gln Gln Leu Lys Glu Tyr Lys Tyr Asn Lys Glu Ala Gln
    625 630 635 640
    Lys Thr Arg Leu Arg Ser Leu Asn Met Pro Arg Ser Thr Tyr Glu Asn
    645 650 655
    Ala Met Lys Glu Val Lys Ala Asp Leu Asn Arg Val Lys Asp Ala Arg
    660 665 670
    Glu Asn Asp Phe Asp Glu Lys Leu Arg Ala Glu Leu Glu Asp Glu Ile
    675 680 685
    Val Arg Tyr Arg Arg Gln Gln Leu Ser Asn Leu His Gln Leu Glu Glu
    690 695 700
    Gln Leu Asp Asp Glu Asp Val Asn Val Gln Glu Arg Gln Met Asp Thr
    705 710 715 720
    Arg His Gly Leu Leu Ser Lys Gln His Glu Met Thr Arg Asp Leu Glu
    725 730 735
    Ile Gln His Leu Asn Glu Leu His Ala Met Lys Lys Arg His Leu Glu
    740 745 750
    Thr Gln His Glu Ala Glu Ser Ala Ser Gln Asn Glu Tyr Thr Gln Arg
    755 760 765
    Gln Gln Asp Glu Leu Arg Lys Lys His Ala Met Gln Ser Arg Gln Gln
    770 775 780
    Pro Arg Asp Leu Lys Ile Gln Glu Ala Gln Ile Arg Lys Gln Tyr Arg
    785 790 795 800
    Gln Val Val Lys Thr Gln Thr Arg Gln Phe Lys Leu Tyr Leu Thr Gln
    805 810 815
    Met Val Gln Val Val Pro Lys Asp Glu Gln Lys Glu Leu Thr Ser Arg
    820 825 830
    Leu Lys Gln Asp Gln Met Gln Lys Val Ala Leu Leu Ala Ser Gln Tyr
    835 840 845
    Glu Ser Gln Ile Lys Lys Met Val Gln Asp Lys Thr Val Lys Leu Glu
    850 855 860
    Ser Trp Gln Glu Asp Glu Gln Arg Val Leu Ser Glu Lys Leu Glu Lys
    865 870 875 880
    Glu Leu Glu Glu Leu Ile Ala Tyr Gln Lys Lys Thr Arg Ala Thr Leu
    885 890 895
    Glu Glu Gln Ile Lys Lys Glu Arg Thr Ala Leu Glu Glu Arg Ile Gly
    900 905 910
    Thr Arg Arg Ala Met Leu Glu Gln Lys Ile Ile Glu Glu Arg Glu Gln
    915 920 925
    Met Gly Glu Met Arg Arg Leu Lys Lys Glu Gln Ile Arg Asp Arg His
    930 935 940
    Ser Gln Glu Arg His Arg Leu Glu Asn His Phe Val Arg Thr Gly Ser
    945 950 955 960
    Thr Ser Arg Ser Ser Gly Gly Ile Ala Pro Gly Val Gly Asn Ser Ser
    965 970 975
    Ser Ile Gln Met Ala Met
    980
    <210> SEQ ID NO 91
    <211> LENGTH: 842
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 91
    Leu Arg Pro Ala Ala Asp Ile Leu Arg Arg Asn Pro Gln Gln Asp Tyr
    1 5 10 15
    Glu Leu Val Gln Arg Val Gly Ser Gly Thr Tyr Gly Asp Val Tyr Lys
    20 25 30
    Ala Arg Asn Val His Thr Gly Glu Leu Ala Ala Val Lys Ile Ile Lys
    35 40 45
    Leu Glu Pro Gly Asp Asp Phe Ser Leu Ile Gln Gln Glu Ile Phe Met
    50 55 60
    Val Lys Glu Cys Lys His Cys Asn Ile Val Ala Tyr Phe Gly Ser Tyr
    65 70 75 80
    Leu Ser Arg Glu Lys Leu Trp Ile Cys Met Glu Tyr Cys Gly Gly Gly
    85 90 95
    Ser Leu Gln Asp Ile Tyr His Val Thr Gly Pro Leu Ser Glu Leu Gln
    100 105 110
    Ile Ala Tyr Val Cys Arg Glu Thr Leu Gln Gly Leu Ala Tyr Leu His
    115 120 125
    Thr Lys Gly Lys Met His Arg Asp Ile Lys Gly Ala Asn Ile Leu Leu
    130 135 140
    Thr Asp His Gly Asp Val Lys Leu Ala Asp Phe Gly Val Ala Ala Lys
    145 150 155 160
    Ile Thr Ala Thr Ile Ala Lys Arg Lys Ser Phe Ile Gly Thr Pro Tyr
    165 170 175
    Trp Met Ala Pro Glu Val Ala Ala Val Glu Lys Asn Gly Gly Tyr Asn
    180 185 190
    Gln Leu Cys Asp Ile Trp Ala Val Gly Ile Thr Ala Ile Glu Leu Gly
    195 200 205
    Glu Leu Gln Pro Pro Met Phe Asp Leu His Pro Met Arg Ala Leu Phe
    210 215 220
    Leu Met Ser Lys Ser Asn Phe Gln Pro Pro Lys Leu Lys Asp Lys Thr
    225 230 235 240
    Lys Trp Ser Ser Thr Phe His Asn Phe Val Lys Ile Ala Leu Thr Lys
    245 250 255
    Asn Pro Lys Lys Arg Pro Thr Ala Glu Arg Leu Leu Thr His Thr Phe
    260 265 270
    Val Ala Gln Pro Gly Leu Ser Arg Ala Leu Ala Val Glu Leu Leu Asp
    275 280 285
    Lys Val Asn Asn Pro Asp Asn His Ala His Tyr Thr Glu Ala Asp Asp
    290 295 300
    Asp Asp Phe Glu Pro His Ala Ile Ile Arg His Thr Ile Arg Ser Thr
    305 310 315 320
    Asn Arg Asn Ala Arg Ala Glu Arg Thr Ala Ser Glu Ile Asn Phe Asp
    325 330 335
    Lys Leu Gln Phe Glu Pro Pro Leu Arg Lys Glu Thr Glu Ala Arg Asp
    340 345 350
    Glu Met Gly Leu Ser Ser Asp Pro Asn Phe Met Leu Gln Trp Asn Pro
    355 360 365
    Phe Val Asp Gly Ala Asn Thr Gly Lys Ser Thr Ser Lys Arg Ala Ile
    370 375 380
    Pro Pro Pro Leu Pro Pro Lys Pro Arg Ile Ser Ser Tyr Pro Glu Asp
    385 390 395 400
    Asn Phe Pro Asp Glu Glu Lys Ala Ser Thr Ile Lys His Cys Pro Asp
    405 410 415
    Ser Glu Ser Arg Ala Pro Gln Ile Leu Arg Arg Gln Ser Ser Pro Ser
    420 425 430
    Cys Gly Pro Val Ala Glu Thr Ser Ser Ile Gly Asn Gly Asp Gly Ile
    435 440 445
    Ser Lys Leu Met Ser Glu Asn Thr Glu Gly Ser Ala Gln Ala Pro Gln
    450 455 460
    Leu Pro Arg Lys Asn Asp Lys Arg Asp Phe Pro Lys Pro Ala Ile Asn
    465 470 475 480
    Gly Leu Pro Pro Thr Pro Lys Val Leu Met Gly Ala Cys Phe Ser Lys
    485 490 495
    Val Phe Asp Gly Cys Pro Leu Lys Ile Asn Cys Ala Thr Ser Trp Ile
    500 505 510
    His Pro Asp Thr Lys Asp Gln Tyr Ile Ile Phe Gly Thr Glu Asp Gly
    515 520 525
    Ile Tyr Thr Leu Asn Leu Asn Glu Leu His Glu Ala Thr Met Glu Gln
    530 535 540
    Leu Phe Pro Arg Lys Cys Thr Trp Leu Tyr Val Ile Asn Asn Thr Leu
    545 550 555 560
    Met Ser Leu Ser Glu Gly Lys Thr Phe Gln Leu Tyr Ser His Asn Leu
    565 570 575
    Ile Ala Leu Phe Glu His Ala Lys Lys Pro Gly Leu Ala Ala His Ile
    580 585 590
    Gln Thr His Arg Phe Pro Asp Arg Ile Leu Pro Arg Lys Phe Ala Leu
    595 600 605
    Thr Thr Lys Ile Pro Asp Thr Lys Gly Cys His Lys Cys Cys Ile Val
    610 615 620
    Arg Asn Pro Tyr Thr Gly His Lys Tyr Leu Cys Gly Ala Leu Gln Ser
    625 630 635 640
    Gly Ile Val Leu Leu Gln Trp Tyr Glu Pro Met Gln Lys Phe Met Leu
    645 650 655
    Ile Lys His Phe Asp Phe Pro Leu Pro Ser Pro Leu Asn Val Phe Glu
    660 665 670
    Met Leu Val Ile Pro Glu Gln Glu Tyr Pro Met Val Cys Val Ala Ile
    675 680 685
    Ser Lys Gly Thr Glu Ser Asn Gln Val Val Gln Phe Glu Thr Ile Asn
    690 695 700
    Leu Asn Ser Ala Ser Ser Trp Phe Thr Glu Ile Gly Ala Gly Ser Gln
    705 710 715 720
    Gln Leu Asp Ser Ile His Val Thr Gln Leu Glu Arg Asp Thr Val Leu
    725 730 735
    Val Cys Leu Asp Lys Phe Val Lys Ile Val Asn Leu Gln Gly Lys Leu
    740 745 750
    Lys Ser Ser Lys Lys Leu Ala Ser Glu Leu Ser Phe Asp Phe Arg Ile
    755 760 765
    Glu Ser Val Val Cys Leu Gln Asp Ser Val Leu Ala Phe Trp Lys His
    770 775 780
    Gly Met Gln Gly Lys Ser Phe Lys Ser Asp Glu Val Thr Gln Glu Ile
    785 790 795 800
    Ser Asp Glu Thr Arg Val Phe Arg Leu Leu Gly Ser Asp Arg Val Val
    805 810 815
    Val Leu Glu Ser Arg Pro Thr Glu Asn Pro Thr Ala His Ser Asn Leu
    820 825 830
    Tyr Ile Leu Ala Gly His Glu Asn Ser Tyr
    835 840
    <210> SEQ ID NO 92
    <211> LENGTH: 911
    <212> TYPE: PRT
    <213> ORGANISM: Murine sp.
    <400> SEQUENCE: 92
    Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
    1 5 10 15
    Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp Leu Asp Pro
    20 25 30
    Asn Asp Val Trp Glu Ile Val Gly Glu Leu Gly Asp Gly Ala Phe Gly
    35 40 45
    Lys Val Tyr Lys Ala Lys Asn Lys Glu Thr Gly Ala Leu Ala Ala Ala
    50 55 60
    Lys Val Ile Glu Thr Lys Ser Glu Glu Glu Leu Glu Asp Tyr Ile Val
    65 70 75 80
    Glu Ile Glu Ile Leu Ala Thr Cys Asp His Pro Tyr Ile Val Lys Leu
    85 90 95
    Leu Gly Ala Tyr Tyr Tyr Asp Gly Lys Leu Trp Ile Met Ile Glu Phe
    100 105 110
    Cys Pro Gly Gly Ala Val Asp Ala Ile Met Leu Glu Leu Asp Arg Gly
    115 120 125
    Leu Thr Glu Pro Gln Ile Gln Val Val Cys Arg Gln Met Leu Glu Ala
    130 135 140
    Leu Asn Phe Leu His Gly Lys Arg Ile Ile His Arg Asp Leu Lys Ala
    145 150 155 160
    Gly Asn Val Leu Met Thr Leu Glu Gly Asp Ile Arg Leu Ala Asp Phe
    165 170 175
    Gly Val Ser Ala Lys Asn Leu Lys Thr Leu Gln Lys Arg Asp Ser Phe
    180 185 190
    Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Leu Cys Glu Thr
    195 200 205
    Met Lys Asp Ala Pro Tyr Asp Tyr Lys Ala Asp Ile Trp Ser Leu Gly
    210 215 220
    Ile Thr Leu Ile Glu Met Ala Gln Ile Glu Pro Pro His His Glu Leu
    225 230 235 240
    Asn Pro Met Arg Val Leu Leu Lys Ile Ala Lys Ser Asp Pro Pro Thr
    245 250 255
    Leu Leu Thr Pro Ser Lys Trp Ser Val Glu Phe Arg Asp Phe Leu Lys
    260 265 270
    Ile Ala Leu Asp Lys Asn Pro Glu Thr Arg Pro Ser Ala Ala Gln Leu
    275 280 285
    Leu Gln His Pro Phe Val Ser Arg Val Thr Ser Asn Lys Ala Leu Arg
    290 295 300
    Glu Leu Val Ala Glu Ala Lys Ala Glu Val Met Glu Glu Ile Glu Asp
    305 310 315 320
    Gly Arg Glu Asp Gly Glu Glu Glu Asp Ala Val Asp Ala Val Pro Pro
    325 330 335
    Leu Val Asn His Thr Gln Asp Ser Ala Asn Val Thr Gln Pro Ser Leu
    340 345 350
    Asp Ser Asn Lys Leu Leu Gln Asp Ser Ser Thr Pro Leu Pro Pro Ser
    355 360 365
    Gln Pro Gln Glu Pro Val Asn Gly Pro Cys Ser Gln Pro Ser Gly Asp
    370 375 380
    Gly Pro Leu Gln Thr Thr Ser Pro Ala Asp Gly Leu Ser Lys Asn Asp
    385 390 395 400
    Asn Asp Leu Lys Val Pro Val Pro Leu Arg Lys Ser Arg Pro Leu Ser
    405 410 415
    Met Asp Ala Arg Ile Gln Met Asp Glu Glu Lys Gln Ile Pro Asp Gln
    420 425 430
    Asp Glu Asn Pro Ser Pro Ala Ala Ser Lys Ser Gln Lys Ala Asn Gln
    435 440 445
    Ser Arg Pro Asn Ser Ser Ala Leu Glu Thr Leu Gly Gly Glu Ala Leu
    450 455 460
    Thr Asn Gly Gly Leu Glu Leu Pro Ser Ser Val Thr Pro Ser His Ser
    465 470 475 480
    Lys Arg Ala Ser Asp Cys Ser Asn Leu Ser Thr Ser Glu Ser Met Asp
    485 490 495
    Tyr Gly Thr Ser Leu Ser Ala Asp Leu Ser Leu Asn Lys Glu Thr Gly
    500 505 510
    Ser Leu Ser Leu Lys Gly Ser Lys Leu His Asn Lys Thr Leu Lys Arg
    515 520 525
    Thr Arg Arg Phe Val Val Asp Gly Val Glu Val Ser Ile Thr Thr Ser
    530 535 540
    Lys Ile Ile Ser Glu Asp Glu Lys Lys Asp Glu Glu Met Arg Phe Leu
    545 550 555 560
    Arg Arg Gln Glu Leu Arg Glu Leu Arg Leu Leu Gln Lys Glu Glu His
    565 570 575
    Arg Asn Gln Thr Gln Leu Ser Ser Lys His Glu Leu Gln Leu Glu Gln
    580 585 590
    Met His Lys Arg Phe Glu Gln Glu Ile Asn Ala Lys Lys Lys Phe Tyr
    595 600 605
    Asp Val Glu Leu Glu Asn Leu Glu Arg Gln Gln Lys Gln Gln Val Glu
    610 615 620
    Lys Met Glu Gln Asp His Ser Val Arg Arg Lys Glu Glu Ala Lys Arg
    625 630 635 640
    Ile Arg Leu Glu Gln Asp Arg Asp Tyr Ala Lys Phe Gln Glu Gln Leu
    645 650 655
    Lys Gln Met Lys Lys Glu Val Lys Ser Glu Val Glu Lys Leu Pro Arg
    660 665 670
    Gln Gln Arg Lys Glu Ser Met Lys Gln Lys Met Glu Glu His Ser Gln
    675 680 685
    Lys Lys Gln Arg Leu Asp Arg Asp Phe Val Ala Lys Gln Lys Glu Asp
    690 695 700
    Leu Glu Leu Ala Met Arg Lys Leu Thr Thr Glu Asn Arg Arg Glu Ile
    705 710 715 720
    Cys Asp Lys Glu Arg Asp Cys Leu Ser Lys Lys Gln Glu Leu Leu Arg
    725 730 735
    Asp Arg Glu Ala Ala Leu Trp Glu Met Glu Glu His Gln Leu Gln Glu
    740 745 750
    Arg His Gln Leu Val Lys Gln Gln Leu Lys Asp Gln Tyr Phe Leu Gln
    755 760 765
    Arg His Asp Leu Leu Arg Lys His Glu Lys Glu Arg Glu Gln Met Gln
    770 775 780
