EP1274859A1 - Specific peptide substrate for human yak1, yak3a, and yak3b and yeast yak1 protein kinases - Google Patents

Abstract

hYAK1, hYAK3a and 3b, and yeast YAK1 polypeptides and polynucleotides, methods for producing such polypeptides by recombinant techniques, and methods of using Ser-164 and these polypeptides and polynucleotides to determine the effect on phosphorylation are disclosed. Also disclosed are methods for utilizing hYAK1, hYAK3a and 3b, and yeast YAK1 polypeptides and polynucleotides in the design of protocols for the treatment of bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthritis, Osteoarthritis, Inflammatory Bowel Disease (IBD), psoriasis, dermatitis, asthma, allergies; infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; HIV-associated cachexia and other immunodeficiency disorders; septic shock; pain; injury; cancers; anorexia; bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension; hypertension; urinary retention; angina pectoris; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourett's syndrome, among others, and diagnostic assays for such conditions.

Description

SPECIFIC PEPTIDE SUBSTRATE FOR HUMAN YAKl, YAK3A, AND YAK3B AND YEAST YAKl PROTEIN KINASES

Related Applications

This application claims priority of U.S. Provisional Application Serial No. 60/163,901, filed on November 5, 1999.

Field of the Invention

This invention relates to a newly identified peptide substrate for the seπne/threonme protein kmase family members, human YAKl, human YAK3a and hYAK3b, and yeast YAKl. More particularly, the peptide of the present invention is derived from the carboxyl terminal end of bovine myehn basic protein (MBP). It is a 15 ammo acid peptide with Ser at position 164 of the bovme MBP sequence as the major site of phosphorylation by hYAKl, hYAK3a and hYAK3b, and yeast YAKl, hereinafter referred to as Serl64. The sequence of Serl64 is:

Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Arg-Arg (SEQ ID NO:9)

155 160 164 169

The invention also relates to a method of screening for inhibitors of human YAKl and

YAK3a and YAK3b, and yeast YAKl protein seπne/threomne kinases, and to inhibiting or activating the action of the above mentioned protein kinases and other YAKl homologs by agents discovered using Ser 164 or related peptide analogs as a method of screening for inhibitors and ' activators. In addition, this invention relates to using the Ser 164 peptide to search for cellular substrates and/or cellular proteins that interact with the YAK family members of STPK's.

Background of the Invention

A number of polypeptide growth factors and hormones mediate their cellular effects through a signal transduction pathway. Transductton of signals from the cell surface receptors for these hgands to mtracellular effectors frequently involves phosphorylation or dephosphorylation of specific protein substrates by regulatory protein seπne/threonme kinases (PSTK) and phosphatases. Seπne/threonme phosphorylation is a major mediator of signal transduction m multicellular organisms. Receptor- bound, membrane-bound and mtracellular PSTKs regulate cell proliferation, cell differentiation and signaling processes in many cell types.

Aberrant protein seπne/threonine kmase activity has been implicated or is suspected in a number of pathologies such as rheumatoid arthπtis, psoπasis, septic shock, bone loss, many cancers and other prohferative diseases. Accordmgly, seπne/threonme kinases and the signal transduction pathways which they are part of are potential targets for drug design.

Several PSTKs are involved m regulation of cell cycling. One example is the cychn- dependent kinases or CDKs (Peter and Herskowitz, Cell 1994: 79, 181-184), which are activated by binding to regulatory proteins called cychns and control passage of the cell through specific cell cycle checkpoints. For example, CDK2 complexed with cyclm E allows cells to progress through the Gl to S phase transition. The complexes of CDKs and cychns are subject to inhibition by low molecular weight proteins such as pl6 (Serrano et al, Nature 1993: 366, 704), which binds to and inhibits CDK4. Deletions or mutations in pi 6 have been implicated in a vaπety of tumors (Kamb et al, Science 1994. 264, 436-440). Therefore, the prohferative state of cells and diseases associated with this state are dependent on the activity of CDKs and their associated regulatory molecules. In diseases such as cancer where inhibition of proliferation is desired, compounds that inhibit CDKs may be useful therapeutic agents. Conversely, activators of CDKs may be useful where enhancement of proliferation is needed, such as in the treatment of immunodeficiency.

The yeast YAKl is a PSTK with sequence homology to CDKs. It was oπgmally identified in S cerevisiae as a mediator of cell cycle arrest caused by mactivation of the cAMP-dependent protein kmase PKA (Garrett et al, Mol Cell Biol. 1991: 11, 4045-4052) YAKl kmase activity is low in cycling yeast but increases dramatically when the cells are arrested pπor to the S-G2 transition. Increased expression of YAKl causes growth arrest m yeast cells deficient in PKA. Therefore, YAKl can act as a cell cycle suppressor in yeast. Frequently, in diseases such as osteoporosis and osteoarthπtis, patients have established lesions of bone or cartilage, respectively. Treatment of such lesions requires an agent that will stimulate new bone or cartilage formation to replace that lost to the disease; therefore, there is a need for drugs that increase the number of osteoblasts or chondrocytes, the cells responsible for bone or cartilage formation, respectively. Similarly, replacement of heart or skeletal muscle depleted by diseases such as myocardial infarction or HIV-assocιated cachexia requires drugs that sϋmulate proliferation of cardiac myocytes or skeletal myoblasts.

This indicates that these protein seπne/threonine kinases have an established, proven history as therapeutic targets. Clearly there is a need for identification and characteπzation of further members of the protein seπne/threonme kmase family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthπtis, Osteoarthπtis, Inflammatory Bowel Disease (IBD), psoπasis, dermatitis, asthma, allergies; infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; HIV-associated cachexia and other immunodeficiency disorders; septic shock, pain, injury; cancers, anorexia, bulimia, Parkinson's disease, cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction, hypotension, hypertension, uπnary retention; angina pectoπs; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, mcludmg anxiety, schizophrenia, manic depression, deliπum, dementia, severe mental retardation and dyskmesias, such as Huntmgton's disease or Gilles dela Tourett's syndrome

Previously, a novel human homolog of yeast YAKl, termed hYAKl, which is expressed in osteoblasts, chondrocytes, cardiac and skeletal muscle, and at lower levels, in placenta and pancreas was descπbed (CREASY et al., EP Publication Number 98301124.8, published February 16, 1998, the disclosure of which is incorporated herein by reference in its entirety). The sequence of hYAKl shares homology with predicted PSTK's from C elegans, S pombe and S cerevisiae and has motifs associated with known protein kinases Inhibitors of hYAKl are expected to stimulate proliferation of cells in which it is expressed

Another novel human homolog of yeast YAKl, termed hYAK3b, which is expressed in testis, skeletal muscle, and m hematopoietic cells that can undergo erythroid differentiation (XIE, and CREASY, U.S. Patent Number 5,965,420, granted October 12, 1999, and U.S. Application Seπal Number 09/359,257, filed July 22, 1999, the disclosures of which are incorporated herein by reference in their entireties). The sequence of hYAK3b shares 79% ammo acid identity with hYAKl in its kmase domain, and shares homology with predicted PSTK's from C elegans, S pombe and S cerevisiae. hYAKl and hYAK3b are members of a novel subfamily of protein kinases with unique structural and enzymatic features. This family had been termed Dyrk (for dual specificity YAK- related kinases) by Dr. Joost and his co-workers. In their publications, hYAKl and hYAK3b were termed Dyrk2 and Dyrk3, respectively (Becker and Joost, Prog. Nucl. Acid Res., 1999, 62, 1-17). Summary of the Invention

In one aspect, the invention relates to a peptide substrate for hYAKl, hYAK3a and hYAK3b and yeast YAKl . This peptide, which has the sequence LGGRDSRSGSPMARR(SEQ ID NO:9), is derived from the carboxyl terminal end of bovine MBP (residues 155-169), and in it the Ser residue at position 164 (underlined) was identified as the major phosphoacceptor residue for hYAKl Hereinafter this peptide is referred to as Ser 164 This same residue was identified as the major phosphoacceptor site also for hYAK3a and 3b and yeast YAKl PSTKs One aspect of the invention relates to methods for using the Ser 164 peptide as a substrate to screen for inhibitors of the kmase activity of hYAKl, hYAK3a, hYAK3b, yeast YAKl, and other members of this family of protem kinases. Such uses include the treatment of bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthπtis, Osteoarthπtis, Inflammatory Bowel Disease (IBD), psoπasis, dermatitis, asthma, allergies; infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; HIV-associated cachexia and other immunodeficiency disorders; septic shock; pain; injury; cancers; anorexia; bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension, hypertension; uπnary retention; angina pectoπs; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, deliπum. dementia, severe mental retardation and dyskmesias, such as Huntington's disease or Gilles dela Tourett's syndrome, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with hYAKl, hYAK3a and 3b, and yeast YAKl imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with mappropπate hYAKl, hYAK3a and 3b, and yeast YAKl activity or levels. Still another aspect of the invention is the use of the SI 64 peptide sequence to identify cellular proteins that are phosphorylated by or interact with hYAKl, hYAK3a and 3b, and yeast YAKl .

Brief Description of the Drawings

Figure 1 shows the determination of hYAKl -catalyzed phosphorylation sites on bovme MBP.

Figure 2 shows the activity of hYAKl, hYAK3b, and yeast YAKl against bovine MBP and the SI 64 peptide.

