Human protein kinases hYAK3

hYAK3 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing hYAK3 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 including testicular cancer; 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 dela Tourett's syndrome., among others, and diagnostic assays for such conditions.

FIELD OF INVENTION 
This invention relates to newly identified polynucleotides, polypeptides 
encoded by them and to the use of such polynucleotides and polypeptides, 
and to their production. More particularly, the polynucleotides and 
polypeptides of the present invention relate to a serine/threonine protein 
kinase, hereinafter referred to as hYAK3. The invention also relates to 
inhibiting or activating the action of such polynucleotides and 
polypeptides. 
BACKGROUND OF THE INVENTION 
A number of polypeptide growth factors and hormones mediate their cellular 
effects through a signal transduction pathway. Transduction of signals 
from the cell surface receptors for these ligands to intracellular 
effectors frequently involves phosphorylation or dephosphorylation of 
specific protein substrates by regulatory protein serine/threonine kinases 
(PSTK) and phosphatases. Serine/threonine phosphorylation is a major 
mediator of signal transduction in multicellular organisms. 
Receptor-bound, membrane-bound and intracellular PSTKs regulate cell 
proliferation, cell differentiation and signalling processes in many cell 
types. 
Aberrant protein serine/threonine kinase activity has been implicated or is 
suspected in a number of pathologies such as rheumatoid arthritis, 
psoriasis, septic shock, bone loss, many cancers and other proliferative 
diseases. Accordingly, serine/threonine kinases and the signal 
transduction pathways which they are part of are potential targets for 
drug design. 
A subset of PSTKs are involved in regulation of cell cycling. These are the 
cyclin-dependent kinases or CDKs (Peter and Herskowitz, Cell 1994: 79, 
181-184). CDKs are activated by binding to regulatory proteins called 
cyclins and control passage of the cell through specific cell cycle 
checkpoints. For example, CDK2 complexed with cyclin E allows cells to 
progress through the G1 to S phase transition. The complexes of CDKs and 
cyclins are subject to inhibition by low molecular weight proteins such as 
p16 (Serrano et al, Nature 1993: 366, 704), which binds to and inhibits 
CDK4. Deletions or mutations in p16 have been implicated in a variety of 
tumors (Kamb et al, Science 1994: 264, 436-440). Therefore, the 
proliferative 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. 
YAK1, a PSTK with sequence homology to CDKs, was originally identified in 
yeast as a mediator of cell cycle arrest caused by inactivation of the 
cAMP-dependent protein kinase PKA (Garrett et al, Mol Cell Biol. 1991: 11, 
4045-4052). YAK1 kinase activity is low in cycling yeast but increases 
dramatically when the cells are arrested prior to the S-G2 transition. 
Increased expression of YAK1 causes growth arrest in yeast cells deficient 
in PKA. Therefore, YAK1 can act as a cell cycle suppressor in yeast. 
Frequently, in disease such as osteoporosis and osteoarthritis, 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 forration, respectively. 
Similarly, replacement of heart or skeletal muscle depleted by diseases 
such as myocardial infarction or HIV-associated cachexia requires drugs 
that stimulate proliferation of cardiac myocytes or skeletal myoblasts. 
The present invention describes a novel human homolog of yeast YAK1 termed 
hYAK3, which is expressed predominantly in testis and skeletal muscle. The 
sequence of hYAK3 shares homology with predicted PSTK's from C. elegans, 
S. pombe and S. cerevisiae and has motifs associated with known protein 
kinases. Inhibitors of hYAK3 are expected to stimulate proliferation of 
cells in which it is expressed. 
This indicates that these serine/threonine protein kinases have an 
established, proven history as therapeutic targets. Clearly there is a 
need for identification and characterization of further members of the 
serine/threonine protein kinase 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 arthritis, Osteoarthritis, Infammatory 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 including testicular 
cancer; 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 dela Tourett's syndrome. 