    Arg Tyr Asn Gln Arg Met Met Glu Gln Leu Lys Val Arg Gln Gln Gln
    785 790 795 800
    Glu Lys Ala Arg Leu Pro Lys Ile Gln Arg Ser Asp Gly Glu Thr Arg
    805 810 815
    Met Ala Met Tyr Lys Lys Ser Leu His Ile Asn Gly Ala Gly Ser Ala
    820 825 830
    Ser Glu Gln Arg Glu Lys Ile Lys Gln Phe Ser Gln Gln Glu Glu Lys
    835 840 845
    Arg Gln Lys Ala Glu Arg Leu Gln Gln Gln Gln Lys His Glu His Gln
    850 855 860
    Met Arg Asp Met Val Ala Gln Cys Glu Ser Asn Met Ser Glu Leu Gln
    865 870 875 880
    Gln Leu Gln Asn Glu Lys Cys Tyr Leu Leu Val Glu His Glu Thr Gln
    885 890 895
    Lys Leu Lys Ala Leu Asp Glu Ser His Asn Gln Ser Leu Lys Glu
    900 905 910
    <210> SEQ ID NO 93
    <211> LENGTH: 545
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 93
    Met Ser Asn Asn Gly Leu Asp Ile Gln Asp Lys Pro Pro Ala Pro Pro
    1 5 10 15
    Met Arg Asn Thr Ser Thr Met Ile Gly Ala Gly Ser Lys Asp Ala Gly
    20 25 30
    Thr Leu Asn His Gly Ser Lys Pro Leu Pro Pro Asn Pro Glu Glu Lys
    35 40 45
    Lys Lys Lys Asp Arg Phe Tyr Arg Ser Ile Leu Pro Gly Asp Lys Thr
    50 55 60
    Asn Lys Lys Lys Glu Lys Glu Arg Pro Glu Ile Ser Leu Pro Ser Asp
    65 70 75 80
    Phe Glu His Thr Ile His Val Gly Phe Asp Ala Val Thr Gly Glu Phe
    85 90 95
    Thr Gly Met Pro Glu Gln Trp Ala Arg Leu Leu Gln Thr Ser Asn Ile
    100 105 110
    Thr Lys Ser Glu Gln Lys Lys Asn Pro Gln Ala Val Leu Asp Val Leu
    115 120 125
    Glu Phe Tyr Asn Ser Lys Lys Thr Ser Asn Ser Gln Lys Tyr Met Ser
    130 135 140
    Phe Thr Asp Lys Ser Ala Glu Asp Tyr Asn Ser Ser Asn Ala Leu Asn
    145 150 155 160
    Val Lys Ala Val Ser Glu Thr Pro Ala Val Pro Pro Val Ser Glu Asp
    165 170 175
    Glu Asp Asp Asp Asp Asp Asp Ala Thr Pro Pro Pro Val Ile Ala Pro
    180 185 190
    Arg Pro Glu His Thr Lys Ser Val Tyr Thr Arg Ser Val Ile Glu Pro
    195 200 205
    Leu Pro Val Thr Pro Thr Arg Asp Val Ala Thr Ser Pro Ile Ser Pro
    210 215 220
    Thr Glu Asn Asn Thr Thr Pro Pro Asp Ala Leu Thr Leu Asn Thr Glu
    225 230 235 240
    Lys Gln Lys Lys Lys Pro Lys Met Ser Asp Glu Glu Ile Leu Glu Lys
    245 250 255
    Leu Arg Ser Ile Val Ser Val Gly Asp Pro Lys Lys Lys Tyr Thr Arg
    260 265 270
    Phe Glu Lys Ile Gly Gln Gly Ala Ser Gly Thr Val Tyr Thr Ala Met
    275 280 285
    Asp Val Ala Thr Gly Gln Glu Val Ala Ile Lys Gln Met Asn Leu Gln
    290 295 300
    Gln Gln Pro Lys Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Arg
    305 310 315 320
    Glu Asn Lys Asn Pro Asn Ile Val Asn Tyr Leu Asp Ser Tyr Leu Val
    325 330 335
    Gly Asp Glu Leu Trp Val Val Met Glu Tyr Leu Ala Gly Gly Ser Leu
    340 345 350
    Thr Asp Val Val Thr Glu Thr Cys Met Asp Glu Gly Gln Ile Ala Ala
    355 360 365
    Val Cys Arg Glu Cys Leu Gln Ala Leu Glu Ser Leu His Ser Asn Gln
    370 375 380
    Val Ile His Arg Asp Ile Lys Ser Asp Asn Ile Leu Leu Gly Met Asp
    385 390 395 400
    Gly Ser Val Lys Leu Thr Asp Phe Gly Phe Cys Ala Gln Ile Thr Pro
    405 410 415
    Glu Gln Ser Lys Arg Ser Thr Met Val Gly Thr Pro Tyr Trp Met Ala
    420 425 430
    Pro Glu Val Val Thr Arg Lys Ala Tyr Gly Pro Lys Val Asp Ile Trp
    435 440 445
    Ser Leu Gly Ile Met Ala Ile Glu Met Ile Glu Gly Glu Pro Pro Tyr
    450 455 460
    Leu Asn Glu Asn Pro Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly
    465 470 475 480
    Thr Pro Glu Leu Gln Asn Pro Glu Lys Leu Ser Ala Ile Phe Arg Asp
    485 490 495
    Phe Leu Asn Arg Cys Leu Glu Met Asp Val Glu Lys Arg Gly Ser Ala
    500 505 510
    Lys Glu Leu Leu Gln His Gln Phe Leu Lys Ile Ala Lys Pro Leu Ser
    515 520 525
    Ser Leu Thr Pro Leu Ile Ala Ala Ala Lys Glu Ala Thr Lys Asn Asn
    530 535 540
    His
    545
    <210> SEQ ID NO 94
    <211> LENGTH: 506
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 94
    Met Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Ser Ala Asn
    1 5 10 15
    His Ser Leu Lys Pro Leu Pro Ser Val Pro Glu Glu Lys Lys Pro Arg
    20 25 30
    His Lys Ile Ile Ser Ile Phe Ser Gly Thr Glu Lys Gly Ser Lys Lys
    35 40 45
    Lys Glu Lys Glu Arg Pro Glu Ile Ser Pro Pro Ser Asp Phe Glu His
    50 55 60
    Thr Ile His Val Gly Phe Asp Thr Val Thr Gly Glu Phe Thr Gly Met
    65 70 75 80
    Pro Glu Gln Trp Ala Arg Leu Leu Gln Thr Ser Asn Ile Thr Lys Leu
    85 90 95
    Glu Gln Lys Lys Asn Pro Gln Ala Val Leu Asp Val Leu Lys Phe Tyr
    100 105 110
    Asp Ser Asn Thr Val Lys Gln Lys Tyr Leu Ser Phe Thr Pro Pro Glu
    115 120 125
    Lys Asp Gly Phe Pro Ser Gly Thr Pro Ala Leu Asn Ala Lys Gly Thr
    130 135 140
    Glu Ala Pro Ala Val Val Thr Glu Glu Glu Asp Asp Asp Glu Glu Thr
    145 150 155 160
    Ala Pro Pro Val Ile Ala Pro Arg Pro Asp His Thr Lys Ser Ile Tyr
    165 170 175
    Thr Arg Ser Val Ile Asp Pro Val Pro Ala Pro Val Gly Asp Ser His
    180 185 190
    Val Asp Gly Ala Ala Lys Ser Leu Asp Lys Gln Lys Lys Lys Thr Lys
    195 200 205
    Met Thr Asp Glu Glu Ile Met Glu Lys Leu Arg Thr Ile Val Ser Ile
    210 215 220
    Gly Asp Pro Lys Lys Lys Tyr Thr Arg Tyr Glu Lys Ile Gly Gln Gly
    225 230 235 240
    Ala Ser Gly Thr Val Phe Thr Ala Thr Asp Val Ala Leu Gly Gln Glu
    245 250 255
    Val Ala Ile Lys Gln Ile Asn Leu Gln Lys Gln Pro Lys Lys Glu Leu
    260 265 270
    Ile Ile Asn Glu Ile Leu Val Met Lys Glu Leu Lys Asn Pro Asn Ile
    275 280 285
    Val Asn Phe Leu Asp Ser Tyr Leu Val Gly Asp Glu Leu Phe Val Val
    290 295 300
    Met Glu Tyr Leu Ala Gly Arg Ser Leu Thr Asp Val Val Thr Glu Thr
    305 310 315 320
    Cys Met Asp Glu Ala Gln Ile Ala Ala Val Cys Arg Glu Cys Leu Gln
    325 330 335
    Ala Leu Glu Phe Leu His Ala Asn Gln Val Ile His Arg Asp Ile Lys
    340 345 350
    Ser Asp Asn Val Leu Leu Gly Met Glu Gly Ser Val Lys Leu Thr Asp
    355 360 365
    Phe Gly Phe Cys Ala Gln Ile Thr Pro Glu Gln Ser Lys Arg Ser Thr
    370 375 380
    Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Thr Arg Lys
    385 390 395 400
    Ala Tyr Gly Pro Lys Val Asp Ile Trp Ser Leu Gly Ile Met Ala Ile
    405 410 415
    Glu Met Val Glu Gly Glu Pro Pro Tyr Leu Asn Glu Asn Pro Leu Arg
    420 425 430
    Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro Glu Leu Gln Asn Pro
    435 440 445
    Glu Lys Leu Ser Pro Ile Phe Arg Asp Phe Leu Asn Arg Cys Leu Glu
    450 455 460
    Met Asp Val Glu Lys Arg Gly Ser Ala Lys Glu Leu Leu Gln His Pro
    465 470 475 480
    Phe Leu Lys Leu Ala Lys Pro Leu Ser Ser Leu Thr Pro Leu Ile Met
    485 490 495
    Ala Ala Lys Glu Ala Met Lys Ser Asn Arg
    500 505
    <210> SEQ ID NO 95
    <211> LENGTH: 544
    <212> TYPE: PRT
    <213> ORGANISM: Murine sp.
    <400> SEQUENCE: 95
    Met Ser Asp Ser Leu Asp Asn Glu Glu Lys Pro Pro Ala Pro Pro Leu
    1 5 10 15
    Arg Met Asn Ser Asn Asn Arg Asp Ser Ser Ala Leu Asn His Ser Ser
    20 25 30
    Lys Pro Leu Pro Met Ala Pro Glu Glu Lys Asn Lys Lys Ala Arg Leu
    35 40 45
    Arg Ser Ile Phe Pro Gly Gly Gly Asp Lys Thr Asn Lys Lys Lys Glu
    50 55 60
    Lys Glu Arg Pro Glu Ile Ser Leu Pro Ser Asp Phe Glu His Thr Ile
    65 70 75 80
    His Val Gly Phe Asp Ala Val Thr Gly Glu Phe Thr Gly Ile Pro Glu
    85 90 95
    Gln Trp Ala Arg Leu Leu Gln Thr Ser Asn Ile Thr Lys Leu Glu Gln
    100 105 110
    Lys Lys Asn Pro Gln Ala Val Leu Asp Val Leu Lys Phe Tyr Asp Ser
    115 120 125
    Lys Glu Thr Val Asn Asn Gln Lys Tyr Met Ser Phe Thr Ser Gly Asp
    130 135 140
    Lys Ser Ala His Gly Tyr Ile Ala Ala His Gln Ser Asn Thr Lys Thr
    145 150 155 160
    Gly Ser Glu Pro Pro Leu Ala Pro Pro Val Ser Glu Glu Glu Asp Glu
    165 170 175
    Glu Glu Glu Glu Glu Glu Asp Asp Asn Glu Pro Pro Pro Val Ile Ala
    180 185 190
    Pro Arg Pro Glu His Thr Lys Ser Ile Tyr Thr Arg Ser Val Val Glu
    195 200 205
    Ser Ile Ala Ser Pro Ala Ala Pro Asn Lys Glu Asp Ile Pro Pro Ser
    210 215 220
    Ala Glu Asn Ala Asn Ser Thr Thr Leu Tyr Arg Asn Thr Asp Arg Gln
    225 230 235 240
    Arg Lys Lys Ser Lys Met Thr Asp Glu Glu Ile Leu Glu Lys Leu Arg
    245 250 255
    Ser Ile Val Ser Val Gly Asp Pro Lys Lys Lys Tyr Thr Arg Leu Glu
    260 265 270
    Lys Ile Gly Gln Gly Ala Ser Gly Thr Val Tyr Thr Ala Leu Asp Ile
    275 280 285
    Ala Thr Gly Gln Glu Val Ala Ile Lys Gln Met Asn Leu Gln Gln Gln
    290 295 300
    Pro Lys Lys Glu Leu Ile Ile Asn Glu Ile Leu Val Met Arg Glu Asn
    305 310 315 320
    Lys Asn Pro Asn Ile Val Asn Tyr Leu Asp Ser Tyr Leu Val Gly Asp
    325 330 335
    Glu Leu Trp Val Val Met Glu Tyr Leu Ala Gly Gly Ser Leu Thr Asp
    340 345 350
    Val Val Thr Glu Thr Cys Met Asp Val Gly Gln Ile Ala Ala Val Cys
    355 360 365
    Arg Glu Cys Leu Gln Ala Leu Asp Phe Leu His Ser Asn Gln Val Ile
    370 375 380
    His Arg Asp Ile Lys Ser Asp Asn Ile Leu Leu Gly Met Asp Gly Ser
    385 390 395 400
    Val Lys Leu Thr Asp Phe Gly Phe Cys Ala Gln Ile Thr Pro Glu Gln
    405 410 415
    Ser Lys Arg Ser Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu
    420 425 430
    Val Val Thr Arg Lys Ala Tyr Gly Pro Lys Val Asp Ile Trp Ser Leu
    435 440 445
    Gly Ile Met Ala Ile Glu Met Val Glu Gly Glu Pro Pro Tyr Leu Asn
    450 455 460
    Glu Asn Pro Leu Arg Ala Leu Tyr Leu Ile Ala Thr Asn Gly Thr Pro
    465 470 475 480
    Glu Leu Gln Asn Pro Glu Arg Leu Ser Ala Val Phe His Asp Phe Leu
    485 490 495
    Asn Arg Cys Leu Glu Met Asp Val Asp Arg Arg Gly Ser Ala Lys Glu
    500 505 510
    Leu Leu Gln His Pro Phe Leu Lys Leu Ala Lys Pro Leu Ser Ser Leu
    515 520 525
    Thr Pro Leu Ile Ile Ala Ala Lys Glu Ala Ile Lys Asn Ser Ser Arg
    530 535 540
    <210> SEQ ID NO 96
    <211> LENGTH: 2110
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 96
    ggccaagacg gtcggggctg cttgctaact ccaggaacag gtttaagttt ttgaaactga 60
    agtaggtcta cacagtagga actcatgtca tttcttgtaa gtaaaccaga gcgaatcagg 120
    cggtgggtct cggaaaagtt cattgttgag ggcttaagag atttggaact atttggagac 180
    caatgatgcg agctcagagt caatagcatc cttctctaaa caggaggtca tgagtagctt 240
    tctgccagag ggagggtgtt acgagctgct cactgtgata ggcaaaggat ttgaggacct 300
    gatgactgtg aatctagcaa ggtacaaacc aacaggagag tacgtgactg tacggaggat 360
    taacctagaa gcttgttcca atgagatggt aacattcttg cagggcgagc tgcatgtctc 420
    caaactcttc aaccatccca atatcgtgcc atatcgagcc acttttattg cagacaatga 480
    gctgtgggtt gtcacatcat tcatggcata cggttctgca aaagatctca tctgtacaca 540
    cttcatggat ggcatgaatg agctggcgat tgcttacatc ctgcaggggg tgctgaaggc 600
    cctcgactac atccaccaca tgggatatgt acacaggagt gtcaaagcca gccacatcct 660
    gatctctgtg gatgggaagg tctacctgtc tggtttgcgc agcaacctca gcatgataag 720
    ccatgggcag cggcagcgag tggtccacga ttttcccaag tacagtgtca aggttctgcc 780
    gtggctcagc cccgaggtcc tccagcagaa tctccagggt tatgatgcca agtctgacat 840
    ctacagtgtg ggaatcacag cctgtgaact ggccaacggc catgtcccct ttaaggatat 900
    gcctgccacc cagatgctgc tagagaaact gaacggcaca gtgccctgcc tgttggatac 960