Figure 3 shows the results of steady state two substrate analysis to determine the kinetic constants of the kmase reaction of purified recombinant hYAKl using the Serl64 as substrate. Description of the Invention

Definitions

The following definitions are provided to facilitate understanding of certain terms used frequently herein.

"hYAKl" refers, among others, generally to a polypeptide having the amino acid sequence set forth m SEQ ID NO:4 or an allehc variant thereof. "hYAKl activity or hYAKl polypeptide activity" or "biological activity of the hYAKl or hYAKl polypeptide" refers to the metabolic or physiologic function of said hYAKl including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and lmmunogenic activities of said hYAKl. "hYAKl gene" refers to a polynucleotide having the nucleotide sequence set forth m SEQ

ID NO:3 or allehc vaπants thereof and/or their complements.

"hYAK3a" refers, among others, generally to a polypeptide having the amino acid sequence set forth m SEQ ID NO: 6 or an allehc variant thereof.

"hYAK3a activity or hYAK3a polypeptide activity" or "biological activity of the hYAK3a or hYAK3a polypeptide" refers to the metabolic or physiologic function of said hYAK3a including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said hYAK3a.

"hYAK3a gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 5 or allehc vaπants thereof and/or their complements. "hYAK3b" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 8 or an allehc vaπant thereof.

"hYAK3b activity or hYAK3b polypeptide activity" or "biological activity of the hYAK3b or hYAK3b polypeptide" refers to the metabolic or physiologic function of said hYAK3b including similar activities or improved activities or these activities with decreased undesirable side-effects. Also mcluded are antigenic and lmmunogenic activities of said hYAK3b

"hYAK3b gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:7 or allehc vaπants thereof and/or their complements.

"Yeast YAKl" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allehc variant thereof. "Yeast YAKl activity or yYAKl polypeptide activity" or "biological activity of the yYAKl or yYAKl polypeptide" refers to the metabolic or physiologic function of said yYAKl including similar activities or improved activities or these activities with decreased undesirable side -effects Also included are antigenic and lmmunogenic activities of said yYAKl.

"Yeast YAKl gene" refers to a polynucleotide having the nucleotide sequence set forth m SEQ ID NO: 1 or allehc vaπants thereof and/or their complements. "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulm expression library.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs m nature, it has been changed or removed from its oπgmal environment, or both. For example, a polynucleotide or a polypeptide naturally present m a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

"Polynucleotide" generally refers to any polyπbonucleotide or polydeoxyπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules compπsmg DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, tπtylated bases and unusual bases such as inosine. A vaπety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well-descπbed in basic texts and m more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present m the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavm, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lod ation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as arginylation, and ubiquitmation See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.

Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al , "Protein Synthesis: Posttranslational Modifications and Aging", Ann NYAcad Sci (1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the vaπant are closely similar overall and, in many regions, identical. A vaπant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, or deletions in any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A vaπant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vaπant, or it may be a variant that is not known to occur naturally. Non-naturally occurπng vaπants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. "Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences In general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared For sequences where there is not an exact coπespondence, a "% identity" may be determined In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length

"Similarity" is a further, more sophisticated measure of the relationship between two polypeptide sequences In general, "similaπty" means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated 'score' from which the "% similaπty" of the two sequences can then be determined.

Methods for comparing the identity and similarity of two or more sequences are well known m the art. Thus for instance, programs available m the Wisconsin Sequence Analysis Package, version 9 1 (Devereux J., et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similaπty between two polypeptide sequences. BESTFIT uses the "local homology" algoπthm of Smith and Waterman (J Mol. Biol , 147:195-197, 1981, Advances m Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similaπty between two sequences. BESTFIT is more suited to compaπng two polynucleotide or two polypeptide sequences that are dissimilar m length, the program assuming that the shorter sequence represents a portion of the longer In compaπson, GAP aligns two sequences, finding a "maximum similarity", according to the algoπthm of Neddleman and Wunsch (J Mo I Biol , 48, 443-453, 1970) GAP is more suited to compaπng sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters "Gap Weight" and "Length Weight" used m each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similaπties are determined when the two sequences being compared are optimally aligned.

Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S.F , et al , J Mol Biol , 215, 403-410, 1990, Altschul S.F., et al , Nucleic Acids Res , 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA and accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in Enzymology, 183: 63-99 (1990); Pearson W R and Lipman D.J., Proc Nat Acad Sci USA, 85 2444-2448 (1988) (available as part of the Wisconsin Sequence Analysis Package). Preferably, the BLOSUM62 ammo acid substitution matrix (Henikoff S. and Hemkoff J.G.,

Proc Nat Acad ci USA, 89: 10915-10919 (1992)) is used in polypeptide sequence compaπsons including where nucleotide sequences are first translated into ammo acid sequences before compaπson.

Preferably, the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore descπbed.

Alternatively, for instance, for the purposes of interpreting the scope of a claim including mention of a "% identity" to a reference polynucleotide, a polynucleotide sequence having, for example, at least 95% identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence. Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These point mutations may occur at the 5 ' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a polynucleotide sequence having at least 95% identity to a reference polynucleotide sequence, up to 5% of the nucleotides of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99% and 100%

For the purposes of interpreting the scope of a claim including mention of a "% identity" to a reference polypeptide, a polypeptide sequence having, for example, at least 95% identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 ammo acids of the reference sequence. Such point mutations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These point mutations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a sequence polypeptide sequence having at least 95% identity to a reference polypeptide sequence, up to 5% of the ammo acids of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99%, and 100%.

A prefeπed meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolated polynucleotide compπsmg a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO.1 , (or SEQ ID NOS: 3, 5, or 7) wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO:l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO:l, or: n_n < x_n - (x_n • y),

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides m SEQ ID

NO: l, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_n and y is rounded down to the nearest integer pπor to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations m this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations. (2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, (or SEQ ID NOS: 4, 6, or 8-18)wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO:2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids m the reference sequence or in one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids m SEQ ID NO:2, or: n_a ≤ x_a - (x_a • y),

wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo acids in SEQ ID

NO:2, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

Peptides of the Invention In one aspect, the present invention relates to the Serl64 peptides. The Serl64 peptides include the peptide of the sequence m Figure 3; as well as peptide analogs which were made to study the structure activity relationship of Ser 164 phosphorylation by these PSTK's. These analogs include but are not limited to peptides 1 to 8 below (modified residues are bolded):

Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Arg-Arg (Serl64) (SEQ ID NO:9) Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Ala-Met-Ala-Arg-Arg (Peptide 1 ) (SEQ ID NO: 10)

Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Thr-Pro-Met-Ala-Arg-Arg (Peptide 2) (SEQ ED NO: 11)

Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Pro-Ala-Arg-Arg (Peptide 3) (SEQ ID NO: 12)

Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Pro-Ser-Pro-Met-Ala-Arg-Arg (Peptide 4) (SEQ ID NO: 13)

Leu-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Pro-Ser-Ala-Met-Ala-Arg-Arg (Peptide 5) (SEQ ID NO: 14) Leu-Gly-Gly-Arg-Asp-Ser-Arg-AIa-Gly-Ser-Pro-Met-Ala-Arg-Arg (Peptide 6) (SEQ ID NO: 15) Leu-Gly-Gly-Arg-Asp-Ala-Arg-Ser-Gly-Ser-Pro-Met-Ala-Arg-Arg (Peptide 7) (SEQ ID NO 16)

Leu-Gly-Gly-Arg-Asp-Ala-Arg-Ala-Gly-Ser-Pro-Met-Ala-Arg-Arg (Peptide 8) (SEQ ID NO: 17)

Also mcluded withm the Ser 164 peptides are truncated peptides analogs that do not have the first three residues (Leu-Gly-Gly). Detection of Serl64 as the site on MBP phosphorylated by the YAKs PSTKs:

By screening several proteins as substrates, we determined that hYAKl, hYAK3a and 3b and yeast YAKl preferred MBP as a substrate over casein, enolase, poly-Gly/Tyr, and several forms of histone. To identify the phosphorylation sites on bovme MBP catalyzed by each of the above mentioned protein kinases, bovme MBP was incubated for 30 minutes at 30 °C with 100 μM ATP, 10 mM MgC12, and purified hYAKl, puπfied hYAK3b, or yeast YAKl immunoprecipitated (using anti-HA Ab) from crude extracts of Saccharomyces cerevisiae transfected with HA-tagged yeast YAKl . Such an in vitro phosphorylated MBP was digested with endoprotemase Lys-C for 2 hours at 38 °C in 50 mM Tπs pH 8.8 An aliquot of the unfractionated protein digest was analyzed by MALDI TOF for the presence of phosphorylated peptides. MALDI mass spectra (Annan, and Carr, (1996) Anal Chem. 68, 3413-3421) were recorded on a Micromass TofSpec SE single-stage, gπdless reflectron time-of-flight mass spectrometer equipped with a time-lag-focusmg source Samples were prepared by mixing the digest with an equal volume of matπx solution which contained two internal mass standards at a concentration of 200 fmol/μl. A 0.5 μl aliquot of this sample/matπx solution was applied to the MALDI target. The matπx solution was 10 μg/ml α- Cyano-4-hydroxycmnamιc acid in 50:50 ethanol.acetomtrile. Samples were then rrradiated by 337 nm photons from a pulsed Laser Science nitrogen laser operating at 5 Hz. Typically, 20-50 laser shots were summed into a single mass spectrum. Potential phosphopeptide precursor ions were selected for post-source-decay (PSD) analysis using a Bradbury-Nielsen ion gate. Phosphopeptides were isolated by reversed phase HPLC using a 1mm l.d. C18 column. Peptides are eluted using acetomtπle:water:0.1% trifluoroacetic acid gradients at 50 μl/rmn. The column eluent was split post detector such that 5 μl/mm was sent to a Micromass QTOF quadrupole time-of-flight mass spectrometer, and 45 μl/min went to a fraction collector taking one minute fractions. Phosphopeptides were sequenced by tandem MS (Verma, et al. (1997) Science, 278, 455-560.) using the QTOF mass spectrometer equipped with a nanoelectrospray source (Wilm, and Mann, (1994) Analytical Chem 66, 4390-4399 1994).