SUMMARY OF THE INVENTION 
In one aspect, the invention relates to hYAK3 polypeptides and recombinant 
materials and methods for their production. Another aspect of the 
invention relates to methods for using such hYAK3 polypeptides and 
polynucleotides. Such uses include 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 including 
testicular cancer; 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 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 hYAK3 imbalance with 
the identified compounds. Yet another aspect of the invention relates to 
diagnostic assays for detecting diseases associated with inappropriate 
hYAK3 activity or levels.

DESCRIPTION OF THE INVENTION 
Definitions 
The following definitions are provided to facilitate understanding of 
certain terms used frequently herein. 
"hYAK3" refers, among others, generally to a polypeptide having the amino 
acid sequence set forth in SEQ ID NO:2 or 4 or an allelic variant thereof. 
"hYAK3 activity or hYAK3 polypeptide activity" or "biological activity of 
the hYAK3 or hYAK3 polypeptide" refers to the metabolic or physiologic 
function of said hYAK3 including similar activities or improved activities 
or these activities with decreased undesirable side-effects. Also included 
are antigenic and immunogenic activities of said hYAK3. 
"hYAK3 gene" refers to a polynucleotide having the nucleotide sequence set 
forth in SEQ ID NO:1 or 3 or allelic variants thereof and/or their 
complements. 
"Antibodies" as used herein includes polyclonal and monoclonal antibodies, 
chimeric, single chain, and humanized antibodies, as well as Fab 
fragments, including the products of an Fab or other immunoglobulin 
expression library. 
"Isolated" means altered "by the hand of man" from the natural state. If an 
"isolated" composition or substance occurs in nature, it has been changed 
or removed from its original environment, or both. For example, a 
polynucleotide or a polypeptide naturally present in 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 polyribonucleotide or 
polydeoxribonucleotide, 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 comprising DNA and 
RNA that may be single-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, tritylated bases and unusual bases such as inosine. A variety of 
modifications has been made to DNA and RNA; thus, "polynucleotide" 
embraces chemically, enzymatically or metabolically 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 
oligonucleotides. 
"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, oligopeptides or oligomers, 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 described in basic 
texts and in more detailed monographs, as well as in a voluminous research 
literature. Modifications can occur anywhere in a polypeptide, including 
the peptide backbone, the amino acid side-chains and the amino or carboxyl 
termini. It will be appreciated that the same type of modification may be 
present in 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 ubiquitination, 
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-ribosylation, amidation, covalent attachment 
of flavin, covalent attachment of a heme moiety, covalent attachment of a 
nucleotide or nucleotide derivative, covalent attachment of a lipid or 
lipid derivative, covalent attachment of phosphotidylinositol, 
cross-linking, cyclization, disulfide bond formation, demethylation, 
formation of covalent cross-links, formation of cystine, formation of 
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor 
formation, hydroxylation, iodination, methylation, myristoylation, 
oxidation, proteolytic processing, phosphorylation, prenylation, 
racemization, selenoylation, sulfation, transfer-RNA mediated addition of 
amino acids to proteins such as arginylation, and ubiquitination. 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 NY Acad 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 in the nucleotide sequence of the variant may or may not alter the 
amino acid sequence of a polypeptide encoded by the reference 
polynucleotide. Nucleotide changes may result in amino acid substitutions, 
additions, deletions, fusions and truncations in the polypeptide encoded 
by the reference sequence, as discussed below. A typical variant of a 
polypeptide differs in amino acid sequence from another, reference 
polypeptide. Generally, differences are limited so that the sequences of 
the reference polypeptide and the variant are closely similar overall and, 
in many regions, identical. A variant and reference polypeptide may differ 
in amino acid sequence by one or more substitutions, additions, deletions 
in any combination. A substituted or inserted amino acid residue may or 
may not be one encoded by the genetic code. A variant of a polynucleotide 
or polypeptide may be a naturally occurring such as an allelic variant, or 
it may be a variant that is not known to occur naturally. Non-naturally 
occurring variants of polynucleotides and polypeptides may be made by 
mutagenesis techniques or by direct synthesis. 