    cagcaccatc cccgctgagg agctgaccat gagcccttcg cgctcagtgg ccaactctgg 1020
    cctgagtgac agcctgacca ccagcacccc ccggccctcc aacggtgact cgccctccca 1080
    cccctaccac cgaaccttct ccccccactt ccaccacttt gtggagcagt gccttcagcg 1140
    caacccggat gccaggccca gtgccagcac cctcctgaac cactctttct tcaagcagat 1200
    caagcgacgt gcctcagagg ctttgcccga attgcttcgt cctgtcaccc ccatcaccaa 1260
    ttttgagggc agccagtctc aggaccacag tggaatcttt ggcctggtaa caaacctgga 1320
    agagctggag gtggacgatt gggagttctg agcctctgca aactgtgcgc attctccagc 1380
    cagggatgca gaggccaccc agaggccctt cctgagggcc ggccacattc ccgccctcct 1440
    gggcagattg ggtagaaagg acattcttcc aggaaagttg actgctgact gattgggaaa 1500
    gaaaatcctg gagagatact tcactgctcc aaggcttttg agacacaagg gaatctcaac 1560
    aaccagggat caggagggtc caaagccgac attcccagtc ctgtgagctc aggtgacctc 1620
    ctccgcagaa gagagatgct gctctggccc tgggagctga attccaagcc cagggtttgg 1680
    ctccttaaac ccgaggaccg ccacctcttc ccagtgcttg cgaccagcct cattctattt 1740
    aactttgctc tcagatgcct cagatgctat aggtcagtga aagggcaagt agtaagctgc 1800
    ctgcctccct tccctcagac ctctccctca taattccaga gaagggcatt tctgtctttt 1860
    taagcacaga ctaaggctgg aacagtccat ccttatccct cttctggctt gggccctgac 1920
    acctaagtct ttcccacggt ttatgtgtgt gcctcattcc tttcccacca agaatccatc 1980
    ttagcgcctc ctgccagctg ccctggtgct ttctccaagg gccatcagtg tcttgcctag 2040
    cttgagggct taagtcctta tgctgtgtta gtttcgttgt cagaacaaat taaaattttc 2100
    agagacgctg 2110
    <210> SEQ ID NO 97
    <211> LENGTH: 373
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 97
    Met Ser Ser Phe Leu Pro Glu Gly Gly Cys Tyr Glu Leu Leu Thr Val
    1 5 10 15
    Ile Gly Lys Gly Phe Glu Asp Leu Met Thr Val Asn Leu Ala Arg Tyr
    20 25 30
    Lys Pro Thr Gly Glu Tyr Val Thr Val Arg Arg Ile Asn Leu Glu Ala
    35 40 45
    Cys Ser Asn Glu Met Val Thr Phe Leu Gln Gly Glu Leu His Val Ser
    50 55 60
    Lys Leu Phe Asn His Pro Asn Ile Val Pro Tyr Arg Ala Thr Phe Ile
    65 70 75 80
    Ala Asp Asn Glu Leu Trp Val Val Thr Ser Phe Met Ala Tyr Gly Ser
    85 90 95
    Ala Lys Asp Leu Ile Cys Thr His Phe Met Asp Gly Met Asn Glu Leu
    100 105 110
    Ala Ile Ala Tyr Ile Leu Gln Gly Val Leu Lys Ala Leu Asp Tyr Ile
    115 120 125
    His His Met Gly Tyr Val His Arg Ser Val Lys Ala Ser His Ile Leu
    130 135 140
    Ile Ser Val Asp Gly Lys Val Tyr Leu Ser Gly Leu Arg Ser Asn Leu
    145 150 155 160
    Ser Met Ile Ser His Gly Gln Arg Gln Arg Val Val His Asp Phe Pro
    165 170 175
    Lys Tyr Ser Val Lys Val Leu Pro Trp Leu Ser Pro Glu Val Leu Gln
    180 185 190
    Gln Asn Leu Gln Gly Tyr Asp Ala Lys Ser Asp Ile Tyr Ser Val Gly
    195 200 205
    Ile Thr Ala Cys Glu Leu Ala Asn Gly His Val Pro Phe Lys Asp Met
    210 215 220
    Pro Ala Thr Gln Met Leu Leu Glu Lys Leu Asn Gly Thr Val Pro Cys
    225 230 235 240
    Leu Leu Asp Thr Ser Thr Ile Pro Ala Glu Glu Leu Thr Met Ser Pro
    245 250 255
    Ser Arg Ser Val Ala Asn Ser Gly Leu Ser Asp Ser Leu Thr Thr Ser
    260 265 270
    Thr Pro Arg Pro Ser Asn Gly Asp Ser Pro Ser His Pro Tyr His Arg
    275 280 285
    Thr Phe Ser Pro His Phe His His Phe Val Glu Gln Cys Leu Gln Arg
    290 295 300
    Asn Pro Asp Ala Arg Pro Ser Ala Ser Thr Leu Leu Asn His Ser Phe
    305 310 315 320
    Phe Lys Gln Ile Lys Arg Arg Ala Ser Glu Ala Leu Pro Glu Leu Leu
    325 330 335
    Arg Pro Val Thr Pro Ile Thr Asn Phe Glu Gly Ser Gln Ser Gln Asp
    340 345 350
    His Ser Gly Ile Phe Gly Leu Val Thr Asn Leu Glu Glu Leu Glu Val
    355 360 365
    Asp Asp Trp Glu Phe
    370
    <210> SEQ ID NO 98
    <211> LENGTH: 2001
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 98
    aaggaagata aaacaaaagc cttctttgga atagatggat ttttgtcact ttctgtgtga 60
    actaaagtga ttcaatgtct cttttggatt gcttctgcac ttcaagaaca caagttgaat 120
    cactcagacc tgaaaaacag tctgaaacca gtatccatca atacttggtt gatgagccaa 180
    ccctttcctg gtcacgtcca tccactagag ccagtgaagt actatgttcc accaacgttt 240
    ctcactatga gctccaagta gaaataggaa gaggatttga caacttgact tctgtccatc 300
    ttgcacggca tactcccacg ggaacactgg taactataaa aattacaaat ctggaaaact 360
    gcaatgaaga acgcctgaaa gctttacaga aagccgtgat tctatcccac tttttccggc 420
    atcccaatat tacaacttat tggacagttt tcactgttgg cagctggctt tgggttattt 480
    ctccatttat ggcctatggt tcagcaagtc aactcttgag gacctatttt cctgaaggaa 540
    tgagtgaaac tttaataaga aacattctct ttggagccgt gagagggttg aactatctgc 600
    accaaaatgg ctgtattcac aggagtatta aagccagcca tatcctcatt tctggtgatg 660
    gcctagtgac cctctctggc ctttcccatc tgcatagttt ggttaagcat ggacagaggc 720
    atagggctgt gtatgatttc ccacagttca gcacatcagt gcagccgtgg ctgagtccag 780
    aactactgag acaggattta catgggtata atgtgaagtc agatatttac agtgttggga 840
    ttacagcatg tgaattagcc agtgggcagg tgcctttcca ggacatgcat agaactcaga 900
    tgctgttaca gaaactgaaa ggtcctcctt atagcccatt ggatatcagt attttccctc 960
    aatcagaatc cagaatgaaa aattcccagt caggtgtaga ctctgggatt ggagaaagtg 1020
    tgcttgtctc cagtggaact cacacagtaa atagtgaccg attacacaca ccatcctcaa 1080
    aaactttctc tcctgccttc tttagcttgg tacagctctg tttgcaacaa gatcctgaga 1140
    aaaggccatc agcaagcagt ttattgtccc atgttttctt caaacagatg aaagaagaaa 1200
    gccaggattc aatactttca ctgttgcctc ctgcttataa caagccatca atatcattgc 1260
    ctccagtgtt accttggact gagccagaat gtgattttcc tgatgaaaaa gactcatact 1320
    gggaattcta gggctgccaa atcattttat gtcctatata cttgacactt tctccttgct 1380
    gctttttctt ctgtatttct aggtacaaat accagaatta tacttgaaaa tacagttggt 1440
    gcactggaga atctattatt taaaaccact ctgttcaaag gggcaccagt ttgtagtccc 1500
    tctgtttcgc acagagtact atgacaagga aacatcagaa ttactaatct agctagtgtc 1560
    atttattctg gaattttttt ctaagctgtg actaactctt tttatctctc aatataattt 1620
    ttgagccagt taattttttt cagtattttg ctgtcccttg ggaatgggcc ctcagaggac 1680
    agtgcttcca agtacatctt ctcccagatt ctctggcctt tttaatgagc tattgttaaa 1740
    ccaacaggct agtttatctt acatcagacc cttttctggt agagggaaaa tgtttgtgct 1800
    ttcccttttt cttctgttaa tacttatggt aacacctaac tgagcctcac tcacattaaa 1860
    tgattcactt gaaatatata cagaaattgt aatttgcttt tttttaaaaa agggggctaa 1920
    agtaacactt tcctacttat gtaaattata gatcctaaat tcacgcaccc cgtgggagct 1980
    caataaagat ttactgaatt g 2001
    <210> SEQ ID NO 99
    <211> LENGTH: 418
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 99
    Met Ser Leu Leu Asp Cys Phe Cys Thr Ser Arg Thr Gln Val Glu Ser
    1 5 10 15
    Leu Arg Pro Glu Lys Gln Ser Glu Thr Ser Ile His Gln Tyr Leu Val
    20 25 30
    Asp Glu Pro Thr Leu Ser Trp Ser Arg Pro Ser Thr Arg Ala Ser Glu
    35 40 45
    Val Leu Cys Ser Thr Asn Val Ser His Tyr Glu Leu Gln Val Glu Ile
    50 55 60
    Gly Arg Gly Phe Asp Asn Leu Thr Ser Val His Leu Ala Arg His Thr
    65 70 75 80
    Pro Thr Gly Thr Leu Val Thr Ile Lys Ile Thr Asn Leu Glu Asn Cys
    85 90 95
    Asn Glu Glu Arg Leu Lys Ala Leu Gln Lys Ala Val Ile Leu Ser His
    100 105 110
    Phe Phe Arg His Pro Asn Ile Thr Thr Tyr Trp Thr Val Phe Thr Val
    115 120 125
    Gly Ser Trp Leu Trp Val Ile Ser Pro Phe Met Ala Tyr Gly Ser Ala
    130 135 140
    Ser Gln Leu Leu Arg Thr Tyr Phe Pro Glu Gly Met Ser Glu Thr Leu
    145 150 155 160
    Ile Arg Asn Ile Leu Phe Gly Ala Val Arg Gly Leu Asn Tyr Leu His
    165 170 175
    Gln Asn Gly Cys Ile His Arg Ser Ile Lys Ala Ser His Ile Leu Ile
    180 185 190
    Ser Gly Asp Gly Leu Val Thr Leu Ser Gly Leu Ser His Leu His Ser
    195 200 205
    Leu Val Lys His Gly Gln Arg His Arg Ala Val Tyr Asp Phe Pro Gln
    210 215 220
    Phe Ser Thr Ser Val Gln Pro Trp Leu Ser Pro Glu Leu Leu Arg Gln
    225 230 235 240
    Asp Leu His Gly Tyr Asn Val Lys Ser Asp Ile Tyr Ser Val Gly Ile
    245 250 255
    Thr Ala Cys Glu Leu Ala Ser Gly Gln Val Pro Phe Gln Asp Met His
    260 265 270
    Arg Thr Gln Met Leu Leu Gln Lys Leu Lys Gly Pro Pro Tyr Ser Pro
    275 280 285
    Leu Asp Ile Ser Ile Phe Pro Gln Ser Glu Ser Arg Met Lys Asn Ser
    290 295 300
    Gln Ser Gly Val Asp Ser Gly Ile Gly Glu Ser Val Leu Val Ser Ser
    305 310 315 320
    Gly Thr His Thr Val Asn Ser Asp Arg Leu His Thr Pro Ser Ser Lys
    325 330 335
    Thr Phe Ser Pro Ala Phe Phe Ser Leu Val Gln Leu Cys Leu Gln Gln
    340 345 350
    Asp Pro Glu Lys Arg Pro Ser Ala Ser Ser Leu Leu Ser His Val Phe
    355 360 365
    Phe Lys Gln Met Lys Glu Glu Ser Gln Asp Ser Ile Leu Ser Leu Leu
    370 375 380
    Pro Pro Ala Tyr Asn Lys Pro Ser Ile Ser Leu Pro Pro Val Leu Pro
    385 390 395 400
    Trp Thr Glu Pro Glu Cys Asp Phe Pro Asp Glu Lys Asp Ser Tyr Trp
    405 410 415
    Glu Phe
    <210> SEQ ID NO 100
    <211> LENGTH: 311
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 100
    tcaacaggga cgattacgag ctgcaggagg tgatcgggag tggagcaact gctgtagtcc 60
    aagcagctta ttgtgcccct aaaaaggaga aagtggcaat caaacggata aaccttgaga 120
    aatgtcaaac tagcatggat gaactcctga aagaaattca agccatgagt caatgccatc 180
    atcctaatat tgtatcttac tacacatctt ttgtggtaaa agatgagctg tggcttgtca 240
    tgaagctgct aagtggaggt tctgttctgg atattattaa gcacattgtg gcaaaagggg 300
    aacacaaaag t 311
    <210> SEQ ID NO 101
    <211> LENGTH: 103
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 101
    Asn Arg Asp Asp Tyr Glu Leu Gln Glu Val Ile Gly Ser Gly Ala Thr
    1 5 10 15
    Ala Val Val Gln Ala Ala Tyr Cys Ala Pro Lys Lys Glu Lys Val Ala
    20 25 30
    Ile Lys Arg Ile Asn Leu Glu Lys Cys Gln Thr Ser Met Asp Glu Leu
    35 40 45
    Leu Lys Glu Ile Gln Ala Met Ser Gln Cys His His Pro Asn Ile Val
    50 55 60
    Ser Tyr Tyr Thr Ser Phe Val Val Lys Asp Glu Leu Trp Leu Val Met
    65 70 75 80
    Lys Leu Leu Ser Gly Gly Ser Val Leu Asp Ile Ile Lys His Ile Val
    85 90 95
    Ala Lys Gly Glu His Lys Ser
    100
    <210> SEQ ID NO 102
    <211> LENGTH: 2806
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 102
    cgggagtgtc cgcggtggtg gcggtgcaag agagctgaag gaggcgcgag ggcgcggagt 60
    tccaggccga gcagttaggc cgcgagcgac tgcggcgccg agccgatgag taacccgaag 120
    cccctagagg agtggtcacc tgcctgaggg cacttctgtc ccaccagcat cagaccaggc 180
    cgcaccgagt ccccggcacc atgtttggga agaggaagaa gcgggtggag atctccgcgc 240
    cgtccaactt cgagcaccgc gtgcacacgg gcttcgacca gcacgagcag aagttcacgg 300
    ggctgccccg ccagtggcag agcctgatcg aggagtcggc tcgccggccc aagcccctcg 360
    tcgaccccgc ctgcatcacc tccatccagc ccggggcccc caagaccatc gtgcggggca 420
    gcaaaggtgc caaagatggg gccctcacgc tgctgctgga cgagtttgag aacatgtcgg 480
    tgacacgctc caactccctg cggagagaca gcccgccgcc gcccgcccgt gcccgccagg 540
    aaaatgggat gccagaggag ccggccacca cggccagagg gggcccaggg aaggcaggca 600
    gccgaggccg gttcgccggt cacagcgagg cgggtggcgg cagtggtgac aggcgacggg 660
    cggggccaga gaagaggccc aagtcttcca gggagggctc agggggtccc caggagtcct 720
    cccgggacaa acgccccctc tccgggcctg atgtcggcac cccccagcct gctggtctgg 780
    ccagtggggc gaaactggca gctggccggc cctttaacac ctacccgagg gctgacacgg 840
    accacccatc ccggggtgcc cagggggagc ctcatgacgt ggcccctaac gggccatcag 900