MALDI-TOF analysis of 5% of the unfractionated endoprotemase Lys-C digests from phosphorylated and nonphosphorylated MBP revealed 2 apparent phosphate-containing peptides at m/z 1571.78 (MH+) and 1682.80 (MH+) (Fig 1 (labelled A)), corresponding to peptides of molecular mass 1570 77 and 1681 79, respectively The 1570 Da species is consistent with the mass of a peptide containing residues 91-104 from MBP plus one mole of phosphate (calculated mass, 1570.78 Da), whereas the 1681 Da peptide was consistent with the mass of the C-termmal Lys-C fragment from MBP incorporating residues 155-169 plus one mole of phosphate (calculated mass, 1681.78). Observation of the characteristic loss of H₃P0₄ (98 Da) from both peptides upon subsequent PSD analysis (Annan, and Can, (1996) Anal Chem 68, 3413-3421) confirmed that these peptides were, m fact, phosphorylated.

The Lys-C digests were fractionated by reverse phase HPLC using MS as an on-line readout of the fractions. Fractions containing the two putative phosphopeptides were analyzed by nanoES, and the appropriate precursor ions sequenced by colhsion-mduced dissociation tandem mass spectrometry. The spectrum obtained for the 1570 9 peptide (Fig. 1 (labelled B)) is consistent with two phosphorylated forms of the sequence NIVTPRTPPPSQGK (SEQ ID NO: 18), which corresponds to residues 91-104 of bovme MBP. The major phosphorylation site was found to be Thr97 The 181.04 Da mass difference between the bg and >η ions indicates the presence of phosphothreonme at this position. A very small amount of phosphorylation was also found at Thr94 The evidence for phosphorylation at this second site was a seπes of b_nΔ ions starting at b4 and ending at bg (The evidence for phosphorylation at either site would converge at the b7 ion.)

The tandem mass spectrum of the 1681 peptide (Fig. 1 (labelled C)) is consistent with a monophosphorylated form of LGGRDSRSGSPMARR (SEQ ID NO:9), corresponding to the C- terminal residues 155-169 of bov e MBP. Although the tandem MS data clearly mdicate that Serl60 is not phosphorylated, it was unable to distinguish between Serl62 or Ser 164 as the sole site of phosphorylation in this peptide. Assignment of the phosphorylation site as Serl64 is based on the presence of an S/TP sequence at this site, which is analogous to the motif found at Thr94 and Thr97. Phosphorylation of bovme MBP on Thr97 by the MAP kinases has been previously reported (Eπkson, et al., (1990) J. Biol. Chem. 265, 19728-19735;Clark-Lewιs, et al., (1991) J Biol. Chem. 266, 11580-11584.). However, we are not aware of any kmase that phosphorylates bovme MBP on Ser 164. Therefore, the phosphorylation pattern of yeast YAKl on MBP appears to be unique. Kinase Assays Using Serl64 as the phosphoacceptor

The source of Ser 164 peptide: It was custom-ordered from California Peptide Research Inc. (Napa, CA), and its puπty was determined by HPLC. The peptide contained 15 ammo acids, and its calculated molecular mass was 1601.82 daltons. Solid sample was dissolved at 5 mM in ice-cold kmase assay buffer (see later), aliquoted, and stored at -20 °C until use.

Figure 1 shows a determination of YAKl -induced phosphorylation sites on myehn basic protein. A, Partial MALDI mass spectrum of an unfractionated Lys-C digest of in vitro phosphorylated MBP. Peptides modified by a single mole of phosphate show an increase in mass of 80 Da. B, ES-CID tandem mass spectrum of the M^⁺ ion (m/z 786.4) from the 1570 Da phosphorylated peptide shown above. Ions marked b_nΔ have the structure b_n-H3P04. C, ES-CID tandem mass spectrum of the M^⁺ ion (m/z

561.6) from the 1681 Da phosphorylated peptide shown above. Ions marked b_n ^ h_ave th_e structure b_n-H₃P0₄ -NH₃.

Figure 2 shows the activity of hYAKl, hYAK3, and yeast YAKl against MBP and the Serl64 peptide. 5 ng puπfied hYAKl and 100 ng purified hYAK3 were used per assay. Anti-HA mAb immune complex kmase assay was performed on 100 μg protein from crude extracts of yeast cells expressing either FL or ΔN yeast YAKl. Concentration of ATP was 100 μM, Serl64 was used at 0.5 mM, and MBP was at 10 μg/reaction (18.5 uM)

Figure 3 shows double reciprocal plots of enzyme velocity vs. ATP or Ser 164 concentration. Steady state two substrate analysis of puπfied hYAKl using SI 64 as phosphoacceptor. 1 ng hYAKl was incubated for 30 minutes at 37 °C m hYAKl reaction mix that contains various concentrations of ATP and Serl64. Each ATP concentration was used with 1, 0.75, 0.5, 0.33, 0.25, 0.15, and 0.05 mM Serl64. Each Serl64 concentration was used with 333, 166, 100, 66.7, 33.3, 16.7, and 6.6 μM ATP. Reciprocal values of enzyme velocity were calculated and plotted against reciprocal values of the concentration of each substrate. Data were analyzed by GraphFit, and calculated kinetic constants are shown.

The source of enzyme:

1) hYAKl : DETl/DET2-tagged full length hYAKl was expressed in Drosophila sf9 cells and puπfied to >95% puπty using Ni column chromatography. The puπfied protein migrated on SDS gels as a single band with an apparent molecular mass of 62 kDa. Samples were stored at -80 °C until use.

2) hYAK3b: Glutathione-S-Transferase (GST)/Factor Xa-tagged hYAK3b was expressed in baculovirus cells and puπfied to about 50% puπty using Glutathione Sepharose 4B column chromatography, followed by Ni-NTA column chromatography. Samples were stored at -80 °C until use. 3) Yeast YAKl : Full length and an ammo-terminally truncated (ammo acids 148-807, termed ΔN) hemagglutmm (HA)-tagged yeast YAKl was each expressed m a strain of S cerevisiae lacking the endogenous YAKl gene and all three PKA genes. Cultures for experiments were grown in liquid Sc-His to an ODgoo °f ^at ^^east 1 0. washed with Sc-His g/r, resuspended m Sc-His g/r to twice the oπgmal volume and grown for 16-24 hours at RT. Cells were washed once with H2O and the pellets stored at -80 °C until use. To prepare lysates, cell pellets were thawed and resuspended at 1 ml/100 ml of original culture m lysis buffer (LB) containing 50 mM Tπs pH 7.5, 150 mM NaCl, 10 μg/ml each aprotin , leupeptin and TLCK, 0.1 mM PMSF, 50 mM NaF, 1 mM NaVanadate, 10 mM β-glycerophosphate. Following the addition of 0.5 ml steπle acid-washed glass beads, cells were disrupted via ten, 30 second intervals of vortexmg. NP40 was added to a 2% final concentration followed by rocking at 4 °C for 30-50 minutes. Lysates were claπfied by high-speed centrifugation, and the supernatants were stored at -80 °C until use. Each form of yeast YAKl was immunoprecipitated from the detergent extracts using anti-HA mAb.

Immune Complex Protein Kmase Assay for Yeast YAKl : Yeast cellular extracts were immunoprecipitated by rocking overnight at 4°C with 4 μg of the anti-HA tag antibody and 100 μl of 20% suspension of protein A agarose (GIBCO-BRL) in LB that contained 1% NP-40. Samples were then washed twice with LB and once with basic kmase assay buffer (25 mM Hepes, pH 7.5; 1 mM DTT; 10 mM β-glycerol phosphate; 0.2 mM NaV). Washed immune complexes were suspended m 20 μl of basic kmase assay buffer that contained 0.1 mM ATP, 3 μCi of [γ-32p]ATP, 10 mM MgCl2, plus either bovme MBP or the Ser 164 peptide. After incubation for 15 minutes at

30°C, the reactions were stopped by adding 20 μl of 0.15 M phosphoric acid. Phosphorylated substrates were isolated by spotting 20 μl of each sample on phosphocellulose (p81) filters. Filters were washed 3 times with 75 mM phosphoric acid followed by 3 times with H2O, and counted for 2p incorporation using a β-scmtillation counter.