"Identity" is a measure of the identity of nucleotide sequences or amino 
acid sequences. In general, the sequences are aligned so that the highest 
order match is obtained. "Identity" per se has an art-recognized meaning 
and can be calculated using published techniques. See, e.g.: 
(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University 
Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, 
Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF 
SEQUENCE DATA, T I, Griffin, A. M., and Griffin, H. G., eds., Humana 
Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von 
Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov, 
M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there 
exist a number of methods to measure identity between two polynucleotide 
or polypeptide sequences, the term "identity" is well known to skilled 
artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 
48:1073). Methods commonly employed to determine identity or similarity 
between two sequences include, but are not limited to, those disclosed in 
Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 
1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073. 
Methods to determine identity and similarity are codified in computer 
programs. Preferred computer program methods to determine identity and 
similarity between two sequences include, but are not limited to, GCS 
program package (Devereux, J., et al., Nucleic Acids Research (1984) 
12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J Molec Biol 
(1990) 215:403). 
Polypeptides of the Invention 
In one aspect, the present invention relates to hYAK3 polypeptides. The 
hYAK3 polypeptides include the polypeptide of SEQ ID NO:2 or 4; as well as 
polypeptides comprising the amino acid sequence of SEQ ID NO: 2 or 4; and 
polypeptides comprising the amino acid sequence which have at least 80% 
identity to that of SEQ ID NO:2 or 4 over its entire length, and still 
more preferably at least 90% identity, and even still more preferably at 
least 95% identity to SEQ ID NO: 2 or 4. Also included within hYAK3 
polypeptides are polypeptides having the amino acid sequence which have at 
least 80% identity to the polypeptide having the amino acid sequence of 
SEQ ID NO:2 or 4 over its entire length, and still more preferably at 
least 90% identity, and still more preferably at least 95% identity to SEQ 
ID NO:2 or 4. Preferably hYAK3 polypeptide exhibit at least one biological 
activity of hYAK3. 
The hYAK3 polypeptides may be in the form of the "mature" protein or may be 
a part of a larger protein such as a fusion protein. It is often 
advantageous to include an additional amino acid sequence which contains 
secretory or leader sequences, pro-sequences, sequences which aid in 
purification such as multiple histidine residues, or an additional 
sequence for stability during recombinant production. 
Biologically active fragments of the hYAK3 polypeptides are also included 
in the invention. A fragment is a polypeptide having an amino acid 
sequence that entirely is the same as part, but not all, of the amino acid 
sequence of the aforementioned hYAK3 polypeptides. As with hYAK3 
polypeptides, fragments may be "free-standing," or comprised within a 
larger polypeptide of which they form a part or region, most preferably as 
a single continuous region. Representative examples of polypeptide 
fragments of the invention, include, for example, fragments from about 
amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of 
hYAK3 polypeptide. In this context "about" includes the particularly 
recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at 
either extreme or at both extremes. 
Preferred fragments include, for example, truncation polypeptides having 
the amino acid sequence of hYAK3 polypeptides, except for deletion of a 
continuous series of residues that includes the amino terminus, or a 
continuous series of residues that includes the carboxyl terminus or 
deletion of two continuous series of residues, one including the amino 
terminus and one including the carboxyl terminus. Also preferred are 
fragments characterized by structural or functional attributes such as 
fragments that comprise alpha-helix and alpha-helix forming regions, 
beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, 
coil and coil-forming regions, hydrophilic regions, hydrophobic regions, 
alpha amphipathic regions, beta amphipathic regions, flexible regions, 
surface-fornming regions, substrate binding region, and high antigenic 
index regions. Biologically active fragments are those that mediate hYAK3 
activity, including those with a similar activity or an improved activity, 
or with a decreased undesirable activity. Also included are those that are 
antigenic or immunogenic in an animal, especially in a human. 
Preferably, all of these polypeptide fragments retain the biological 
activity of the hYAK3, including antigenic activity. Variants of the 
defined sequence and fragments also formn part of the present invention. 
Preferred variants are those that vary from the referents by conservative 
amino acid substitutions--i.e., those that substitute a residue with 
another of like characteristics. Typical such substitutions are among Ala, 
Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and 
Glu; among Asn and Gln; and among the basic residues Lys and Arg; or 
aromatic residues Phe and Tyr. Particularly preferred are variants in 
which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or 
added in any combination. 