    cggggggcct ggccatcccc cagtcctcct cctcctcctc ccggcctccc acccgagccc 960
    gaggtgcccc cagccctgga gtgctgggac cccacgcctc agagccccag ctggcccctc 1020
    cagcctgcac ccccgccgcc cctgctgttc ctgggccccc tggcccccgc tcaccacagc 1080
    gggagccaca gcgagtatcc catgagcagt tccgggctgc cctgcagctg gtggtggacc 1140
    caggcgaccc ccgctcctac ctggacaact tcatcaagat tggcgagggc tccacgggca 1200
    tcgtgtgcat cgccaccgtg cgcagctcgg gcaagctggt ggccgtcaag aagatggacc 1260
    tgcgcaagca gcagaggcgc gagctgctct tcaacgaggt ggtaatcatg agggactacc 1320
    agcacgagaa tgtggtggag atgtacaaca gctacctggt gggggacgag ctctgggtgg 1380
    tcatggagtt cctggaagga ggcgccctca ccgacatcgt cacccacacc aggatgaacg 1440
    aggagcagat cgcggccgtg tgccttgcag tgctgcaggc cctgtcggtg ctccacgccc 1500
    agggcgtcat ccaccgggac atcaagagcg actcgatcct gctgacccat gatggcaggg 1560
    tgaagctgtc agactttggg ttctgcgccc aggtgagcaa ggaagtgccc cgaaggaagt 1620
    cgctggtcgg cacgccctac tggatggccc cagagctcat ctcccgcctt ccctacgggc 1680
    cagaggtaga catctggtcg ctggggataa tggtgattga gatggtggac ggagagcccc 1740
    cctacttcaa cgagccaccc ctcaaagcca tgaagatgat tcgggacaac ctgccacccc 1800
    gactgaagaa cctgcacaag gtgtcgccat ccctgaaggg cttcctggac cgcctgctgg 1860
    tgcgagaccc tgcccagcgg gccacggcag ccgagctgct gaagcaccca ttcctggcca 1920
    aggcagggcc gcctgccagc atcgtgcccc tcatgcgcca gaaccgcacc agatgaggcc 1980
    cagcgccctt cccctcaacc aaagagcccc cccgggtcac ccccgcccca ctgaggccag 2040
    tagggggcca ggcctcccac tcctcccagc ccgggagatg ctccgcgtgg caccaccctc 2100
    cttgctgggg gtagatgaga ccctactact gaactccagt tttgatctcg tgacttttag 2160
    aaaaacacag ggactcgtgg gagcaagcga ggctcccagg acccccaccc tctgggacag 2220
    gccctccccc atgttcttct gtctccagga agggcagcgg ccctcccatc actggaagtc 2280
    tgcagtgggg gtcgctgggg gtggagagaa cactaagagg tgaacatgta tgagtgtgtg 2340
    cacgcgtgtg agtgtgcatg tgtgtgtgtg tgcaaaggtc cagccacccc gtcctccagc 2400
    ccgcaagggg tgtctggcgc cttgcctgac acccagcccc ctctccccct gagccattgt 2460
    gggggtcgat catgaatgtc cgaagagtgg ccttttcccg tagccctgcg ccccctttct 2520
    gtggctggat ggggagacag gtcagggccc cccaccctct ccagcccctg cagcaaatga 2580
    ctactgcacc tggacagcct cctcttttct agaagtctat ttatattgtc attttataac 2640
    actctagccc ctgcccttat tgggggacag atggtccctg tcctgcgggg tggccctggc 2700
    agaaccactg cctgaagaac caggttcctg cccggtcagc gcagccccag cccgcccacc 2760
    cctgcctcga gttagtttta caattaaaac attgtcttgt tttgtg 2806
    <210> SEQ ID NO 103
    <211> LENGTH: 591
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 103
    Met Phe Gly Lys Arg Lys Lys Arg Val Glu Ile Ser Ala Pro Ser Asn
    1 5 10 15
    Phe Glu His Arg Val His Thr Gly Phe Asp Gln His Glu Gln Lys Phe
    20 25 30
    Thr Gly Leu Pro Arg Gln Trp Gln Ser Leu Ile Glu Glu Ser Ala Arg
    35 40 45
    Arg Pro Lys Pro Leu Val Asp Pro Ala Cys Ile Thr Ser Ile Gln Pro
    50 55 60
    Gly Ala Pro Lys Thr Ile Val Arg Gly Ser Lys Gly Ala Lys Asp Gly
    65 70 75 80
    Ala Leu Thr Leu Leu Leu Asp Glu Phe Glu Asn Met Ser Val Thr Arg
    85 90 95
    Ser Asn Ser Leu Arg Arg Asp Ser Pro Pro Pro Pro Ala Arg Ala Arg
    100 105 110
    Gln Glu Asn Gly Met Pro Glu Glu Pro Ala Thr Thr Ala Arg Gly Gly
    115 120 125
    Pro Gly Lys Ala Gly Ser Arg Gly Arg Phe Ala Gly His Ser Glu Ala
    130 135 140
    Gly Gly Gly Ser Gly Asp Arg Arg Arg Ala Gly Pro Glu Lys Arg Pro
    145 150 155 160
    Lys Ser Ser Arg Glu Gly Ser Gly Gly Pro Gln Glu Ser Ser Arg Asp
    165 170 175
    Lys Arg Pro Leu Ser Gly Pro Asp Val Gly Thr Pro Gln Pro Ala Gly
    180 185 190
    Leu Ala Ser Gly Ala Lys Leu Ala Ala Gly Arg Pro Phe Asn Thr Tyr
    195 200 205
    Pro Arg Ala Asp Thr Asp His Pro Ser Arg Gly Ala Gln Gly Glu Pro
    210 215 220
    His Asp Val Ala Pro Asn Gly Pro Ser Ala Gly Gly Leu Ala Ile Pro
    225 230 235 240
    Gln Ser Ser Ser Ser Ser Ser Arg Pro Pro Thr Arg Ala Arg Gly Ala
    245 250 255
    Pro Ser Pro Gly Val Leu Gly Pro His Ala Ser Glu Pro Gln Leu Ala
    260 265 270
    Pro Pro Ala Cys Thr Pro Ala Ala Pro Ala Val Pro Gly Pro Pro Gly
    275 280 285
    Pro Arg Ser Pro Gln Arg Glu Pro Gln Arg Val Ser His Glu Gln Phe
    290 295 300
    Arg Ala Ala Leu Gln Leu Val Val Asp Pro Gly Asp Pro Arg Ser Tyr
    305 310 315 320
    Leu Asp Asn Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val Cys
    325 330 335
    Ile Ala Thr Val Arg Ser Ser Gly Lys Leu Val Ala Val Lys Lys Met
    340 345 350
    Asp Leu Arg Lys Gln Gln Arg Arg Glu Leu Leu Phe Asn Glu Val Val
    355 360 365
    Ile Met Arg Asp Tyr Gln His Glu Asn Val Val Glu Met Tyr Asn Ser
    370 375 380
    Tyr Leu Val Gly Asp Glu Leu Trp Val Val Met Glu Phe Leu Glu Gly
    385 390 395 400
    Gly Ala Leu Thr Asp Ile Val Thr His Thr Arg Met Asn Glu Glu Gln
    405 410 415
    Ile Ala Ala Val Cys Leu Ala Val Leu Gln Ala Leu Ser Val Leu His
    420 425 430
    Ala Gln Gly Val Ile His Arg Asp Ile Lys Ser Asp Ser Ile Leu Leu
    435 440 445
    Thr His Asp Gly Arg Val Lys Leu Ser Asp Phe Gly Phe Cys Ala Gln
    450 455 460
    Val Ser Lys Glu Val Pro Arg Arg Lys Ser Leu Val Gly Thr Pro Tyr
    465 470 475 480
    Trp Met Ala Pro Glu Leu Ile Ser Arg Leu Pro Tyr Gly Pro Glu Val
    485 490 495
    Asp Ile Trp Ser Leu Gly Ile Met Val Ile Glu Met Val Asp Gly Glu
    500 505 510
    Pro Pro Tyr Phe Asn Glu Pro Pro Leu Lys Ala Met Lys Met Ile Arg
    515 520 525
    Asp Asn Leu Pro Pro Arg Leu Lys Asn Leu His Lys Val Ser Pro Ser
    530 535 540
    Leu Lys Gly Phe Leu Asp Arg Leu Leu Val Arg Asp Pro Ala Gln Arg
    545 550 555 560
    Ala Thr Ala Ala Glu Leu Leu Lys His Pro Phe Leu Ala Lys Ala Gly
    565 570 575
    Pro Pro Ala Ser Ile Val Pro Leu Met Arg Gln Asn Arg Thr Arg
    580 585 590
    <210> SEQ ID NO 104
    <211> LENGTH: 3684
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 104
    atggcgggac ctgggggctg gagggacagg gaggtcacgg atctgggcca cctgccggat 60
    ccaactggaa tattctcact agataaaacc attggccttg gtacttatgg cagaatctat 120
    ttgggacttc atgagaagac tggtgcattt acagctgtta aagtgatgaa cgctcgtaag 180
    gatgaggaag aggatctcag gactgaactc aaccttctga ggaagtactc tttccacaaa 240
    aacattgtgt ccttctatgg agcatttttc aagctgagtc cccctggtca gcggcaccaa 300
    ctttggatgg tgatggagtt atgtgcagca ggttcggtca ctgatgtagt gagaatgacc 360
    agtaatcaga gtttaaaaga agattggatt gcttatatct gccgagaaat ccttcagggc 420
    ttagctcacc ttcacgcaca ccgagtaatt caccgggaca tcaaaggtca gaatgtgctg 480
    ctgactcata atgctgaagt aaaactggtt gattttggag tgagtgccca ggtgagcaga 540
    actaatggaa gaaggaatag tttcattggg acaccatact ggatggcacc tgaggtgatt 600
    gactgtgatg aggacccaag acgctcctat gattacagaa gtgatgtgtg gtctgtggga 660
    attactgcca ttgaaatggc tgaaggagcc cctcctctgt gtaaccttca acccttggaa 720
    gctctcttcg ttattttgcg ggaatctgct cccacagtca aatccagcgg atggtcccgt 780
    aagttccaca atttcatgga aaagtgtacg ataaaaaatt tcctgtttcg tcctacttct 840
    gcaaacatgc ttcaacaccc atttgttcgg gatataaaaa atgaacgaca tgttgttgag 900
    tcattaacaa ggcatcttac tggaatcatt aaaaaaagac agaaaaaaga acaggcacgg 960
    gagaaaaaat caaaagtttc tactctgagg caagcactgg caaaaagact atcaccaaag 1020
    aggttcaggg caaagtcatc atggagacct gaaaagcttg aactctcgga tttagaagcc 1080
    cgcaggcaaa ggcgccaacg cagatgggaa gatatcttta atcagcatga ggaagaattg 1140
    agacaagttg ataaagacaa agaagatgaa tcatcagaca atgatgaagt atttcattcg 1200
    attcaggctg aagtccagat agagccattg aagccataca tttcaaatcc taaaaaaatt 1260
    gaggttcaag agagatctcc ttctgtgcct aacaaccagg atcatgcaca tcatgtcaag 1320
    ttctcttcaa gcgttcctca gcggtctctt ttggaacaag ctcagaagcc cattgacatc 1380
    agacaaagga gttcgcaaaa tcgtcaaaat tggctggcag catcaggtga ttcaaagcac 1440
    aaaattttag caggcaaaac acagagctac tgtttaacaa tttatatttc agaagtcaag 1500
    aaagaagaat ttcaagaagg aatgaatcaa aagtgtcagg gagcccaagt aggattagga 1560
    cctgaaggcc attgtatttg gcaattgggt gaatcttctt ctgaggaaga aagtcctgtg 1620
    actggaagga ggtctcagtc atcaccacct tattctacta ttgatcagaa gttgctggtt 1680
    gacatccatg ttccagatgg atttaaagta ggaaaaatat caccccctgt atacttgaca 1740
    aacgaatggg taggctataa tgcactctct gaaatcttcc ggaatgattg gttaactccg 1800
    gcacctgtca ttcagccacc tgaagaggat ggtgattatg ttgaactcta tgatgccagt 1860
    gctgatactg atggtgatga tgatgatgag tctaatgata cttttgaaga tacctatgat 1920
    catgccaatg gcaatgatga cttggataac caggttgatc aggctaatga tgtttgtaaa 1980
    gaccatgatg atgacaacaa taagtttgtt gatgatgtaa ataataatta ttatgaggcg 2040
    cctagttgtc caagggcaag ctatggcaga gatggaagct gcaagcaaga tggttatgat 2100
    ggaagtcgtg gaaaagagga agcctacaga ggctatggaa gccatacagc caatagaagc 2160
    catggaggaa gtgcagccag tgaggacaat gcagccattg gagatcagga agaacatgca 2220
    gccaatatag gcagtgaaag aagaggcagt gagggtgatg gaggtaaggg agtcgttcga 2280
    accagtgaag agagtggagc ccttggactc aatggagaag aaaattgctc agagacagat 2340
    ggtccaggat tgaagagacc tgcgtctcag gactttgaat atctacagga ggagccaggt 2400
    ggtggaaatg aggcctcaaa tgccattgac tcaggtgctg caccgtcagc acctgatcat 2460
    gagagtgaca ataaggacat atcagaatca tcaacacaat cagatttttc tgccaatcac 2520
    tcatctcctt ccaaaggttc tgggatgtct gctgatgcta actttgccag tgccatctta 2580
    tacgctggat tcgtagaagt acctgaggaa tcacctaagc aaccctctga agtcaatgtt 2640
    aacccactct atgtctctcc tgcatgtaaa aaaccactaa tccacatgta tgaaaaggag 2700
    ttcacttctg agatctgctg tggttctttg tggggagtca atttgctgtt gggaacccga 2760
    tctaatctat atctgatgga cagaagtgga aaggctgaca ttactaaact tataaggcga 2820
    agaccattcc gccagattca agtcttagag ccactcaatt tgctgattac catctcaggt 2880
    cataagaaca gacttcgggt gtatcatctg acctggttga ggaacaagat tttgaataat 2940
    gatccagaaa gtaaaagaag gcaagaagaa atgctgaaga cagaggaagc ctgcaaagct 3000
    attgataagt taacaggctg tgaacacttc agtgtcctcc aacatgaaga aacaacatat 3060
    attgcaattg ctttgaaatc atcaattcac ctttatgcat gggcaccaaa gtcctttgat 3120
    gaaagcactg ctattaaagt atttccaaca cttgatcata agccagtgac agttgacctg 3180
    gctattggtt ctgaaaaaag actaaagatt ttcttcagct cagcagatgg atatcacctc 3240
    atcgatgcag aatctgaggt tatgtctgat gtgaccctgc caaagaatcc cctggaaatc 3300
    attataccac agaatatcat cattttacct gattgcttgg gaattggcat gatgctcacc 3360
    ttcaatgctg aagccctctc tgtggaagca aatgaacaac tcttcaagaa gatccttgaa 3420
    atgtggaaag acataccatc ttctatagct tttgaatgta cacagcgaac cacaggatgg 3480
    ggccaaaagg ccattgaagt gcgctctttg caatccaggg ttctggaaag tgagctgaag 3540
    cgcaggtcaa ttaagaagct gagattcctg tgcacccggg gtgacaagct gttctttacc 3600
    tctaccctgc gcaatcacca cagccgggtt tacttcatga cacttggaaa acttgaagag 3660
    ctccaaagca attatgatgt ctaa 3684
    <210> SEQ ID NO 105
    <211> LENGTH: 1227
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 105
    Met Ala Gly Pro Gly Gly Trp Arg Asp Arg Glu Val Thr Asp Leu Gly
    1 5 10 15
    His Leu Pro Asp Pro Thr Gly Ile Phe Ser Leu Asp Lys Thr Ile Gly
    20 25 30
    Leu Gly Thr Tyr Gly Arg Ile Tyr Leu Gly Leu His Glu Lys Thr Gly
    35 40 45
    Ala Phe Thr Ala Val Lys Val Met Asn Ala Arg Lys Asp Glu Glu Glu