Kinase assay of puπfied hYAKl and hYAK3: The assay was performed in 96 well Mmisorp plates (Costar, Catalog No 3356). Reaction (in 30 μl volume) mixtures contained in final concentrations, 25 mM Hepes buffer, pH 7.5; 0.2 mM sodium vandate; 10 mM MgCl2; 1 mM DTT;

10 mM β-glycerol phosphate; 0.1% BSA; 0.1 mM ATP, 2.5 μCi of [γ-³²P]ATP; puπfied hYAKl (1-5 ng/assay), or puπfied hYAK3b (50-100 ng/assay); and either bovme MBP or the Serl 64 peptide used at the concentrations indicated below and in the descriptions of the figures. Reactions were incubated for 20 minutes at 37°C, and were stopped by adding 10 μl of 0.3 M phosphoric acid. Phosphorylated substrates were isolated by spotting 20 μl of the reaction on p81 filters, and processed as detailed earlier.

This same assay can be performed on a FlashPlate format m which the plate is coated with MBP or with the SI 64 peptide by incubation overnight at 4 °C in 100 μl of either substrate dissolved in Sodium Carbonate buffer, pH 8.8. When coating with MBP, a solution of 100 μg/ml MBP was used to coat wells with 100 μl (10 μg) MBP per well. When coating with Serl64, a solution of 0.4 mg/ml (0.25 mM) was used to coat wells with 100 μl (40 μg) Serl64 per well. An example of a FlashPlate assay protocol and typical results are given below:

FlashPlate (FP) Protocol 1. Coat Maxisorp plates with MBP or Serl 64 as above.

2. Wash plates once with kmase assay buffer (KB): 25 mM Hepes, pH 7.5; 0.2 mM NaV; 10 mM β- glycerol phosphate; 1 mM Na pyrophosphate

3. Add enzyme (Ni-hYAKl , diluted m KB), DMSO or inhibitors (in KB) and keep on ice for 30 minutes 4. Add KB containing Mg/ATP to a [final] of 0.1 mM [γ-³³P]ATP and 10 mM MgCl₂

5. Incubate with shaking, 1-2 hrs, at room temperature

6. Aspirate and wash 6 times with 0.5 ml KB

7. Read ³³P incorporation m FP reader

8. Blank = No enzyme added 9. Reaction volume: 25, 50 or 100 μl

10. 0.5 or 1.0 μCi ³³P/0.1 mM ATP

11. MBP-FP better than basic FP (in house coating)

12. 37 °C incubation was not better (several time points)

13. Other incubation times at room temperature were not better

Results: Each kmase phosphorylated the Serl 64 peptide to much higher specific activity than MBP (Figure 2). Steady state kinetic constants of the hYAKl reaction were generated by varymg both substrates simultaneously and fitting enzyme velocity as a function of each substrate concentration. Double reciprocal plots (1 V vs. l/[Substrate]) with SI 64 peptide as the phosphate acceptor are shown in Figure 3. GraphFit analysis of the results generated the following steady state kinetic constants: K_/w[ATP]=42 +7 μM .

K_/w[S164]=160 ±14 μM.

V_max ^~ 51 ±6 μmol/mg.

k_cβ,=160 +19 min^-1.

Typical results of FlashPlate are shown below FlashPlate Typical results

Signal to noise ratio: >7 fold

Blanks: 30-80 cpm [Ni-hYAKl]: As low as 20 ng/reaction (5 nM) for 100 μl reactions

As low as 8 ng/reaction (5 nM) for 25 μl reactions

Kinase inhibitors: Potency comparable to tube assay:

SKF-108752 IC50: 0.19 μM (0.1 μg hYAKl)

0.13 μM; 0.16 μM (0.3 μg hYAKl)

K252a IC50: 0.552 μM; 0.427 μM (0.3 μg hYAKl) Specific Activity: At 20 ng enzyme, MBP gave 5S ±3 (n = 6), and Ser 164 gave 484 ±63 (n = 6) nmol/mg protein

DMSO: No effect up to 3%

Variability: <10% (between wells and from plate to plate)

Screening Assays The hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides of the present invention may be employed in a screening process for compounds, which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides of the present invention. Thus, polypeptides of the invention may also be used to assess or identify agonists or antagonists from, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures. These agonists or antagonists may be natural substrates, hgands, receptors, etc., as the case may be, of the polypeptides of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Coligan et al , Current Protocols in Immunology 1(2): Chapter 5 (1991). hYAKl, hYAK3a and 3b , and yeast YAKl polypeptides are ubiquitous in the mammalian host and are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide on the one hand and which can inhibit the function of hYAKl , hYAK3a and 3b, and yeast YAKl polypeptide on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthπtis, Osteoarthπtis, Inflammatory Bowel Disease (IBD), psoπasis, dermatitis, asthma, allergies; infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HTV-1 or HIV-2; HlV-associated cachexia and other immunodeficiency disorders; septic shock; pam; injury; cancers; anorexia; bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension; hypertension; uπnary retention; angina pectoπs; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, dehπum, dementia, severe mental retardation and dyskmesias, such as Huntington's disease or Gilles dela Tourett's syndrome. Antagonists may be employed for a vaπety of therapeutic and prophylactic purposes for such conditions as bone loss including osteoporosis; inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthπtis, Osteoarthπtis, Inflammatory Bowel Disease (IBD), psoπasis, dermatitis, asthma, allergies; infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; HlV-associated cachexia and other immunodeficiency disorders; septic shock; pam; injury; cancers; anorexia, bulimia; Parkinson's disease; cardiovascular disease including restenosis, atherosclerosis, acute heart failure, myocardial infarction; hypotension; hypertension; uπnary retention; angina pectoπs; ulcers; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, dehπum, dementia, severe mental retardation and dyskmesias, such as Huntington's disease or Gilles dela Tourett's syndrome.

In general, such screenmg procedures may involve using appropπate cells which express the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides or respond to hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide of the present invention. Such cells include cells from mammals, yeast, Drosoph a or E coh Cells which express the hYAKl , hYAK3a and 3b, and yeast YAKl polypeptides (or cell membrane containing the expressed polypeptides) or respond to hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The ability of the cells, which were contacted with the candidate compounds, is compared with the same cells which were not contacted for hYAKl, hYAK3a and 3b, and yeast YAKl activity.

The knowledge that the hYAKl, hYAK3a and 3b, and yeast YAKl encodes a protein kmase suggests that recombinant forms can be used to establish a protein kmase activity. Typically this would involve the direct incubation of hYAKl, hYAK3a and 3b, and yeast YAKl with a protein or peptide substrate in the presence of γ-32p. ATP, followed by the measurement of radioactivity incorporated into the substrate by separation and counting. Separation methods include lmmunoprecipitation, conjugation of substrate to a bead allowing separation by centπfugation or determination of incorporation by scintillation proximity assay, SDS-PAGE followed by autoradiography or biosensor analysis. While the specific substrates are not yet known, candidates include hYAKl , hYAK3a and 3b, and yeast YAKl themselves (autophosphorylation), myehn basic protein, casem, histone and HSP27. Other substances might be discovered by incubating hYAKl, hYAK3a and 3b, and yeast YAKl with random peptides conjugated to solid supports or displayed on the surface of phage or by incubation of hYAKl, hYAK3a and 3b, and yeast YAKl with mammalian cell lysates and γ-32p. ATP, followed by separation of the labelled target proteins, and sequencmg. The protein kmase activity of hYAKl , hYAK3a and 3b, and yeast YAKl may require incubation with a specific upstream effector. This may be achieved by preincubatmg hYAKl, hYAK3a and 3b, and yeast YAKl with lysates from a vaπety of stimulated eukaryotic cells and ATP. These assays permit the discovery and modification of compounds which inhibit hYAKl, hYAK3a and 3b, and yeast YAKl kmase activity in vitro and would be expected to have effects on proliferation of osteoblasts, chondrocytes, cardiac myocytes or skeletal myoblasts. Any inhibitors so identified would be expected to have up-regulatory effects on proliferation and be useful as a therapeutic for the treatment and prevention of diseases such as osteoporosis, osteoarthπtis, cardiomyopathy and cachexia.

This invention contemplates the treatment and/or amelioration of such diseases by adrmmsteπng a hYAKl, hYAK3a and 3b, or yeast YAKl inhibiting amount of a compound. Without wishing to be bound by any particular theory of the functioning of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides of this invention, it is believed that among the useful inhibitors of hYAKl, hYAK3a and 3b, and yeast YAKl function are those compounds which inhibit the kmase activity of the hYAKl, hYAK3a and 3b, and yeast YAKl . Other sites of inhibition are, of course, possible owing to its position in a signal transduction cascade. Therefore, inhibiting the interaction of hYAKl, hYAK3a and 3b, and yeast YAKl with one or more of its upstream or downstream modulators/substrates is also contemplated by this invention. Inhibitors of protein-protein interactions between hYAKl, hYAK3a and 3b, and yeast YAKl and other factors could lead to the development of pharmaceutical agents for the modulation of hYAKl, hYAK3a and 3b, and yeast YAKl activity

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide, using detection systems appropriate to the cells bearing the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Standard methods for conducting such screening assays are well understood in the art. Examples of potential hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide antagonists include antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, etc., as the case may be, of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide, e.g., a fragment of the hgands, substrates, receptors, or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Prophylactic and Therapeutic Methods

This invention provides methods of treating an abnormal condition related to both an excess of and insufficient amounts of hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide activity.

If the activity of hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides is m excess, several approaches are available. One approach compπses administeπng to a subject an inhibitor compound (antagonist) as hereinabove descπbed along with a pharmaceutically acceptable earner in an amount effective to inhibit activation by blocking binding of hgands to the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides, or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides still capable of binding the hgand m competition with endogenous hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides may be administered. Typical embodiments of such competitors compπse fragments of the hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides.