    50 55 60
    Asp Leu Arg Thr Glu Leu Asn Leu Leu Arg Lys Tyr Ser Phe His Lys
    65 70 75 80
    Asn Ile Val Ser Phe Tyr Gly Ala Phe Phe Lys Leu Ser Pro Pro Gly
    85 90 95
    Gln Arg His Gln Leu Trp Met Val Met Glu Leu Cys Ala Ala Gly Ser
    100 105 110
    Val Thr Asp Val Val Arg Met Thr Ser Asn Gln Ser Leu Lys Glu Asp
    115 120 125
    Trp Ile Ala Tyr Ile Cys Arg Glu Ile Leu Gln Gly Leu Ala His Leu
    130 135 140
    His Ala His Arg Val Ile His Arg Asp Ile Lys Gly Gln Asn Val Leu
    145 150 155 160
    Leu Thr His Asn Ala Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala
    165 170 175
    Gln Val Ser Arg Thr Asn Gly Arg Arg Asn Ser Phe Ile Gly Thr Pro
    180 185 190
    Tyr Trp Met Ala Pro Glu Val Ile Asp Cys Asp Glu Asp Pro Arg Arg
    195 200 205
    Ser Tyr Asp Tyr Arg Ser Asp Val Trp Ser Val Gly Ile Thr Ala Ile
    210 215 220
    Glu Met Ala Glu Gly Ala Pro Pro Leu Cys Asn Leu Gln Pro Leu Glu
    225 230 235 240
    Ala Leu Phe Val Ile Leu Arg Glu Ser Ala Pro Thr Val Lys Ser Ser
    245 250 255
    Gly Trp Ser Arg Lys Phe His Asn Phe Met Glu Lys Cys Thr Ile Lys
    260 265 270
    Asn Phe Leu Phe Arg Pro Thr Ser Ala Asn Met Leu Gln His Pro Phe
    275 280 285
    Val Arg Asp Ile Lys Asn Glu Arg His Val Val Glu Ser Leu Thr Arg
    290 295 300
    His Leu Thr Gly Ile Ile Lys Lys Arg Gln Lys Lys Glu Gln Ala Arg
    305 310 315 320
    Glu Lys Lys Ser Lys Val Ser Thr Leu Arg Gln Ala Leu Ala Lys Arg
    325 330 335
    Leu Ser Pro Lys Arg Phe Arg Ala Lys Ser Ser Trp Arg Pro Glu Lys
    340 345 350
    Leu Glu Leu Ser Asp Leu Glu Ala Arg Arg Gln Arg Arg Gln Arg Arg
    355 360 365
    Trp Glu Asp Ile Phe Asn Gln His Glu Glu Glu Leu Arg Gln Val Asp
    370 375 380
    Lys Asp Lys Glu Asp Glu Ser Ser Asp Asn Asp Glu Val Phe His Ser
    385 390 395 400
    Ile Gln Ala Glu Val Gln Ile Glu Pro Leu Lys Pro Tyr Ile Ser Asn
    405 410 415
    Pro Lys Lys Ile Glu Val Gln Glu Arg Ser Pro Ser Val Pro Asn Asn
    420 425 430
    Gln Asp His Ala His His Val Lys Phe Ser Ser Ser Val Pro Gln Arg
    435 440 445
    Ser Leu Leu Glu Gln Ala Gln Lys Pro Ile Asp Ile Arg Gln Arg Ser
    450 455 460
    Ser Gln Asn Arg Gln Asn Trp Leu Ala Ala Ser Gly Asp Ser Lys His
    465 470 475 480
    Lys Ile Leu Ala Gly Lys Thr Gln Ser Tyr Cys Leu Thr Ile Tyr Ile
    485 490 495
    Ser Glu Val Lys Lys Glu Glu Phe Gln Glu Gly Met Asn Gln Lys Cys
    500 505 510
    Gln Gly Ala Gln Val Gly Leu Gly Pro Glu Gly His Cys Ile Trp Gln
    515 520 525
    Leu Gly Glu Ser Ser Ser Glu Glu Glu Ser Pro Val Thr Gly Arg Arg
    530 535 540
    Ser Gln Ser Ser Pro Pro Tyr Ser Thr Ile Asp Gln Lys Leu Leu Val
    545 550 555 560
    Asp Ile His Val Pro Asp Gly Phe Lys Val Gly Lys Ile Ser Pro Pro
    565 570 575
    Val Tyr Leu Thr Asn Glu Trp Val Gly Tyr Asn Ala Leu Ser Glu Ile
    580 585 590
    Phe Arg Asn Asp Trp Leu Thr Pro Ala Pro Val Ile Gln Pro Pro Glu
    595 600 605
    Glu Asp Gly Asp Tyr Val Glu Leu Tyr Asp Ala Ser Ala Asp Thr Asp
    610 615 620
    Gly Asp Asp Asp Asp Glu Ser Asn Asp Thr Phe Glu Asp Thr Tyr Asp
    625 630 635 640
    His Ala Asn Gly Asn Asp Asp Leu Asp Asn Gln Val Asp Gln Ala Asn
    645 650 655
    Asp Val Cys Lys Asp His Asp Asp Asp Asn Asn Lys Phe Val Asp Asp
    660 665 670
    Val Asn Asn Asn Tyr Tyr Glu Ala Pro Ser Cys Pro Arg Ala Ser Tyr
    675 680 685
    Gly Arg Asp Gly Ser Cys Lys Gln Asp Gly Tyr Asp Gly Ser Arg Gly
    690 695 700
    Lys Glu Glu Ala Tyr Arg Gly Tyr Gly Ser His Thr Ala Asn Arg Ser
    705 710 715 720
    His Gly Gly Ser Ala Ala Ser Glu Asp Asn Ala Ala Ile Gly Asp Gln
    725 730 735
    Glu Glu His Ala Ala Asn Ile Gly Ser Glu Arg Arg Gly Ser Glu Gly
    740 745 750
    Asp Gly Gly Lys Gly Val Val Arg Thr Ser Glu Glu Ser Gly Ala Leu
    755 760 765
    Gly Leu Asn Gly Glu Glu Asn Cys Ser Glu Thr Asp Gly Pro Gly Leu
    770 775 780
    Lys Arg Pro Ala Ser Gln Asp Phe Glu Tyr Leu Gln Glu Glu Pro Gly
    785 790 795 800
    Gly Gly Asn Glu Ala Ser Asn Ala Ile Asp Ser Gly Ala Ala Pro Ser
    805 810 815
    Ala Pro Asp His Glu Ser Asp Asn Lys Asp Ile Ser Glu Ser Ser Thr
    820 825 830
    Gln Ser Asp Phe Ser Ala Asn His Ser Ser Pro Ser Lys Gly Ser Gly
    835 840 845
    Met Ser Ala Asp Ala Asn Phe Ala Ser Ala Ile Leu Tyr Ala Gly Phe
    850 855 860
    Val Glu Val Pro Glu Glu Ser Pro Lys Gln Pro Ser Glu Val Asn Val
    865 870 875 880
    Asn Pro Leu Tyr Val Ser Pro Ala Cys Lys Lys Pro Leu Ile His Met
    885 890 895
    Tyr Glu Lys Glu Phe Thr Ser Glu Ile Cys Cys Gly Ser Leu Trp Gly
    900 905 910
    Val Asn Leu Leu Leu Gly Thr Arg Ser Asn Leu Tyr Leu Met Asp Arg
    915 920 925
    Ser Gly Lys Ala Asp Ile Thr Lys Leu Ile Arg Arg Arg Pro Phe Arg
    930 935 940
    Gln Ile Gln Val Leu Glu Pro Leu Asn Leu Leu Ile Thr Ile Ser Gly
    945 950 955 960
    His Lys Asn Arg Leu Arg Val Tyr His Leu Thr Trp Leu Arg Asn Lys
    965 970 975
    Ile Leu Asn Asn Asp Pro Glu Ser Lys Arg Arg Gln Glu Glu Met Leu
    980 985 990
    Lys Thr Glu Glu Ala Cys Lys Ala Ile Asp Lys Leu Thr Gly Cys Glu
    995 1000 1005
    His Phe Ser Val Leu Gln His Glu Glu Thr Thr Tyr Ile Ala Ile Ala
    1010 1015 1020
    Leu Lys Ser Ser Ile His Leu Tyr Ala Trp Ala Pro Lys Ser Phe Asp
    1025 1030 1035 1040
    Glu Ser Thr Ala Ile Lys Val Phe Pro Thr Leu Asp His Lys Pro Val
    1045 1050 1055
    Thr Val Asp Leu Ala Ile Gly Ser Glu Lys Arg Leu Lys Ile Phe Phe
    1060 1065 1070
    Ser Ser Ala Asp Gly Tyr His Leu Ile Asp Ala Glu Ser Glu Val Met
    1075 1080 1085
    Ser Asp Val Thr Leu Pro Lys Asn Pro Leu Glu Ile Ile Ile Pro Gln
    1090 1095 1100
    Asn Ile Ile Ile Leu Pro Asp Cys Leu Gly Ile Gly Met Met Leu Thr
    1105 1110 1115 1120
    Phe Asn Ala Glu Ala Leu Ser Val Glu Ala Asn Glu Gln Leu Phe Lys
    1125 1130 1135
    Lys Ile Leu Glu Met Trp Lys Asp Ile Pro Ser Ser Ile Ala Phe Glu
    1140 1145 1150
    Cys Thr Gln Arg Thr Thr Gly Trp Gly Gln Lys Ala Ile Glu Val Arg
    1155 1160 1165
    Ser Leu Gln Ser Arg Val Leu Glu Ser Glu Leu Lys Arg Arg Ser Ile
    1170 1175 1180
    Lys Lys Leu Arg Phe Leu Cys Thr Arg Gly Asp Lys Leu Phe Phe Thr
    1185 1190 1195 1200
    Ser Thr Leu Arg Asn His His Ser Arg Val Tyr Phe Met Thr Leu Gly
    1205 1210 1215
    Lys Leu Glu Glu Leu Gln Ser Asn Tyr Asp Val
    1220 1225
    SEQ ID NO 106
    LENGTH: 2962
    TYPE: DNA
    ORGANISM: Homo sapiens
    SEQUENCE: 106
    cgaagccaca gcccgagccc gagcccgagc ccgagccggc gccaccgcgc ccccggccat 60
    ggcttttgcc aatttccgcc gcatcctgcg cctgtctacc ttcgagaaga gaaagtcccg 120
    cgaatatgag cacgtccgcc gcgacctgga ccccaacgag gtgtgggaga tcgtgggcga 180
    gctgggcgac ggcgccttcg gcaaggttta caaggccaag aataaggaga cgggtgcttt 240
    ggctgcggcc aaagtcattg aaaccaagag tgaggaggag ctggaggact acatcgtgga 300
    gattgagatc ctggccacct gcgaccaccc ctacattgtg aagctcctgg gagcctacta 360
    tcacgacggg aagctgtgga tcatgattga gttctgtcca gggggagccg tggacgccat 420
    catgctggag ctggacagag gcctcacgga gccccagata caggtggttt gccgccagat 480
    gctagaagcc ctcaacttcc tgcacagcaa gaggatcatc caccgagatc tgaaagctgg 540
    caacgtgctg atgaccctcg agggagacat caggctggct gactttggtg tgtctgccaa 600
    gaatctgaag actctacaga aacgagattc cttcatcggc acgccttact ggatggcccc 660
    cgaggtggtc atgtgtgaga ccatgaaaga cacgccctac gactacaaag ccgacatctg 720
    gtccctgggc atcacgctga ttgagatggc ccagatcgag ccgccacacc acgagctcaa 780
    ccccatgcgg gtcctgctaa agatcgccaa gtcggaccct cccacgctgc tcacgccctc 840
    caagtggtct gtagagttcc gtgacttcct gaagatagcc ctggataaga acccagaaac 900
    ccgacccagt gccgcgcagc tgctggagca tcccttcgtc agcagcatca ccagtaacaa 960
    ggctctgcgg gagctggtgg ctgaggccaa ggccgaggtg atggaagaga tcgaagacgg 1020
    ccgggatgag ggggaagagg aggacgccgt ggatgccgcc tccaccctgg agaaccatac 1080
    tcagaactcc tctgaggtga gtccgccaag cctcaatgct gacaagcctc tcgaggagtc 1140
    accttccacc ccgctggcac ccagccagtc tcaggacagt gtgaatgagc cctgcagcca 1200
    gccctctggg gacagatccc tccaaaccac cagtccccca gtcgtggccc ctggaaatga 1260
    gaacggcctg gcagtgcctg tgcccctgcg gaagtcccga cccgtgtcaa tggatgccag 1320
    aattcaggta gcccaggaga agcaagttgc tgagcagggt ggggacctca gcccagcagc 1380
    caacagatct caaaaggcca gccagagccg gcccaacagc agcgccctgg agaccttggg 1440
    tggggagaag ctggccaatg gcagcctgga gccacctgcc caggcagctc cagggccttc 1500
    caagagggac tcggactgca gcagcctctg cacctctgag agcatggact atggtaccaa 1560
    tctctccact gacctgtcgc tgaacaaaga gatgggctct ctgtccatca aggacccgaa 1620
    actgtacaaa aaaaccctca agcggacacg caaatttgtg gtggatggtg tggaggtgag 1680
    catcaccacc tccaagatca tcagcgaaga tgagaagaag gatgaggaga tgagatttct 1740
    caggcgccag gaactccgag agcttcggct gctccagaaa gaagagcatc ggaaccagac 1800
    ccagctgagt aacaagcatg agctgcagct ggagcaaatg cataaacgtt ttgaacagga 1860
    aatcaacgcc aagaagaagt tctttgacac ggaattagag aacctggagc gtcagcaaaa 1920
    gcagcaagtg gagaagatgg agcaagacca tgccgtgcgc cgccgggagg aggccaggcg 1980
    gatccgcctg gagcaggatc gggactacac caggttccaa gagcagctca aactgatgaa 2040
    gaaagaggtg aagaacgagg tggagaagct cccccgacag cagcggaagg aaagcatgaa 2100
    gcagaagatg gaggagcaca cgcagaaaaa gcagcttctt gaccgggact ttgtagccaa 2160
    gcagaaggag gacctggagc tggccatgaa gaggctcacc accgacaaca ggcgggagat 2220
    ctgtgacaag gagcgcgagt gcctcatgaa gaagcaggag ctccttcgag accgggaagc 2280
    agccctgtgg gagatggaag agcaccagct gcaggagagg caccagctgg tgaagcagca 2340
    gctcaaagac cagtacttcc tccagcggca cgagctgctg cgcaagcatg agaaggagcg 2400
    ggagcagatg cagcgctaca accagcgcat gatagagcag ctgaaggtgc ggcagcaaca 2460
    ggaaaaggcg cggctgccca agatccagag gagtgagggc aagacgcgca tggccatgta 2520
    caagaagagc ctccacatca acggcggggg cagcgcagct gagcagcgtg agaagatcaa 2580
    gcagttctcc cagcaggagg agaagaggca gaagtcggag cggctgcagc aacagcagaa 2640
    acacgagaac cagatgcggg acatgctggc gcagtgcgag agcaacatga gcgagctgca 2700
    gcagctgcag aatgaaaagt gccacctcct ggtagagcac gaaacccaga aactgaaggc 2760
    cctggatgag agccataacc agaacctgaa ggaatggcgg gacaagcttc ggccgcgcaa 2820
    gaaggctctg gaagaggatc tgaaccagaa gaagcgggag caggagatgt tcttcaagct 2880
    gagcgaggag gcggagtgcc caaacccctc caccccaagc aaggccgcca agttcttccc 2940
    ctacagctct ggggatgctt cc 2962
    <210> SEQ ID NO 107
    <211> LENGTH: 968
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 107
    Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
    1 5 10 15
    Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp Leu Asp Pro
    20 25 30
    Asn Glu Val Trp Glu Ile Val Gly Glu Leu Gly Asp Gly Ala Phe Gly
    35 40 45
    Lys Val Tyr Lys Ala Lys Asn Lys Glu Thr Gly Ala Leu Ala Ala Ala
    50 55 60
    Lys Val Ile Glu Thr Lys Ser Glu Glu Glu Leu Glu Asp Tyr Ile Val
    65 70 75 80
    Glu Ile Glu Ile Leu Ala Thr Cys Asp His Pro Tyr Ile Val Lys Leu
    85 90 95
    Leu Gly Ala Tyr Tyr His Asp Gly Lys Leu Trp Ile Met Ile Glu Phe
    100 105 110
    Cys Pro Gly Gly Ala Val Asp Ala Ile Met Leu Glu Leu Asp Arg Gly
    115 120 125
    Leu Thr Glu Pro Gln Ile Gln Val Val Cys Arg Gln Met Leu Glu Ala
    130 135 140
    Leu Asn Phe Leu His Ser Lys Arg Ile Ile His Arg Asp Leu Lys Ala
    145 150 155 160
    Gly Asn Val Leu Met Thr Leu Glu Gly Asp Ile Arg Leu Ala Asp Phe
    165 170 175
    Gly Val Ser Ala Lys Asn Leu Lys Thr Leu Gln Lys Arg Asp Ser Phe
    180 185 190
    Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Met Cys Glu Thr
    195 200 205
    Met Lys Asp Thr Pro Tyr Asp Tyr Lys Ala Asp Ile Trp Ser Leu Gly
    210 215 220
    Ile Thr Leu Ile Glu Met Ala Gln Ile Glu Pro Pro His His Glu Leu
    225 230 235 240
    Asn Pro Met Arg Val Leu Leu Lys Ile Ala Lys Ser Asp Pro Pro Thr
    245 250 255
    Leu Leu Thr Pro Ser Lys Trp Ser Val Glu Phe Arg Asp Phe Leu Lys
    260 265 270
    Ile Ala Leu Asp Lys Asn Pro Glu Thr Arg Pro Ser Ala Ala Gln Leu
    275 280 285
    Leu Glu His Pro Phe Val Ser Ser Ile Thr Ser Asn Lys Ala Leu Arg
    290 295 300
    Glu Leu Val Ala Glu Ala Lys Ala Glu Val Met Glu Glu Ile Glu Asp
    305 310 315 320
    Gly Arg Asp Glu Gly Glu Glu Glu Asp Ala Val Asp Ala Ala Ser Thr
    325 330 335
    Leu Glu Asn His Thr Gln Asn Ser Ser Glu Val Ser Pro Pro Ser Leu
    340 345 350
    Asn Ala Asp Lys Pro Leu Glu Glu Ser Pro Ser Thr Pro Leu Ala Pro
    355 360 365
    Ser Gln Ser Gln Asp Ser Val Asn Glu Pro Cys Ser Gln Pro Ser Gly
    370 375 380
    Asp Arg Ser Leu Gln Thr Thr Ser Pro Pro Val Val Ala Pro Gly Asn
    385 390 395 400
    Glu Asn Gly Leu Ala Val Pro Val Pro Leu Arg Lys Ser Arg Pro Val
    405 410 415
    Ser Met Asp Ala Arg Ile Gln Val Ala Gln Glu Lys Gln Val Ala Glu
    420 425 430
    Gln Gly Gly Asp Leu Ser Pro Ala Ala Asn Arg Ser Gln Lys Ala Ser
    435 440 445
    Gln Ser Arg Pro Asn Ser Ser Ala Leu Glu Thr Leu Gly Gly Glu Lys
    450 455 460
    Leu Ala Asn Gly Ser Leu Glu Pro Pro Ala Gln Ala Ala Pro Gly Pro
    465 470 475 480
    Ser Lys Arg Asp Ser Asp Cys Ser Ser Leu Cys Thr Ser Glu Ser Met
    485 490 495
    Asp Tyr Gly Thr Asn Leu Ser Thr Asp Leu Ser Leu Asn Lys Glu Met
    500 505 510
    Gly Ser Leu Ser Ile Lys Asp Pro Lys Leu Tyr Lys Lys Thr Leu Lys
    515 520 525
    Arg Thr Arg Lys Phe Val Val Asp Gly Val Glu Val Ser Ile Thr Thr
    530 535 540
    Ser Lys Ile Ile Ser Glu Asp Glu Lys Lys Asp Glu Glu Met Arg Phe
    545 550 555 560
    Leu Arg Arg Gln Glu Leu Arg Glu Leu Arg Leu Leu Gln Lys Glu Glu
    565 570 575
    His Arg Asn Gln Thr Gln Leu Ser Asn Lys His Glu Leu Gln Leu Glu
    580 585 590
    Gln Met His Lys Arg Phe Glu Gln Glu Ile Asn Ala Lys Lys Lys Phe
    595 600 605
    Phe Asp Thr Glu Leu Glu Asn Leu Glu Arg Gln Gln Lys Gln Gln Val
    610 615 620
    Glu Lys Met Glu Gln Asp His Ala Val Arg Arg Arg Glu Glu Ala Arg
    625 630 635 640
    Arg Ile Arg Leu Glu Gln Asp Arg Asp Tyr Thr Arg Phe Gln Glu Gln
    645 650 655
    Leu Lys Leu Met Lys Lys Glu Val Lys Asn Glu Val Glu Lys Leu Pro
    660 665 670
    Arg Gln Gln Arg Lys Glu Ser Met Lys Gln Lys Met Glu Glu His Thr
    675 680 685
    Gln Lys Lys Gln Leu Leu Asp Arg Asp Phe Val Ala Lys Gln Lys Glu
    690 695 700
    Asp Leu Glu Leu Ala Met Lys Arg Leu Thr Thr Asp Asn Arg Arg Glu
    705 710 715 720
    Ile Cys Asp Lys Glu Arg Glu Cys Leu Met Lys Lys Gln Glu Leu Leu
    725 730 735
    Arg Asp Arg Glu Ala Ala Leu Trp Glu Met Glu Glu His Gln Leu Gln
    740 745 750
    Glu Arg His Gln Leu Val Lys Gln Gln Leu Lys Asp Gln Tyr Phe Leu
    755 760 765
    Gln Arg His Glu Leu Leu Arg Lys His Glu Lys Glu Arg Glu Gln Met
    770 775 780
    Gln Arg Tyr Asn Gln Arg Met Ile Glu Gln Leu Lys Val Arg Gln Gln
    785 790 795 800
    Gln Glu Lys Ala Arg Leu Pro Lys Ile Gln Arg Ser Glu Gly Lys Thr
    805 810 815
    Arg Met Ala Met Tyr Lys Lys Ser Leu His Ile Asn Gly Gly Gly Ser
    820 825 830
    Ala Ala Glu Gln Arg Glu Lys Ile Lys Gln Phe Ser Gln Gln Glu Glu
    835 840 845
    Lys Arg Gln Lys Ser Glu Arg Leu Gln Gln Gln Gln Lys His Glu Asn
    850 855 860
    Gln Met Arg Asp Met Leu Ala Gln Cys Glu Ser Asn Met Ser Glu Leu
    865 870 875 880
    Gln Gln Leu Gln Asn Glu Lys Cys His Leu Leu Val Glu His Glu Thr
    885 890 895
    Gln Lys Leu Lys Ala Leu Asp Glu Ser His Asn Gln Asn Leu Lys Glu
    900 905 910
    Trp Arg Asp Lys Leu Arg Pro Arg Lys Lys Ala Leu Glu Glu Asp Leu
    915 920 925
    Asn Gln Lys Lys Arg Glu Gln Glu Met Phe Phe Lys Leu Ser Glu Glu
    930 935 940
    Ala Glu Cys Pro Asn Pro Ser Thr Pro Ser Lys Ala Ala Lys Phe Phe
    945 950 955 960
    Pro Tyr Ser Ser Gly Asp Ala Ser
    965
    <210> SEQ ID NO 108
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (2)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (4)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (6)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (8)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (10)
    <223> OTHER INFORMATION: Any amino acid
    <400> SEQUENCE: 108
    Leu Xaa Leu Xaa Leu Xaa Leu Xaa Leu Xaa Leu
    1 5 10
    <210> SEQ ID NO 109
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 109
    gcagcaagtg gagaagatgg 20
    <210> SEQ ID NO 110
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 110
    ggaagcatcc ccagagctgt ag 22
    <210> SEQ ID NO 111
    <211> LENGTH: 12
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 111
    Glu Lys Phe Gln Lys Cys Ser Ala Asp Glu Ser Pro
    1 5 10
    <210> SEQ ID NO 112
    <211> LENGTH: 16
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 112
    Ser Ile Ser Asn Ser Glu Leu Phe Pro Thr Thr Asp Pro Val Gly Thr
    1 5 10 15
    <210> SEQ ID NO 113
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 113
    Leu Asp Phe Pro Lys Glu Asp Tyr Arg
    1 5
    <210> SEQ ID NO 114
    <211> LENGTH: 12
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 114
    His Gly Asp Pro Arg Pro Glu Pro Arg Pro Thr Gln
    1 5 10
    <210> SEQ ID NO 115
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 115
    Pro Ser Thr Asn Arg Ala Gly Ser Leu Lys Asp Pro Glu Cys
    1 5 10
    <210> SEQ ID NO 116
    <211> LENGTH: 19
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 116
    Asp Pro Arg Thr Arg Ala Ser Asp Pro Gln Ser Pro Pro Gln Val Ser
    1 5 10 15
    Arg His Lys
    <210> SEQ ID NO 117
    <211> LENGTH: 16
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 117
    Cys Leu Val Pro Leu Ile Gln Leu Tyr Arg Lys Gln Thr Ser Thr Cys
    1 5 10 15
    <210> SEQ ID NO 118
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 118
    Pro Leu Met Arg Gln Asn Arg Thr Arg
    1 5
    <210> SEQ ID NO 119
    <211> LENGTH: 16
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 119
    Ser Gly Asp Arg Arg Arg Ala Gly Pro Glu Lys Arg Pro Lys Ser Ser
    1 5 10 15
    <210> SEQ ID NO 120
    <211> LENGTH: 16
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 120
    Cys Arg Arg Lys Ser Leu Val Gly Thr Pro Tyr Trp Met Ala Pro Glu
    1 5 10 15
    <210> SEQ ID NO 121
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 121
    Arg Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro Pro Tyr
    1 5 10 15
    <210> SEQ ID NO 122
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 122
    Lys Arg Lys Ser Phe Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu
    1 5 10 15
    <210> SEQ ID NO 123
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 123
    Lys Arg Asn Thr Phe Val Gly Thr Pro Phe Trp Met Ala Pro Glu
    1 5 10 15
    <210> SEQ ID NO 124
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 124
    Pro Ala Asn Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu
    1 5 10 15
    <210> SEQ ID NO 125
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 125
    Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu
    1 5 10 15
    <210> SEQ ID NO 126
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 126
    Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu
    1 5 10 15
    <210> SEQ ID NO 127
    <211> LENGTH: 18
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 127
    Arg Asn Lys Val Arg Lys Thr Phe Val Gly Thr Pro Cys Trp Met Ala
    1 5 10 15
    Pro Glu
    <210> SEQ ID NO 128
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 128
    ctcccatttc ctagcaaaat ca 22
    <210> SEQ ID NO 129
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 129
    agaggcagta ttgtcagatg ta 22
    <210> SEQ ID NO 130
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 130
    ccacacatgc gtatctctgt tg 22
    <210> SEQ ID NO 131
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 131
    ttgctagaat tcacatcagg taca 24
    <210> SEQ ID NO 132
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 132
    atccctggat cacactgctt ct 22
    <210> SEQ ID NO 133
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 133
    caaggtgttc tttgcctctg tt 22
    <210> SEQ ID NO 134
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 134
    agatggactg tactgggagg g 21
    <210> SEQ ID NO 135
    <211> LENGTH: 22
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 135
    agaagagcac ttggcactta tc 22
    <210> SEQ ID NO 136
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 136
    catcatgaac tggtgacggg 20
    <210> SEQ ID NO 137
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 137
    ccagtgaaat caaaccagta aaa 23
    <210> SEQ ID NO 138
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 138
    caaaacctgg ccgtctcttc tatt 24
    <210> SEQ ID NO 139
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 139
    atttgtgcta ctgggattct gtg 23
    <210> SEQ ID NO 140
    <211> LENGTH: 24
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 140
    gaatagcggt accatgatag aata 24
    <210> SEQ ID NO 141
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 141
    taccaaaaag agccaaaagt gtg 23
    <210> SEQ ID NO 142
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 142
    ctcagtattc tctccaaaga ttg 23
    <210> SEQ ID NO 143
    <211> LENGTH: 23
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 143
    gatgttctct ccattctgta aag 23
    <210> SEQ ID NO 144
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 144
    catcactgga agtctgcagt g 21
    <210> SEQ ID NO 145
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 145
    caggtgcagt agtcatttgc 20
    <210> SEQ ID NO 146
    <211> LENGTH: 20
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 146
    ggagctgtcg tattccagtc 20
    <210> SEQ ID NO 147
    <211> LENGTH: 21
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Primer
    <400> SEQUENCE: 147
    aacccctcaa gacccgttta g 21
    <210> SEQ ID NO 148
    <211> LENGTH: 4
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    motif
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (2)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (3)
    <223> OTHER INFORMATION: Any amino acid