In still another approach, expression of the gene encoding endogenous hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988). Alternatively, ohgonucleotides which form triple helices with the gene can be supplied. See. for example, Lee et al, Nucleic Acids Res (1979) 6:3073; Cooney et al , Science (1988) 241 :456; Dervan et al , Science (1991) 251:1360. These ohgomers can be admmιstered »er 5e or the relevant ohgomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of hYAKl, hYAK3a and 3b, and yeast YAKl and its activity, several approaches are also available. One approach compπses administering to a subject a therapeutically effective amount of a compound which activates hYAKl, hYAK3a and 3b, and yeast YAKl polypeptide, i.e., an agonist as descπbed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of hYAKl, hYAK3a and 3b, and yeast YAKl by the relevant cells m the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packagmg cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engmeenng cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therem) m Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).

Formulation and Administration

Peptides, such as the soluble form of hYAKl, hYAK3a and 3b, and yeast YAKl polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner. Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient. Such earners include but are not limited to, salme, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration, and is well withm the skill of the art. The invention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may be employed alone or m conjunction with other compounds, such as therapeutic compounds.

Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intrapeπtoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteπc or encapsulated formulations, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected m view of the vanety of compounds available and the differing efficiencies of vanous routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Vanations m these dosage levels can be adjusted using standard empiπcal routines for optimization, as is well understood m the art.

Polypeptides used m treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.

Examples

The examples below are earned out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise descπbed in detail. The examples illustrate, but do not limit the invention. Example 1

A partial clone (ATG-355, HGS EST # 454640) was initially identified through random searches of the Human Genome Sciences database. This partial clone (~ lkb) showed significant homology to YAKl from S cerevisiae. To get the full length cDNA A total of 1M plaques were screened from a Human Osteoblast cDNA library (Stratagene, LaJolla CA) using the insert of the above partial clone as a probe. Library screening procedure is described by (Elgin, et al. Strategies 4: 8-9, 1991). The probes were α -32P labeled, using Random Pnmed Labeling Kit (Boheπnger Manheim, Germany, Cat. # 1585584 ) and punfied by running over Sephadex G-50 columns

(Pharmacia Biotech. Cat # 17-0855-02) The hybπdization and washing conditions were according to ( J. Sambrook, E.F Fntch and T. Mamatis (1989) A Laboratory Manual Second. Ed. Vol. 1 pp. 2.69- 2.81 Cold Spnng Harbor Laboratory Press, Cold Spnng Harbor, New York). Five clones were isolated by plaque puπfication and a fragments containing the inserts were excised and sequenced. The longest insert was a 2.7 kb fragment containing the 3' untranslated region and part of the coding sequence of hYAKl . A second probe prepared by EcoRI-BamHI digestion of this insert followed by labelling with 32P was used to screen a commercially available human heart cDNA library (Stratagene #936207). An additional five clones were plaque puπfied, excised into phagemids and sequenced Fasta analysis show this peptide to have high homology to a putative senne/threomne kmase of unknown function from C elegans (F49E 11.1 ) .

To confirm that the cDNA was full length, Human Leukocyte "Marathon Ready" cDNA (Clontech, Palo Alto, CA) was used as a template to amplify a fragment corresponding to the 5' region of hYAKl using a 5' anchor pnmer-1 (Clontech) and a reverse gene specific pπmer. This fragment was T/A cloned into pCR2.1 (Invitrogen), and multiple isolates were sequenced. An m-frame stop codon was identified upstream of the predicted initiation codon confirming that the full-length cDNA had been obtained.

Northern analysis was earned out to determine the distnbution of hYAKl mRNA in human tissues. A fragment contammg the 3' untranslated region of hYAKl was isolated from SEQ ID NO: 1 using standard techniques. The isolated fragment was radiolabelled with α-32p-dATP using a randomly pnmed labelling kit. Membranes contammg mRNA from multiple human tissues (Clontech #7760-1) were hybπdized with the probe and washed under high stπngency conditions as directed. Hybπdized mRNA was visualized by exposing the membranes for 4 days to X-ray film. Three major transcπpts of 2 6, 7 and 10 kb were present and were expressed most prominently in heart and skeletal muscle, but were present to a lesser degree in pancreas, placenta and brain. All three transcπpts appeared absent from kidney. SEQUENCE INFORMATION SEQ ID NO :1

ATGAACTCAT CCAATAATAA CGACTCGTCC AGCTCCAATA GCAACATGAA TAACTCCTTG AGCCCGACCC TTGTGACCCA CAGTGATGCT AGTATGGGCT CGGGTAGAGC AAGCCCAGAC AATAGCCATA TGGGGAGAGG TATATGGAAT CCATCGTACG TAAATCAAGG CTCTCAAAGG TCTCCACAGC AGCAGCATCA GAATCATCAC CAGCAGCAGC AGCAGCAGCA ACAACAACAA CAACAGAATT CTCAATTTTG CTTTGTTAAC CCTTGGAATG AGGAAAAAGT AACTAATTCT CAACAAAACC TGGTGTATCC CCCTCAATAC GATGACTTAA ACAGTAACGA AAGTCTAGAT GCGTACAGAC GACGTAAATC TAGTCTCGTT GTACCTCCAG CCAGGGCACC TGCCCCAAAT CCTTTCCAGT ACGATAGTTA TCCCGCTTAC ACCAGCTCTA ATACGAGTTT GGCAGGAAAT AGCAGTGGCC AGTATCCTTC TGGCTATCAA CAACAACAAC AGCAAGTATA CCAGCAGGGC GCTATCCATC CTTCCCAATT TGGATCCAGA TTTGTTCCCT CCCTTTATGA TCGTCAAGAT TTCCAAAGAA GGCAGAGTCT GGCTGCAACT AATTATTCGT CCAATTTTTC TTCCCTAAAT TCAAATACTA ATCAAGGAAC AAACTCCATA CCAGTCATGT CACCTTACAG GAGGCTTAGC GCATATCCTC CTAGTACAAG TCCCCCACTA CAACCCCCTT TCAAGCAGTT ACGAAGAGAT GAAGTACAAG GTCAGAAATT GTCCATTCCT CAGATGCAAC TTTGCAACTC TAAAAACGAT CTTCAACCTG TCTTAAATGC TACTCCAAAG TTTAGACGTG CGTCGCTCAA TTCCAAGACG ATATCTCCCC TAGTCAGTGT CACGAAAAGC CTGATTACCA CATATTCTTT ATGTTCTCCA GAATTTACCT ACCAAACGTC CAAAAACCCC AAGAGAGTAC TTACGAAGCC CAGTGAAGGG AAATGTAACA ACGGATTCGA CAACATAAAC AGTGACTATA TTCTTTATGT AAATGACGTT TTGGGTGTAG AGCAGAACAG AAAGTACCTT GTGCTAGACA TTTTGGGTCA AGGTACATTT GGTCAAGTGG TCAAATGTCA AAATTTGCTG ACGAAAGAGA TATTGGCTGT AAAAGTGGTT AAATCGAGGA CAGAATATTT GACTCAAAGT ATAACGGAGG CTAAAATTTT AGAGCTACTG AATCAAAAGA TAGACCCTAC TAATAAACAT CATTTTTTAA GGATGTATGA CTCCTTTGTC CACAAGAACC ACTTATGTTT AGTGTTTGAA TTACTAAGCA ATAATTTGTA CGAATTATTA AAACAAAACA AATTTCATGG GCTCTCTATA CAACTTATCA GAACATTCAC CACTCAAATA CTGGATTCGT TATGCGTTTT GAAGGAAAGC AAACTGATTC ATTGCGATCT GAAGCCAGAG AATATCTTGC TCTGCGCACC TGATAAGCCG GAATTGAAAA TTATTGATTT TGGCTCATCT TGCGAAGAGG CAAGAACCGT TTATACATAC ATCCAGTCCA GGTTTTATCG TGCTCCTGAA ATTATACTGG GTATACCGTA TTCAACCAGT ATTGACATGT GGTCGTTAGG TTGTATTGTT GCTGAATTAT TTTTGGGTAT ACCGATCTTC CCAGGCGCTT CTGAATATAA CCAATTAACA AGAATAATAG ACACGCTTGG ATATCCTCCA TCGTGGATGA TAGATATGGG TAAAAACTCT GGAAAATTTA TGAAGAAATT GGCACCAGAA GAAAGTTCTT CTTCTACACA AAAGCATCGT ATGAAAACTA TTGAAGAGTT TTGCAGAGAA TACAATATAG TGGAAAAGCC CAGTAAACAA TATTTTAAGT GGAGAAAGTT ACCAGATATT ATTAGAAACT ACAGGTATCC TAAAAGCATA CAGAACTCCC AAGAACTTAT CGACCAAGAA ATGCAGAATA GGGAGTGTTT GATCCACTTT TTAGGCGGTG TGCTAAATTT GAACCCGTTA GAAAGATGGA CACCACAACA AGCTATGCTA CACCCCTTCA TAACAAAGCA GGAGTTTACA GGTGAGTGGT TTCCTCCAGG ATCGTCTTTA CCGGGTCCTT CAGAAAAACA TGACGATGCA AAAGGCCAGC AAAGTGAATA TGGAAGTGCG AACGACTCTA GTAACAATGC AGGCCACAAC TATGTCTATA ATCCTAGCTC TGCCACTGGT GGTGCTGATA GCGTCGACAT TGGTGCTATC AGTAAAAGGA AGGAGAATAC ATCTGGCGAC ATCTCCAATA ATTTTGCTGT TACTCATTCT GTTCAAGAAG GGCCAACAAG CGCGTTCAAT AAACTTCACA TTGTCGAAGA ATAA