    <400> SEQUENCE: 148
    Pro Xaa Xaa Pro
    1
    <210> SEQ ID NO 149
    <211> LENGTH: 50
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    leucine zipper
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (2)..(7)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (9)..(14)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (16)..(21)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (23)..(42)
    <223> OTHER INFORMATION: Any amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (44)..(49)
    <223> OTHER INFORMATION: Any amino acid
    <400> SEQUENCE: 149
    Leu Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa
    1 5 10 15
    Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
    20 25 30
    Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa
    35 40 45
    Xaa Leu
    50
    <210> SEQ ID NO 150
    <211> LENGTH: 13
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic
    Cdc42/Rac-binding motif
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (2)
    <223> OTHER INFORMATION: Variable amino acid
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (4)..(9)
    <223> OTHER INFORMATION: Four to six variable amino acids
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (11)..(12)
    <223> OTHER INFORMATION: Variable amino acid
    <400> SEQUENCE: 150
    Ser Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa His Xaa Xaa His
    1 5 10
    <210> SEQ ID NO 151
    <211> LENGTH: 787
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 151
    Ile Ile Glu Leu Ala Glu Arg Lys Pro Pro Leu Phe Asn Met Asn Ala
    1 5 10 15
    Met Ser Ala Leu Tyr His Ile Ala Gln Asn Glu Ser Pro Thr Leu Gln
    20 25 30
    Ser Asn Glu Trp Ser Asp Tyr Phe Arg Asn Phe Val Asp Ser Cys Leu
    35 40 45
    Gln Lys Ile Pro Gln Asp Arg Pro Thr Ser Glu Glu Leu Leu Lys His
    50 55 60
    Ile Phe Val Leu Arg Glu Arg Pro Glu Thr Val Leu Ile Asp Leu Ile
    65 70 75 80
    Gln Arg Thr Lys Asp Ala Val Arg Glu Leu Asp Asn Leu Gln Tyr Arg
    85 90 95
    Lys Met Lys Lys Leu Leu Phe Gln Glu Ala His Asn Gly Pro Ala Tyr
    100 105 110
    Glu Ala Gln Glu Glu Glu Glu Glu Gln Asp His Gly Val Gly Arg Thr
    115 120 125
    Gly Thr Val Asn Ser Val Gly Ser Asn Gln Ser Ile Pro Ser Met Ser
    130 135 140
    Ile Ser Ala Ser Ser Gln Ser Ser Ser Val Asn Ser Leu Pro Asp Val
    145 150 155 160
    Ser Asp Asp Lys Ser Glu Leu Asp Met Met Glu Gly Asp His Thr Val
    165 170 175
    Met Ser Asn Ser Ser Val Ile His Leu Lys Pro Glu Glu Glu Asn Tyr
    180 185 190
    Arg Glu Glu Gly Asp Pro Arg Thr Arg Ala Ser Asp Pro Gln Ser Pro
    195 200 205
    Pro Gln Val Ser Arg His Lys Ser His Tyr Arg Asn Arg Glu His Phe
    210 215 220
    Ala Thr Ile Arg Thr Ala Ser Leu Val Thr Arg Gln Met Gln Glu His
    225 230 235 240
    Glu Gln Asp Ser Glu Leu Arg Glu Gln Met Ser Gly Tyr Lys Arg Met
    245 250 255
    Arg Arg Gln His Gln Lys Gln Leu Met Thr Leu Glu Asn Lys Leu Lys
    260 265 270
    Ala Glu Met Asp Glu His Arg Leu Arg Leu Asp Lys Cys Leu Glu Thr
    275 280 285
    Gly Arg Asn Asn Phe Ala Ala Glu Met Glu Lys Leu Ile Lys Lys His
    290 295 300
    Gln Ala Ala Met Glu Lys Glu Ala Lys Val Met Ser Asn Glu Glu Lys
    305 310 315 320
    Lys Phe Gln Gln His Ile Gln Ala Gln Gln Lys Lys Glu Leu Asn Ser
    325 330 335
    Phe Leu Glu Ser Gln Lys Arg Glu Tyr Lys Leu Arg Lys Glu Gln Leu
    340 345 350
    Lys Glu Glu Leu Asn Glu Asn Gln Ser Thr Pro Lys Lys Glu Lys Gln
    355 360 365
    Glu Trp Leu Ser Lys Gln Lys Glu Asn Ile Gln His Phe Gln Ala Glu
    370 375 380
    Glu Glu Ala Asn Leu Leu Arg Arg Gln Arg Gln Tyr Leu Glu Leu Glu
    385 390 395 400
    Cys Arg Arg Phe Lys Arg Arg Met Leu Leu Gly Arg His Asn Leu Glu
    405 410 415
    Gln Asp Leu Val Arg Glu Glu Leu Asn Lys Arg Gln Thr Gln Lys Asp
    420 425 430
    Leu Glu His Ala Met Leu Leu Arg Gln His Glu Ser Met Gln Glu Leu
    435 440 445
    Glu Phe Arg His Leu Asn Thr Ile Gln Lys Met Arg Cys Glu Leu Ile
    450 455 460
    Arg Leu Gln His Gln Thr Glu Leu Thr Asn Gln Leu Glu Tyr Asn Lys
    465 470 475 480
    Arg Arg Glu Arg Glu Leu Arg Arg Lys His Val Met Glu Val Arg Gln
    485 490 495
    Gln Pro Lys Ser Leu Lys Ser Lys Glu Leu Gln Ile Lys Lys Gln Phe
    500 505 510
    Gln Asp Thr Cys Lys Ile Gln Thr Arg Gln Tyr Lys Ala Leu Arg Asn
    515 520 525
    His Leu Leu Glu Thr Thr Pro Lys Ser Glu His Lys Ala Val Leu Lys
    530 535 540
    Arg Leu Lys Glu Glu Gln Thr Arg Lys Leu Ala Ile Leu Ala Glu Gln
    545 550 555 560
    Tyr Asp His Ser Ile Asn Glu Met Leu Ser Thr Gln Ala Leu Arg Leu
    565 570 575
    Asp Glu Ala Gln Glu Ala Glu Cys Gln Val Leu Lys Met Gln Leu Gln
    580 585 590
    Gln Glu Leu Glu Leu Leu Asn Ala Tyr Gln Ser Lys Ile Lys Met Gln
    595 600 605
    Ala Glu Ala Gln His Asp Arg Glu Leu Arg Glu Leu Glu Gln Arg Val
    610 615 620
    Ser Leu Arg Arg Ala Leu Leu Glu Gln Lys Ile Glu Glu Glu Met Leu
    625 630 635 640
    Ala Leu Gln Asn Glu Arg Thr Glu Arg Ile Arg Ser Leu Leu Glu Arg
    645 650 655
    Gln Ala Arg Glu Ile Glu Ala Phe Asp Ser Glu Ser Met Arg Leu Gly
    660 665 670
    Phe Ser Asn Met Val Leu Ser Asn Leu Ser Pro Glu Ala Phe Ser His
    675 680 685
    Ser Tyr Pro Gly Ala Ser Gly Trp Ser His Asn Pro Thr Gly Gly Pro
    690 695 700
    Gly Pro His Trp Gly His Pro Met Gly Gly Pro Pro Gln Ala Trp Gly
    705 710 715 720
    His Pro Met Gln Gly Gly Pro Gln Pro Trp Gly His Pro Ser Gly Pro
    725 730 735
    Met Gln Gly Val Pro Arg Gly Ser Ser Met Gly Val Arg Asn Ser Pro
    740 745 750
    Gln Ala Leu Arg Arg Thr Ala Ser Gly Gly Arg Thr Glu Gln Gly Met
    755 760 765
    Ser Arg Ser Thr Ser Val Thr Ser Gln Ile Ser Asn Gly Ser His Met
    770 775 780
    Ser Tyr Thr
    785
    <210> SEQ ID NO 152
    <211> LENGTH: 338
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: MOD_RES
    <222> LOCATION: (2)
    <223> OTHER INFORMATION: Any amino acid
    <400> SEQUENCE: 152
    His Xaa Glu Tyr Val Pro Val Arg Arg Ile Asn Leu Glu Ala Cys Ser
    1 5 10 15
    Asn Glu Met Val Thr Ser Cys Arg Ala Ser Cys Met Phe Gln Thr Leu
    20 25 30
    Asn His Pro Asn Ile Val Pro Tyr Arg Ala Thr Leu Ile Ala Asp Asn
    35 40 45
    Glu Leu Trp Val Val Thr Ser Phe Met Ala Tyr Gly Ser Ala Lys Asp
    50 55 60
    Leu Ile Cys Thr His Phe Met Asp Gly Met Asn Glu Leu Ala Ile Ala
    65 70 75 80
    Tyr Ile Leu Gln Gly Val Leu Lys Ala Leu Asp Tyr Ile His His Asn
    85 90 95
    Gly Tyr Val His Arg Ser Val Lys Ala Ser His Ile Leu Ile Ser Val
    100 105 110
    Asp Gly Lys Val Tyr Leu Ser Gly Leu Arg Ser Asn Leu Ser Met Ile
    115 120 125
    Ser His Gly Gln Arg Gln Arg Val Val His Asp Phe Pro Lys Tyr Ser
    130 135 140
    Val Lys Val Leu Pro Trp Leu Ser Pro Glu Val Leu Gln Gln Asn Leu
    145 150 155 160
    Gln Gly Tyr Asp Ala Lys Ser Asp Ile Tyr Ser Val Gly Ile Thr Ala
    165 170 175
    Cys Glu Leu Ala Asn Gly His Val Pro Phe Lys Asp Met Pro Ala Thr
    180 185 190
    Gln Met Leu Leu Glu Lys Leu Asn Gly Thr Val Pro Cys Leu Leu Asp
    195 200 205
    Thr Ser Thr Ile Pro Ala Glu Glu Leu Thr Met Ser Pro Ser Arg Ser
    210 215 220
    Val Ala Asn Ser Gly Leu Ser Asp Ser Leu Thr Thr Ser Thr Pro Arg
    225 230 235 240
    Pro Ser Asn Gly Asp Ser Pro Ser His Pro Tyr His Arg Thr Phe Ser
    245 250 255
    Pro His Phe His His Phe Val Glu Gln Cys Leu Gln Arg Asn Pro Asp
    260 265 270
    Ala Arg Pro Ser Ala Ser Thr Leu Leu Asn His Ser Phe Phe Lys Gln
    275 280 285
    Ile Lys Arg Arg Ala Ser Glu Ala Leu Pro Glu Leu Leu Arg Pro Val
    290 295 300
    Thr Pro Ile Thr Asn Phe Glu Gly Ser Gln Ser Gln Asp His Ser Gly
    305 310 315 320
    Ile Phe Gly Leu Val Thr Asn Leu Glu Glu Leu Glu Val Asp Asp Trp
    325 330 335
    Glu Phe
    <210> SEQ ID NO 153
    <211> LENGTH: 546
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 153
    Met Ala Glu Pro Ser Gly Ser Val His Val Gln Leu Pro Gln Gln Ala
    1 5 10 15
    Ala Pro Val Thr Ala Ala Ala Ala Ala Ala Pro Ala Ala Ala Thr Ala
    20 25 30
    Ala Pro Ala Pro Ala Ala Pro Ala Ala Pro Ala Pro Ala Pro Ala Pro
    35 40 45
    Ala Pro Ala Ala Gln Ala Val Gly Trp Pro Ile Cys Arg Asp Ala Tyr
    50 55 60
    Glu Leu Gln Glu Val Ile Gly Ser Gly Ala Thr Ala Val Val Gln Ala
    65 70 75 80
    Ala Leu Cys Lys Pro Arg Gln Glu Arg Val Ala Ile Lys Arg Ile Asn
    85 90 95
    Leu Glu Lys Cys Gln Thr Ser Met Asp Glu Leu Leu Lys Glu Ile Gln
    100 105 110
    Ala Met Ser Gln Cys Ser His Pro Asn Val Val Thr Tyr Tyr Thr Ser
    115 120 125
    Phe Val Val Lys Asp Glu Leu Trp Leu Val Met Lys Leu Leu Ser Gly
    130 135 140
    Gly Ser Met Leu Asp Ile Ile Lys Tyr Ile Val Asn Arg Gly Glu His
    145 150 155 160
    Lys Asn Gly Val Leu Glu Glu Ala Ile Ile Ala Thr Ile Leu Lys Glu
    165 170 175
    Val Leu Glu Gly Leu Asp Tyr Leu His Arg Asn Gly Gln Ile His Arg
    180 185 190
    Asp Leu Lys Ala Gly Asn Ile Leu Leu Gly Glu Asp Gly Ser Val Gln
    195 200 205
    Ile Ala Asp Phe Gly Val Ser Ala Phe Leu Ala Thr Gly Gly Asp Val
    210 215 220
    Thr Arg Asn Lys Val Arg Lys Thr Phe Val Gly Thr Pro Cys Trp Met
    225 230 235 240
    Ala Pro Glu Val Met Glu Gln Val Arg Gly Tyr Asp Phe Lys Ala Asp
    245 250 255
    Met Trp Ser Phe Gly Ile Thr Ala Ile Glu Leu Ala Thr Gly Ala Ala
    260 265 270
    Pro Tyr His Lys Tyr Pro Pro Met Lys Val Leu Met Leu Thr Leu Gln
    275 280 285
    Asn Asp Pro Pro Thr Leu Glu Thr Gly Val Glu Asp Lys Glu Met Met
    290 295 300
    Lys Lys Tyr Gly Lys Ser Phe Arg Lys Leu Leu Ser Leu Cys Leu Gln
    305 310 315 320
    Lys Asp Pro Ser Lys Arg Pro Thr Ala Ala Glu Leu Leu Lys Cys Lys
    325 330 335
    Phe Phe Gln Lys Ala Lys Asn Arg Glu Tyr Leu Ile Glu Lys Leu Leu
    340 345 350
    Thr Arg Thr Pro Asp Ile Ala Gln Arg Ala Lys Lys Val Arg Arg Val
    355 360 365
    Pro Gly Ser Ser Gly His Leu His Lys Thr Glu Asp Gly Asp Trp Glu
    370 375 380
    Trp Ser Asp Asp Glu Met Asp Glu Lys Ser Glu Glu Gly Lys Ala Ala
    385 390 395 400
    Phe Ser Gln Glu Lys Ser Arg Arg Val Lys Glu Glu Asn Pro Glu Ile
    405 410 415
    Ala Val Ser Ala Ser Thr Ile Pro Glu Gln Ile Gln Ser Leu Ser Val
    420 425 430
    His Asp Ser Gln Gly Pro Pro Asn Ala Asn Glu Asp Tyr Arg Glu Ala
    435 440 445
    Ser Ser Cys Ala Val Asn Leu Val Leu Arg Leu Arg Asn Ser Arg Lys
    450 455 460
    Glu Leu Asn Asp Ile Arg Phe Glu Phe Thr Pro Gly Arg Asp Thr Ala
    465 470 475 480
    Asp Gly Val Ser Gln Glu Leu Phe Ser Ala Gly Leu Val Asp Gly His
    485 490 495
    Asp Val Val Ile Val Ala Ala Asn Leu Gln Lys Ile Val Asp Asp Pro
    500 505 510
    Lys Ala Leu Lys Thr Leu Thr Phe Lys Leu Ala Ser Gly Cys Asp Gly
    515 520 525
    Ser Glu Ile Pro Asp Glu Val Lys Leu Ile Gly Phe Ala Gln Leu Ser
    530 535 540
    Val Ser
    545
    <210> SEQ ID NO 154
    <211> LENGTH: 966
    <212> TYPE: PRT
    <213> ORGANISM: Murine sp.