SEQ ID NO:2

MNSSNNNDSS SSNSNMN SL SPT VTHSDA S GSGRASPD NSHMGRGI N PSYVNQGSQR

SPQQQHQNHH QQQQQQQQQQ QQNSQFCFVN P NEEKVTNS QQNLVYPPQY DDLNSNESLD AYRRRKSSLV VPPARAPAPN PFQYDSYPAY TSSNTSLAGN SSGQYPSGYQ QQQQQVYQQG

AIHPSQFGSR FVPSLYDRQD FQRRQSLAAT NYSSNFSS N SNTNQGTNSI PVMSPYRRLS

AYPPSTSPPL QPPFKQLRRD EVQGQKLSIP QMQLCNSK D LQPVLNATPK FRRASLNSKT ISPLVSVTKS LITTYS CSP EFTYQTSKNP KRV TKPSEG KCN GFDNIN SDYILYVNDV

LGVEQNRKYL VLDILGQGTF GQWKCQNLL TKEILAVKW KSRTEYLTQS ITEAKILELL

NQKIDPTNKH HFLRMYDSFV HKNHLCLVFE LLSNNLYEL KQNKFHGLSI QLIRTFTTQI

LDSLCV KES KLIHCDLKPE NIL CAPDKP ELKIIDFGSS CEEARTVYTY IQSRFYRAPE

IILGIPYSTS IDM SLGCIV AELFLGIPIF PGASEYNQ T RIIDTLGYPP SWMIDMGKNS GKFMKKLAPE ESSSSTQKHR MKTIEEFCRE YNIVEKPSKQ YFK RKLPDI IRNYRYPKSI

QNSQE IDQE MQNRECLIHF LGGV NLNPL ERWTPQQAML HPFITKQEFT GE FPPGSSL

PGPSEKHDDA KGQQSEYGSA NDSS NAGHN YVΥ PSSATG GADSVDIGAI SKRKENTSGD

ISN FAVTHS VQEGPTSAFN KLHIVEE

SEQ ID NO:3

GGAAACCTTC GGCCGCCGCT CCCGCCGCCT ACCCGACCGA TTGGCGGCAG TAAGCACACA ATGAATGATC ACCTGCATGT CGGCAGCCAC GCTCACGGAC AGATCCAGGT TCAACAGTTG TTTGAGGATA ACAGTAACAA GCGGACAGTG CTCACGACAC AACCAAATGG GCTTACAACA GTGGGCAAAA CGGGCTTGCC AGTGGTGCCA GAGCGGCAGC TGGACAGCAT TCATAGACGG CAGGGGAGCT CCACCTCTCT AAAGTCCATG GAAGGCATGG GGAAGGTGAA AGCCACCCCC ATGACACCTG AACAAGCAAT GAAGCAATAC ATGCAAAAAC TCACAGCCTT CGAACACCAT GAGATTTTCA GCTACCCTGA AATATATTTC TTGGGTCTAA ATGCTAAGAA GCGCCAGGGC ATGACAGGTG GGCCCAACAA TGGTGGCTAT GATGATGACC AGGGATCATA TGTGCAGGTG CCCCACGATC ACGTGGCTTA CAGGTATGAG GTCCTCAAGG TCATTGGGAA GGGGAGCTTT GGGCAGGTGG TCAAGGCCTA CGATCACAAA GTCCACCAGC ACGTGGCCCT AAAGATGGTG CGGAATGAGA AGCGCTTCCA CCGGCAAGCA GCGGAGGAGA TCCGAATCCT GGAACACCTG CGGAAGCAGG ACAAGGATAA CACAATGAAT GTCATCCATA TGCTGGAGAA TTTCACCTTC CGCAACCACA TCTGCATGAC GTTTGAGCTG CTGAGCATGA ACCTCTATAA GCTCATCAAG AAGAATAAAT TCCAGGGCTT CAGTCTGCCT TTGGTTCGCA AGTTTGCCCA CTCGATTCTG CAGTGCTTGG ATGCTTTGCA CAAAAACAGA ATAATTCACT GTGACCTTAA GCCCGAGAAC ATTTTGTTAA AGCAGCAGGG TAGAAGCGGT ATTAAAGTAA TTGATTTTGG CTCCAGTTGT TACGAGCATC AGCGTGTCTA CACGTACATC CAGTCGCGTT TTTACCGGGC TCCAGAAGTG ATCCTTGGGG CCAGGTATGG CATGCCCATT GATATGTGGA GCCTGGGCTG CATTTTAGCA GAGCTCCTGA CGGGTTACCC CCTCTTGCCT GGGGAAGATG AAGGGGACCA GCTGGCCTGT ATGATTGAAC TGTTGGGCAT GCCCTCACAG AAACTGCTGG ATGCATCCAA ACGAGCCAAA AATTTTGTGA GCTCCAAGGG TTATCCCCGT TACTGCACTG TCACGACTCT CTCAGATGGC TCTGTGGTCC TAAACGGAGG CCGTTCCCGG AGGGGGAAAC TGAGGGGCCC ACCGGAGAGC AGAGAGTGGG GTAACGCGCT GAAGGGGTGT GATGATCCCC TTTTCCTTGA CTTCTTAAAA CAGTGTTTAG AGTGGGATCC TGCAGTGCGC ATGACCCCAG GCCAGGCTTT GCGGCACCCC TGGCTGAGGA GGCGGTTGCC AAAGCCTCCC ACCGGGGAGA AAACGTCAGT GAAAAGGATA ACTGAGAGCA CCGGTGCTAT CACATCTATA TCCAAGTTAC CTCCACCTTC TAGCTCAGCT TCCAAACTGA GGACTAATTT GGCGCAGATG ACAGATGCCA ATGGGAATAT TCAGCAGAGG ACAGTGTTGC CAAAACTTGT TAGCTGAGCT CACGTCCCCT GATGCTGGTA ACCTGAAAGA TACGACATTG CTGAGCCTTA CTGGGTTGAA AAGGAGTAGC TCAGACCTGT TTTTATTTGC TCAATAACTC TACTCATTTG TATCTTTTCA GCACTTAATT TTAATGTAAG AAAGTTGTTC ATTTTGTTTT TATAAAATAC ATGAGGACAA TGCTTTAAGT TTTTATACTT TCAGAAACTT TTTGTGTTCT AAAAGTACAA TGAGCCTTAC TGTATTTAGT GTGGCAGAAT AATAACATCA ATGGCAGGCC ACTGATTACT TCATGACTGC CACGCATTTA CAGATTGGTG TCAAAGACAT TCACTATGTT TTTATGGTTC ATGTTATATC CTCCCCAGGG TGACAGCCCC TTAAGGCCCT CCTTTTCCCT CCATGCTCCA GGTCCATGCA CAGGTGTAGC ATGTC

SEQ ID NO:4

Met Asn Asp His Leu His Val Gly Ser His Ala His Gly Gin lie Gin Val Gin Gin Leu Phe Glu Asp Asn Ser Asn Lys Arg Thr Val Leu Thr Thr Gin Pro Asn Gly Leu Thr Thr Val Gly Lys Thr Gly Leu Pro Val Val Pro Glu Arg Gin Leu Asp Ser lie His Arg Arg Gin Gly Ser Ser Thr Ser Leu Lys Ser Met Glu Gly Met Gly Lys Val Lys Ala Thr Pro Met Thr Pro Glu Gin Ala Met Lys Gin Tyr Met Gin Lys Leu Thr Ala Phe Glu His His Glu lie Phe Ser Tyr Pro Glu lie Tyr Phe Leu Gly Leu Asn Ala Lys Lys Arg Gin Gly Met Thr Gly Gly Pro Asn Asn Gly Gly Tyr Asp Asp Asp Gin Gly Ser Tyr Val Gin Val Pro His Asp His Val Ala Tyr Arg Tyr Glu Val Leu Lys Val lie Gly Lys Gly Ser Phe Gly Gin Val Val Lys Ala Tyr Asp His Lys Val His Gin His Val Ala Leu Lys Met Val Arg Asn Glu Lys Arg Phe His Arg Gin Ala Ala Glu Glu lie Arg lie Leu Glu His Leu Arg Lys Gin Asp Lys Asp Asn Thr Met Asn Val lie His Met Leu Glu Asn Phe Thr Phe Arg Asn His lie Cys Met Thr Phe Glu Leu Leu Ser Met Asn Leu Tyr Lys Leu lie Lys Lys Asn Lys Phe Gin Gly Phe Ser Leu Pro Leu Val Arg Lys Phe Ala His Ser lie Leu Gin Cys Leu Asp Ala Leu His Lys Asn Arg lie lie His Cys Asp Leu Lys Pro Glu Asn lie Leu Leu Lys Gin Gin Gly Arg Ser Gly lie Lys Val lie Asp Phe Gly Ser Ser Cys Tyr Glu His Gin Arg Val Tyr Thr Tyr lie Gin Ser Arg Phe Tyr Arg Ala Pro Glu Val lie Leu Gly Ala Arg Tyr Gly Met Pro lie Asp Met Trp Ser Leu Gly Cys lie Leu Ala Glu Leu Leu Thr Gly Tyr Pro Leu Leu Pro Gly Glu Asp Glu Gly Asp Gin Leu Ala Cys Met lie Glu Leu Leu Gly Met Pro Ser Gin Lys Leu Leu Asp Ala Ser Lys Arg Ala Lys Asn Phe Val Ser Ser Lys Gly Tyr Pro Arg Tyr Cys Thr Val Thr Thr Leu Ser Asp Gly Ser Val Val Leu Asn Gly Gly Arg Ser Arg Arg Gly Lys Leu Arg Gly Pro Pro Glu Ser Arg Glu Trp Gly Asn Ala Leu Lys Gly Cys Asp Asp Pro Leu Phe Leu Asp Phe Leu Lys Gin Cys Leu Glu Trp Asp Pro Ala Val Arg Met Thr Pro Gly Gin Ala Leu Arg His Pro Trp Leu Arg Arg Arg Leu Pro Lys Pro Pro Thr Gly Glu Lys Thr Ser Val Lys Arg lie Thr Glu Ser Thr Gly Ala lie Thr Ser lie Ser Lys Leu Pro Pro Pro Ser Ser Ser Ala Ser Lys Leu Arg Thr Asn Leu Ala Gin Met Thr Asp Ala Asn Gly Asn lie Gin Gin Arg Thr Val Leu Pro Lys Leu Val Ser

SEQ ID NO:5

GGAGCGAAAT GCGCTGAGCT GCAGTGTCTG GTCGAGAGTA CCCGTGGGAG CGTCGCGCCG CGGAGGCAGC CGTCCCGGCG TAGGTGGCGT GGCCGACCGG ACCCCCAACT GGCGCCTCTC CCCGCGCGGG GTCCCGAGCT AGGAGATGGG AGGCACAGCT CGTGGGCCTG GGCGGAAGGA TGCGGGGCCG CCTGGGGCCG GGCTCCCGCC CCAGCAGCGG AGGTTGGGGG ATGGTGTCTA TGACACCTTC ATGATGATAG ATGAAACCAA ATGTCCCCCC TGTTCAAATG TACTCTGCAA TCCTTCTGAA CCACCTCCAC CCAGAAGACT AAATATGACC ACTGAGCAGT TTACAGGAGA TCATACTCAG CACTTTTTGG ATGGAGGTGA GATGAAGGTA GAACAGCTGT TTCAAGAATT TGGCAACAGA AAATCCAATA CTATTCAGTC AGATGGCATC AGTGACTCTG AAAAATGCTC TCCTACTGTT TCTCAGGGTA AAAGTTCAGA TTGCTTGAAT ACAGTAAAAT CCAACAGTTC ATCCAAGGCA CCCAAAGTGG TGCCTCTGAC TCCAGAACAA GCCCTGAAGC AATATAAACA CCACCTCACT GCCTATGAGA AACTGGAAAT AATTAATTAT CCAGAAATTT ACTTTGTAGG TCCAAATGCC AAGAAAAGAC ATGGAGTTAT TGGTGGTCCC AATAATGGAG GGTATGATGA TGCAGATGGG GCCTATATTC ATGTACCTCG AGACCATCTA GCTTATCGAT ATGAGGTGCT GAAAATTATT GGCAAGGGGA GTTTTGGGCA GGTGGCCAGG GTCTATGATC ACAAACTTCG ACAGTACGTG GCCCTAAAAA TGGTGCGCAA TGAGAAGCGC TTTCATCGTC AAGCAGCTGA GGAGATCCGG ATTTTGGAGC ATCTTAAGAA ACAGGATAAA ACTGGTAGTA TGAACGTTAT CCACATGCTG GAAAGTTTCA CATTCCGGAA CCATGTTTGC ATGGCCTTTG AATTGCTGAG CATAGACCTT TATGAGCTGA TTAAAAAAAA TAAGTTTCAG GGTTTTAGCG TCCAGTTGGT ACGCAAGTTT GCCCAGTCCA TCTTGCAATC TTTGGATGCC CTCCACAAAA ATAAGATTAT TCACTGCGAT CTGAAGCCAG AAAACATTCT CCTGAAACAC CACGGGCGCA GTTCAACCAA GGTCATTGAC TTTGGGTCCA GCTGTTTCGA GTACCAGAAG CTCTACACATATATCCAGTC TCGGTTCTAC AGAGCTCCAG AAATCATCTT AGGAAGCCGC TACAGCACAC CAATTGACAT ATGGAGTTTT GGCTGCATCC TTGCAGAACT TTTAACAGGA CAGCCTCTCT TCCCTGGAGA GGATGAAGGA GACCAGTTGG CCTGCATGAT GGAGCTTCTA GGGATGCCAC CACCAAAACT TCTGGAGCAA TCCAAACGTG CCAAGTACTT TATTAATTCC AAGGGCATAC CCCGCTACTG CTCTGTGACT ACCCAGGCAG ATGGGAGGGT TGTGCTTGTG GGGGGTCGCT CACGTAGGGG TAAAAAGCGG GGTCCCCCAG GCAGCAAAGA CTGGGGGACA GCACTGAAAG GGTGTGATGA CTACTTGTTT ATAGAGTTCT TGAAAAGGTG TCTTCACTGG GACCCCTCTG CCCGCTTGAC CCCAGCTCAA GCATTAAGAC ACCCTTGGAT TAGCAAGTCT GTCCCCAGAC CTCTCACCAC CATAGACAAG GTGTCAGGGA AACGGGTAGT TAATCCTGCA AGTGCTTTCC AGGGATTGGG TTCTAAGCTG CCTCCAGTTG TTGGAATAGC CAATAAGCTT AAAGCTAACT TAATGTCAGA AACCAATGGT AGTATACCCC TATGCAGTGT ATTGCCAAAA CTGATTAGCT AGTGGACAGA GATATGCCCA GAGATGCATA TGTGTATATT TTTATGATCT TACAAACCTG CAAATGGAAA AAATGCAAGC CCATTGGTGG ATGTTTTTGT TAGAGTAGAC TTTTTTTAAA CAAGACAAAA CATTTTTATA TGATTATAAA A

SEQ ID NO:6

MGGTARGPGR KDAGPPGAGL PPQQRRLGDG VYDTFMMIDE TKCPPCSNVL CNPSEPPPPR RLNMTTEQFT GDHTQHFLDG GEMKVEQLFQ EFGNRKSNTI QSDGISDSEK CSPTVSQGKS SDCLNTVKSN SSSKAPKWPLTPEQALKQY KHHLTAYEKL EIINYPEIYF VGPNAKKRHG VIGGPNNGGY DDADGAYIHV PRDHLAYRYE VLKIIGKGSF GQVARVYDHK LRQYVALKMV RNEKRFHRQA AEEIRILEHL KKQDKTGSMN VIHMLESFTF RNHVCMAFEL LSIDLYELIK KNKFQGFSVQ LVRKFAQSIL QSLDALHKNK IIHCDLKPEN ILLKHHGRSS TKVIDFGSSC FEYQKLYTYI QSRFYRAPEI ILGSRYSTPI DIWSFGCILA ELLTGQPLFP GEDEGDQLAC MMELLGMPPP KLLEQSKRAK YFINSKGIPR YCSVTTQADG RWLVGGRSR RGKKRGPPGS KD GTALKGC DDYLFIEFLK RCLHWDPSAR LTPAQALRHP WISKSVPRPL TTIDKVSGKR WNPASAFQG LGSKLPPWG IANKLKA LM SETNGSIPLC SVLPKLIS