    <400> SEQUENCE: 154
    Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
    1 5 10 15
    Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp Leu Asp Pro
    20 25 30
    Asn Asp Val Trp Glu Ile Val Gly Glu Leu Gly Asp Gly Ala Phe Gly
    35 40 45
    Lys Val Tyr Lys Ala Lys Asn Lys Glu Thr Gly Ala Leu Ala Ala Ala
    50 55 60
    Lys Val Ile Glu Thr Lys Ser Glu Glu Glu Leu Glu Asp Tyr Ile Val
    65 70 75 80
    Glu Ile Glu Ile Leu Ala Thr Cys Asp His Pro Tyr Ile Val Lys Leu
    85 90 95
    Leu Gly Ala Tyr Tyr Tyr Asp Gly Lys Leu Trp Ile Met Ile Glu Phe
    100 105 110
    Cys Pro Gly Gly Ala Val Asp Ala Ile Met Leu Glu Leu Asp Arg Gly
    115 120 125
    Leu Thr Glu Pro Gln Ile Gln Val Val Cys Arg Gln Met Leu Glu Ala
    130 135 140
    Leu Asn Phe Leu His Gly Lys Arg Ile Ile His Arg Asp Leu Lys Ala
    145 150 155 160
    Gly Asn Val Leu Met Thr Leu Glu Gly Asp Ile Arg Leu Ala Asp Phe
    165 170 175
    Gly Val Ser Ala Lys Asn Leu Lys Thr Leu Gln Lys Arg Asp Ser Phe
    180 185 190
    Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Leu Cys Glu Thr
    195 200 205
    Met Lys Asp Ala Pro Tyr Asp Tyr Lys Ala Asp Ile Trp Ser Leu Gly
    210 215 220
    Ile Thr Leu Ile Glu Met Ala Gln Ile Glu Pro Pro His His Glu Leu
    225 230 235 240
    Asn Pro Met Arg Val Leu Leu Lys Ile Ala Lys Ser Asp Pro Pro Thr
    245 250 255
    Leu Leu Thr Pro Ser Lys Trp Ser Val Glu Phe Arg Asp Phe Leu Lys
    260 265 270
    Ile Ala Leu Asp Lys Asn Pro Glu Thr Arg Pro Ser Ala Ala Gln Leu
    275 280 285
    Leu Gln His Pro Phe Val Ser Arg Val Thr Ser Asn Lys Ala Leu Arg
    290 295 300
    Glu Leu Val Ala Glu Ala Lys Ala Glu Val Met Glu Glu Ile Glu Asp
    305 310 315 320
    Gly Arg Glu Asp Gly Glu Glu Glu Asp Ala Val Asp Ala Val Pro Pro
    325 330 335
    Leu Val Asn His Thr Gln Asp Ser Ala Asn Val Thr Gln Pro Ser Leu
    340 345 350
    Asp Ser Asn Lys Leu Leu Gln Asp Ser Ser Thr Pro Leu Pro Pro Ser
    355 360 365
    Gln Pro Gln Glu Pro Val Asn Gly Pro Cys Ser Gln Pro Ser Gly Asp
    370 375 380
    Gly Pro Leu Gln Thr Thr Ser Pro Ala Asp Gly Leu Ser Lys Asn Asp
    385 390 395 400
    Asn Asp Leu Lys Val Pro Val Pro Leu Arg Lys Ser Arg Pro Leu Ser
    405 410 415
    Met Asp Ala Arg Ile Gln Met Asp Glu Glu Lys Gln Ile Pro Asp Gln
    420 425 430
    Asp Glu Asn Pro Ser Pro Ala Ala Ser Lys Ser Gln Lys Ala Asn Gln
    435 440 445
    Ser Arg Pro Asn Ser Ser Ala Leu Glu Thr Leu Gly Gly Glu Ala Leu
    450 455 460
    Thr Asn Gly Gly Leu Glu Leu Pro Ser Ser Val Thr Pro Ser His Ser
    465 470 475 480
    Lys Arg Ala Ser Asp Cys Ser Asn Leu Ser Thr Ser Glu Ser Met Asp
    485 490 495
    Tyr Gly Thr Ser Leu Ser Ala Asp Leu Ser Leu Asn Lys Glu Thr Gly
    500 505 510
    Ser Leu Ser Leu Lys Gly Ser Lys Leu His Asn Lys Thr Leu Lys Arg
    515 520 525
    Thr Arg Arg Phe Val Val Asp Gly Val Glu Val Ser Ile Thr Thr Ser
    530 535 540
    Lys Ile Ile Ser Glu Asp Glu Lys Lys Asp Glu Glu Met Arg Phe Leu
    545 550 555 560
    Arg Arg Gln Glu Leu Arg Glu Leu Arg Leu Leu Gln Lys Glu Glu His
    565 570 575
    Arg Asn Gln Thr Gln Leu Ser Ser Lys His Glu Leu Gln Leu Glu Gln
    580 585 590
    Met His Lys Arg Phe Glu Gln Glu Ile Asn Ala Lys Lys Lys Phe Tyr
    595 600 605
    Asp Val Glu Leu Glu Asn Leu Glu Arg Gln Gln Lys Gln Gln Val Glu
    610 615 620
    Lys Met Glu Gln Asp His Ser Val Arg Arg Lys Glu Glu Ala Lys Arg
    625 630 635 640
    Ile Arg Leu Glu Gln Asp Arg Asp Tyr Ala Lys Phe Gln Glu Gln Leu
    645 650 655
    Lys Gln Met Lys Lys Glu Val Lys Ser Glu Val Glu Lys Leu Pro Arg
    660 665 670
    Gln Gln Arg Lys Glu Ser Met Lys Gln Lys Met Glu Glu His Ser Gln
    675 680 685
    Lys Lys Gln Arg Leu Asp Arg Asp Phe Val Ala Lys Gln Lys Glu Asp
    690 695 700
    Leu Glu Leu Ala Met Arg Lys Leu Thr Thr Glu Asn Arg Arg Glu Ile
    705 710 715 720
    Cys Asp Lys Glu Arg Asp Cys Leu Ser Lys Lys Gln Glu Leu Leu Arg
    725 730 735
    Asp Arg Glu Ala Ala Leu Trp Glu Met Glu Glu His Gln Leu Gln Glu
    740 745 750
    Arg His Gln Leu Val Lys Gln Gln Leu Lys Asp Gln Tyr Phe Leu Gln
    755 760 765
    Arg His Asp Leu Leu Arg Lys His Glu Lys Glu Arg Glu Gln Met Gln
    770 775 780
    Arg Tyr Asn Gln Arg Met Met Glu Gln Leu Lys Val Arg Gln Gln Gln
    785 790 795 800
    Glu Lys Ala Arg Leu Pro Lys Ile Gln Arg Ser Asp Gly Glu Thr Arg
    805 810 815
    Met Ala Met Tyr Lys Lys Ser Leu His Ile Asn Gly Ala Gly Ser Ala
    820 825 830
    Ser Glu Gln Arg Glu Lys Ile Lys Gln Phe Ser Gln Gln Glu Glu Lys
    835 840 845
    Arg Gln Lys Ala Glu Arg Leu Gln Gln Gln Gln Lys His Glu His Gln
    850 855 860
    Met Arg Asp Met Val Ala Gln Cys Glu Ser Asn Met Ser Glu Leu Gln
    865 870 875 880
    Gln Leu Gln Asn Glu Lys Cys Tyr Leu Leu Val Glu His Glu Thr Gln
    885 890 895
    Lys Leu Lys Ala Leu Asp Glu Ser His Asn Gln Ser Leu Lys Glu Trp
    900 905 910
    Arg Asp Lys Leu Arg Pro Arg Lys Lys Ala Leu Glu Glu Asp Leu Asn
    915 920 925
    Gln Lys Lys Arg Glu Gln Glu Met Phe Phe Lys Leu Ser Glu Glu Ala
    930 935 940
    Glu Pro Arg Pro Thr Thr Pro Ser Lys Ala Ser Asn Phe Phe Pro Tyr
    945 950 955 960
    Ser Ser Gly Asp Ala Ser
    965
    <210> SEQ ID NO 155
    <211> LENGTH: 968
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <400> SEQUENCE: 155
    Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu
    1 5 10 15
    Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp Leu Asp Pro
    20 25 30
    Asn Glu Val Trp Glu Ile Val Gly Glu Leu Gly Asp Gly Ala Phe Gly
    35 40 45
    Lys Val Tyr Lys Ala Lys Asn Lys Glu Thr Gly Ala Leu Ala Ala Ala
    50 55 60
    Lys Val Ile Glu Thr Lys Ser Glu Glu Glu Leu Glu Asp Tyr Ile Val
    65 70 75 80
    Glu Ile Glu Ile Leu Ala Thr Cys Asp His Pro Tyr Ile Val Lys Leu
    85 90 95
    Leu Gly Ala Tyr Tyr His Asp Gly Lys Leu Trp Ile Met Ile Glu Phe
    100 105 110
    Cys Pro Gly Gly Ala Val Asp Ala Ile Met Leu Glu Leu Asp Arg Gly
    115 120 125
    Leu Thr Glu Pro Gln Ile Gln Val Val Cys Arg Gln Met Leu Glu Ala
    130 135 140
    Leu Asn Phe Leu His Ser Lys Arg Ile Ile His Arg Asp Leu Lys Ala
    145 150 155 160
    Gly Asn Val Leu Met Thr Leu Glu Gly Asp Ile Arg Leu Ala Asp Phe
    165 170 175
    Gly Val Ser Ala Lys Asn Leu Lys Thr Leu Gln Lys Arg Asp Ser Phe
    180 185 190
    Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Met Cys Glu Thr
    195 200 205
    Met Lys Asp Thr Pro Tyr Asp Tyr Lys Ala Asp Ile Trp Ser Leu Gly
    210 215 220
    Ile Thr Leu Ile Glu Met Ala Gln Ile Glu Pro Pro His His Glu Leu
    225 230 235 240
    Asn Pro Met Arg Val Leu Leu Lys Ile Ala Lys Ser Asp Pro Pro Thr
    245 250 255
    Leu Leu Thr Pro Ser Lys Trp Ser Val Glu Phe Arg Asp Phe Leu Lys
    260 265 270
    Ile Ala Leu Asp Lys Asn Pro Glu Thr Arg Pro Ser Ala Ala Gln Leu
    275 280 285
    Leu Glu His Pro Phe Val Ser Ser Ile Thr Ser Asn Lys Ala Leu Arg
    290 295 300
    Glu Leu Val Ala Glu Ala Lys Ala Glu Val Met Glu Glu Ile Glu Asp
    305 310 315 320
    Gly Arg Asp Glu Gly Glu Glu Glu Asp Ala Val Asp Ala Ala Ser Thr
    325 330 335
    Leu Glu Asn His Thr Gln Asn Ser Ser Glu Val Ser Pro Pro Ser Leu
    340 345 350
    Asn Ala Asp Lys Pro Leu Glu Glu Ser Pro Ser Thr Pro Leu Ala Pro
    355 360 365
    Ser Gln Ser Gln Asp Ser Val Asn Glu Pro Cys Ser Gln Pro Ser Gly
    370 375 380
    Asp Arg Ser Leu Gln Thr Thr Ser Pro Pro Val Val Ala Pro Gly Asn
    385 390 395 400
    Glu Asn Gly Leu Ala Val Pro Val Pro Leu Arg Lys Ser Arg Pro Val
    405 410 415
    Ser Met Asp Ala Arg Ile Gln Val Ala Gln Glu Lys Gln Val Ala Glu
    420 425 430
    Gln Gly Gly Asp Leu Ser Pro Ala Ala Asn Arg Ser Gln Lys Ala Ser
    435 440 445
    Gln Ser Arg Pro Asn Ser Ser Ala Leu Glu Thr Leu Gly Gly Glu Lys
    450 455 460
    Leu Ala Asn Gly Ser Leu Glu Pro Pro Ala Gln Ala Ala Pro Gly Pro
    465 470 475 480
    Ser Lys Arg Asp Ser Asp Cys Ser Ser Leu Cys Thr Ser Glu Ser Met
    485 490 495
    Asp Tyr Gly Thr Asn Leu Ser Thr Asp Leu Ser Leu Asn Lys Glu Met
    500 505 510
    Gly Ser Leu Ser Ile Lys Asp Pro Lys Leu Tyr Lys Lys Thr Leu Lys
    515 520 525
    Arg Thr Arg Lys Phe Val Val Asp Gly Val Glu Val Ser Ile Thr Thr
    530 535 540
    Ser Lys Ile Ile Ser Glu Asp Glu Lys Lys Asp Glu Glu Met Arg Phe
    545 550 555 560
    Leu Arg Arg Gln Glu Leu Arg Glu Leu Arg Leu Leu Gln Lys Glu Glu
    565 570 575
    His Arg Asn Gln Thr Gln Leu Ser Asn Lys His Glu Leu Gln Leu Glu
    580 585 590
    Gln Met His Lys Arg Phe Glu Gln Glu Ile Asn Ala Lys Lys Lys Phe
    595 600 605
    Phe Asp Thr Glu Leu Glu Asn Leu Glu Arg Gln Gln Lys Gln Gln Val
    610 615 620
    Glu Lys Met Glu Gln Asp His Ala Val Arg Arg Arg Glu Glu Ala Arg
    625 630 635 640
    Arg Ile Arg Leu Glu Gln Asp Arg Asp Tyr Thr Arg Phe Gln Glu Gln
    645 650 655
    Leu Lys Leu Met Lys Lys Glu Val Lys Asn Glu Val Glu Lys Leu Pro
    660 665 670
    Arg Gln Gln Arg Lys Glu Ser Met Lys Gln Lys Met Glu Glu His Thr
    675 680 685
    Gln Lys Lys Gln Leu Leu Asp Arg Asp Phe Val Ala Lys Gln Lys Glu
    690 695 700
    Asp Leu Glu Leu Ala Met Lys Arg Leu Thr Thr Asp Asn Arg Arg Glu
    705 710 715 720
    Ile Cys Asp Lys Glu Arg Glu Cys Leu Met Lys Lys Gln Glu Leu Leu
    725 730 735
    Arg Asp Arg Glu Ala Ala Leu Trp Glu Met Glu Glu His Gln Leu Gln
    740 745 750
    Glu Arg His Gln Leu Val Lys Gln Gln Leu Lys Asp Gln Tyr Phe Leu
    755 760 765
    Gln Arg His Glu Leu Leu Arg Lys His Glu Lys Glu Arg Glu Gln Met
    770 775 780
    Gln Arg Tyr Asn Gln Arg Met Ile Glu Gln Leu Lys Val Arg Gln Gln
    785 790 795 800
    Gln Glu Lys Ala Arg Leu Pro Lys Ile Gln Arg Ser Glu Gly Lys Thr
    805 810 815
    Arg Met Ala Met Tyr Lys Lys Ser Leu His Ile Asn Gly Gly Gly Ser
    820 825 830
    Ala Ala Glu Gln Arg Glu Lys Ile Lys Gln Phe Ser Gln Gln Glu Glu
    835 840 845
    Lys Arg Gln Lys Ser Glu Arg Leu Gln Gln Gln Gln Lys His Glu Asn
    850 855 860
    Gln Met Arg Asp Met Leu Ala Gln Cys Glu Ser Asn Met Ser Glu Leu
    865 870 875 880
    Gln Gln Leu Gln Asn Glu Lys Cys His Leu Leu Val Glu His Glu Thr
    885 890 895
    Gln Lys Leu Lys Ala Leu Asp Glu Ser His Asn Gln Asn Leu Lys Glu
    900 905 910
    Trp Arg Asp Lys Leu Arg Pro Arg Lys Lys Ala Leu Glu Glu Asp Leu
    915 920 925
    Asn Gln Lys Lys Arg Glu Gln Glu Met Phe Phe Lys Leu Ser Glu Glu
    930 935 940
    Ala Glu Cys Pro Asn Pro Ser Thr Pro Ser Lys Ala Ala Lys Phe Phe
    945 950 955 960
    Pro Tyr Ser Ser Gly Asp Ala Ser
    965

Claims (8)

What is claimed is:
1. A method for identifying a substance that modulates kinase activity comprising the steps of: (a) contacting a PAK5 kinase polypeptide or a catalytic fragment thereof with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide.
2. The method of claim 1, wherein said polypeptide or catalytic fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 30.
3. The method of claim 1, wherein said polypeptide or catalytic fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 103.
4. A method for identifying a substance that modulates kinase activity in a cell comprising the steps of: (a) expressing a PAK5 kinase polypeptide or a catalytic fragment thereof in a cell; (b) adding a test substance to said cell; and (c) monitoring a change in cell phenotype or the interaction between said polypeptide and a natural binding partner.
5. The method of claim 4, wherein said polypeptide or catalytic fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 30.
6. The method of claim 4, wherein said polypeptide or catalytic fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 103.
7. A method of detecting an agonist or antagonist of PAK5 kinase activity or kinase binding partner activity comprising (a) incubating cells that express PAK5 in the presence of a compound and (b) detecting changes in said kinase activity or said kinase binding partner activity.
8. The method of claim 3 wherein said compound is present in serum, body fluid, or a cell extract.
US10/725,329 1998-04-14 2003-12-02 PAK5 screening methods Abandoned US20040224323A1 (en)

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US10/725,329 Abandoned US20040224323A1 (en) 1998-04-14 2003-12-02 PAK5 screening methods
US10/725,121 Abandoned US20050142625A1 (en) 1998-04-14 2003-12-02 Antibodies against PAK5
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US6013500A (en) * 1998-05-21 2000-01-11 The Trustees Of Columbia University In The City Of New York PAK4, a novel gene encoding a serine/threonine kinase

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US20040091992A1 (en) * 1998-05-21 2004-05-13 The Trustees Of Columbia University In The City Of New York PAK4 - related antibodies
US7473766B2 (en) 1998-05-21 2009-01-06 The Trustees Of Columbia University In The City Of New York PAK4-related antibodies
US7615539B2 (en) * 2003-09-25 2009-11-10 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates

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US20070072261A1 (en) 2007-03-29
US20050142625A1 (en) 2005-06-30
ATE302269T1 (en) 2005-09-15
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US6656716B1 (en) 2003-12-02
ES2248997T3 (en) 2006-03-16
EP1073723A2 (en) 2001-02-07
AU3642499A (en) 1999-11-01
US6680170B2 (en) 2004-01-20
DE69926742D1 (en) 2005-09-22
DE69926742T2 (en) 2006-06-14
WO1999053036A9 (en) 2000-02-17
US20030050230A1 (en) 2003-03-13
EP1073723B1 (en) 2005-08-17
WO1999053036A3 (en) 2000-05-11
WO1999053036A2 (en) 1999-10-21
DK1073723T3 (en) 2006-01-02
CA2369172A1 (en) 1999-10-21

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