SEQ ID NO:7

CGGCGCTGGC AAGCGAAGCT TGGGGGTGGG GAGGTAGAGT GAGCCCTCAG TAGGAGGGAC GAGGGCAGGG GTCTGACTGC CTCCCCGGGA CCGCCCCCAC CTCCTCTCTA TCAGGGCCCC CTCCCCCCAT CCCTGTCTCA CCGGGCGCGG GGGACGGGGC TAGAGCGGAG TTAGAGCAAG AAGAATTTCC ACCCCTGGAT TCCCTCTGAA ACCCTAGATC GGGGTATATG TTAAGGGATT ACGAAAATCT AGGACTTTTT GTGGGGCTTT TTATTAAAGG GGGGGAGCCC GGGAGCAATA CCTTGGAAAG AAGCCCTGTT GCTTAGAGCG GATAACCAAC GGCTGAACTC TTGGGGTTTG CTGTGAGGGG TGCGGTCTAG CTTCGAATGT ACAGTGGTGG AGCCACAGTG TTAAAGAACA GAGAAGTGAT CCTTAATCAT TTAGAATTTT GCCTCCACCA TCCACCAGAA AATGAAGTGG AAAGAGAAGT TGGGGGATGG TGTCTATGAC ACCTTCATGA TGATAGATGA AACCAAATGT CCCCCCTGTT CAAATGTACT CTGCAATCCT TCTGAACCAC CTCCACCCAG AAGACTAAAT ATGACCACTG AGCAGTTTAC AGGAGATCAT ACTCAGCACT TTTTGGATGG AGGTGAGATG AAGGTAGAAC AGCTGTTTCA AGAATTTGGC AACAGAAAAT CCAATACTAT TCAGTCAGAT GGCATCAGTG ACTCTGAAAA ATGCTCTCCT ACTGTTTCTC AGGGTAAAAG TTCAGATTGC TTGAATACAG TAAAATCCAA CAGTTCATCC AAGGCACCCA AAGTGGTGCC TCTGACTCCA GAACAAGCCC TGAAGCAATA TAAACACCAC CTCACTGCCT ATGAGAAACT GGAAATAATT AATTATCCAG AAATTTACTT TGTAGGTCCA AATGCCAAGA AAAGACATGG AGTTATTGGT GGTCCCAATA ATGGAGGGTA TGATGATGCA GATGGGGCCT ATATTCATGT ACCTCGAGAC CATCTAGCTT ATCGATATGA GGTGCTGAAA ATTATTGGCA AGGGGAGTTT TGGGCAGGTG GCCAGGGTCT ATGATCACAA ACTTCGACAG TACGTGGCCC TAAAAATGGT GCGCAATGAG AAGCGCTTTC ATCGTCAAGC AGCTGAGGAG ATCCGGATTT TGGAGCATCT TAAGAAACAG GATAAAACTG GTAGTATGAA CGTTATCCAC ATGCTGGAAA GTTTCACATT CCGGAACCAT GTTTGCATGG CCTTTGAATT GCTGAGCATA GACCTTTATG AGCTGATTAA AAAAAATAAG TTTCAGGGTT TTAGCGTCCA GTTGGTACGC AAGTTTGCCC AGTCCATCTT GCAATCTTTG GATGCCCTCC ACAAAAATAA GATTATTCAC TGCGATCTGA AGCCAGAAAA CATTCTCCTG AAACACCACG GGCGCAGTTC AACCAAGGTC ATTGACTTTG GGTCCAGCTG TTTCGAGTAC CAGAAGCTCT ACACATATAT CCAGTCTCGG TTCTACAGAG CTCCAGAAAT CATCTTAGGA AGCCGCTACA GCACACCAAT TGACATATGG AGTTTTGGCT GCATCCTTGC AGAACTTTTA ACAGGACAGC CTCTCTTCCC TGGAGAGGAT GAAGGAGACC AGTTGGCCTC CATGATGGAG CTTCTAGGGA TGCCACCACC AAAACTTCTG GAGCAATCCA AACGTGCCAA GTACTTTATT AATTCCAAGG GCATACCCCG CTACTGCTCT GTGACTACCC AGGCAGATGG GAGGGTTGTG CTTGTGGGGG GTCGCTCACG TAGGGGTAAA AAGCGGGGTC CCCCAGGCAG CAAAGACTGG GGGACAGCAC TGAAAGGGTG TGATGACTAC TTGTTTATAG AGTTCTTGAA AAGGTGTCTT CACTGGGACC CCTCTGCCCG CTTGACCCCA GCTCAAGCAT TAAGACACCC TTGGATTAGC AAGTCTGTCC CCAGACCTCT CACCACCATA GACAAGGTGT CAGGGAAACG GGTAGTTAAT CCTGCAAGTG CTTTCCAGGG ATTGGGTTCT AAGCTGCCTC CAGTTGTTGG AATAGCCAAT AAGCTTAAAG CTAACTTAAT GTCAGAAACC AATGGTAGTA TACCCCTATG CAGTGTATTG CCAAAACTGA TTAGCTAGTG GACAGAGATA TGCCCAGAGA TGCATATGTG TATATTTTTA TGATCTTACA AACCTGCAAA TGGAAAAAAT GCAAGCCCAT TGGTGGATGT TTTTGTTAGA GTAGACTTTT TTTAAACAAG ACAAAACATT TTTATATGAT TATAAAA

SEQ ID NO:8

Met Lys Trp Lys Glu Lys Leu Gly Asp Gly Val Tyr Asp Thr Phe Met Met lie Asp Glu Thr Lys Cys Pro Pro Cys Ser Asn Val Leu Cys Asn Pro Ser Glu Pro Pro Pro Pro Arg Arg Leu Asn Met Thr Thr Glu Gin Phe Thr Gly Asp His Thr Gin His Phe Leu Asp Gly Gly Glu Met Lys Val Glu Gin Leu Phe Gin Glu Phe Gly Asn Arg Lys Ser Asn Thr lie Gin Ser Asp Gly lie Ser Asp Ser Glu Lys Cys Ser Pro Thr Val Ser Gin Gly Lys Ser Ser Asp Cys Leu Asn Thr Val Lys Ser Asn Ser Ser Ser Lys Ala Pro Lys Val Val Pro Leu Thr Pro Glu Gin Ala Leu Lys Gin Tyr Lys His His Leu Thr Ala Tyr Glu Lys Leu Glu lie lie Asn Tyr Pro Glu lie Tyr Phe Val Gly Pro Asn Ala Lys Lys Arg His Gly Val lie Gly Gly Pro Asn Asn Gly Gly Tyr Asp Asp Ala Asp Gly Ala Tyr lie His Val Pro Arg Asp His Leu Ala Tyr Arg Tyr Glu Val Leu Lys lie lie Gly Lys Gly Ser Phe Gly Gin Val Ala Arg Val Tyr Asp His Lys Leu Arg Gin Tyr Val Ala Leu Lys Met Val Arg Asn Glu Lys Arg Phe His Arg Gin Ala Ala Glu Glu lie Arg lie Leu Glu His Leu Lys Lys Gin Asp Lys Thr Gly Ser Met Asn Val lie His Met Leu Glu Ser Phe Thr Phe Arg Asn His Val Cys Met Ala Phe Glu Leu Leu Ser lie Asp Leu Tyr Glu Leu lie Lys Lys Asn Lys Phe Gin Gly Phe Ser Val Gin Leu Val Arg Lys Phe Ala Gin Ser lie Leu Gin Ser Leu Asp Ala Leu His Lys Asn Lys lie lie His Cys Asp Leu Lys Pro Glu Asn lie Leu Leu Lys His His Gly Arg Ser Ser Thr Lys Val lie Asp Phe Gly Ser Ser Cys Phe Glu Tyr Gin Lys Leu Tyr Thr Tyr lie Gin Ser Arg Phe Tyr Arg Ala Pro Glu lie lie Leu Gly Ser Arg Tyr Ser Thr Pro lie Asp lie Trp Ser Phe Gly Cys lie Leu Ala Glu Leu Leu Thr Gly Gin Pro Leu Phe Pro Gly Glu Asp Glu Gly Asp Gin Leu Ala Ser Met Met Glu Leu Leu Gly Met Pro Pro Pro Lys Leu Leu Glu Gln Ser Lys Arg Ala Lys Tyr Phe lie Asn Ser Lys Gly lie Pro Arg Tyr Cys

Ser Val Thr Thr Gin Ala Asp Gly Arg Val Val Leu Val Gly Gly Arg Ser Arg

Arg Gly Lys Lys Arg Gly Pro Pro Gly Ser Lys Asp Trp Gly Thr Ala Leu Lys

Gly Cys Asp Asp Tyr Leu Phe lie Glu Phe Leu Lys Arg Cys Leu His Trp Asp

Pro Ser Ala Arg Leu Thr Pro Ala Gin Ala Leu Arg His Pro Trp lie Ser Lys

Ser Val Pro Arg Pro Leu Thr Thr lie Asp Lys Val Ser Gly Lys Arg Val Val

Asn Pro Ala Ser Ala Phe Gin Gly Leu Gly Ser Lys Leu Pro Pro Val Val Gly lie Ala Asn Lys Leu Lys Ala Asn Leu Met Ser Glu Thr Asn Gly Ser lie Pro

Leu Cys Ser Val Leu Pro Lys Leu lie Ser

SEQ ID NO:9

Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro Met Ala Arg Arg

SEQ ID NO:10

Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Ala Met Ala Arg Arg (Peptide 1)

SEQ ID NO:11

Leu Gly Gly Arg Asp Ser Arg Ser Gly Thr Pro Met Ala Arg Arg (Peptide 2)

SEQ ID NO:12

Leu Gly Gly Arg Asp Ser Arg Ser Gly Ser Pro Pro Ala Arg Arg (Peptide 3)

SEQ ID NO:13

Leu Gly Gly Arg Asp Ser Arg Ser Pro Ser Pro Met Ala Arg Arg (Peptide 4)

SEQID NO:14

Leu Gly Gly Arg Asp Ser Arg Ser Pro Ser Ala Met Ala Arg Arg (Peptide 5)

SEQIDNO:15

Leu Gly Gly Arg Asp Ser Arg Ala Gly Ser Pro Met Ala Arg Arg (peptide 6)

SEQIDNO:16

Leu Gly Gly Arg Asp Ala Arg Ser Gly Ser Pro Met Ala Arg Arg (Peptide 7)

SEQIDNO:17 Leu Gly Gly Arg Asp Ala Arg Ala Gly Ser Pro Met Ala Arg Arg (peptide 8)

SEQ ID NO:18

NIVTPRTPPPSQGK

Claims

What is claimed is:

1. A method for screening for an agent that modulates a YAK polypeptide kmase activity comprising: a. contracting a candidate agent with a YAK polypeptide, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact; and b. subsequently measuπng the ability of the candidate agent to modulate the ability of the polypeptide to phosphorylate the substrate.

2. The method of claim 1 wherein the YAK polypeptide has an ammo acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID

NO:8.

3. The method of claim 2 wherein the substrate comprises the SER-164 polypeptide.