The hYAK3 polypeptides of the invention can be prepared in any suitable 
manner. Such polypeptides include isolated naturally occurring 
polypeptides, recombinantly produced polypeptides, synthetically produced 
polypeptides, or polypeptides produced by a combination of these methods. 
Means for preparing such polypeptides are well understood in the art. 
Polynucleotides of the Invention 
Another aspect of the invention relates to hYAK3 polynucleotides. hYAK3 
polynucleotides include isolated polynucleotides which encode the hYAK3 
polypeptides and fragments, and polynucleotides closely related thereto. 
More specifically, hYAK3 polynucleotide of the invention include a 
polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO:1 
or 3 encoding a hYAK3 polypeptide of SEQ ID NO: 2 or 4, and polynucleotide 
having the particular sequence of SEQ ID NO: 1 or 3. hYAK3 polynucleotides 
further include a polynucleotide comprising a nucleotide sequence that has 
at least 80% identity to a nucleotide sequence encoding the hYAK3 
polypeptide of SEQ ID NO:2 or 4 over its entire length, and a 
polynucleotide that is at least 80% identical to that having SEQ ID NO:1 
or 3 over its entire length. In this regard, polynucleotides at least 90% 
identical are particularly preferred, and those with at least 95% are 
especially preferred. Furthermore, those with at least 97% are highly 
preferred and those with at least 98-99% are most highly preferred, with 
at least 99% being the most preferred. Also included under hYAK3 
polynucleotides are a nucleotide sequence which has sufficient identity to 
a nucleotide sequence contained in SEQ ID NO:1 or 3 to hybridize under 
conditions useable for amplification or for use as a probe or marker. The 
invention also provides polynucleotides which are complementary to such 
hYAK3 polynucleotides. 
hYAK3 of the invention is structurally related to other proteins of the 
serine/threonine protein kinase family, as shown by the results of 
sequencing the cDNA encoding human hYAK3. The cDNA sequence contains an 
open reading frame encoding a polypeptide of 588/568 (.alpha. and .beta. 
forms, respectively) amino acids. Amino acid of sequence of FIGS. 1 and 2 
(SEQ ID NOS:2 and 4, respectively) has about 65% identity (using FASTA) in 
402 amino acid residues with C. elegans protein Kinase F49E11.1. 
Furthermore, hYAK3 is 49% identical to S. pombe protein kinase S2F7.03c 
over 315 amino acids (Barrell et al., Schizosaccahromyces pombe chromosome 
I sequencing project, 1995) and 46% identical to S. cerevisiae protein 
kinase YAK1 over 286 amino acids (Garrett and Broach, Genes & Develop. 
3:1336-1348, 1989). Nucleotide sequence of FIGS. 1 and 2 (SEQ ID NOS:1 and 
4, respectively) has about 64% identity (using FASTA) in 672 nucleotide 
residues with C. elegans protein kinase F49E11.1. 
One polynucleotide of the present invention encoding hYAK3 may be obtained 
using standard cloning and screening, from a cDNA library derived from 
mRNA in cells of human testis and skeletal muscle using the expressed 
sequence tag (EST) analysis (Adams, M. D., et al. Science (1991) 
252:1651-1656; Adams, M.D. et al., Nature, (1992) 355:632-634; Adams, M. 
D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides of the 
invention can also be obtained from natural sources such as genomic DNA 
libraries or can be synthesized using well known and commercially 
available techniques. 
The nucleotide sequence encoding hYAK3 polypeptide of SEQ ID NO:2 or 4 may 
be identical over its entire length to the coding sequence set forth in 
FIGS. 1 or 2 (SEQ ID NO:1 or 3), or may be a degenerate form of this 
nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or 4, or may 
be highly identical to a nucleotide sequence that encodes the polypeptide 
of SEQ ID NO: 2 or 4. Preferably, the polynucleotides of the invention 
comprise a nucleotide sequence that is highly identical, at least 80% 
identical, with a nucleotide sequence encoding a hYAK3 polypeptide, or at 
least 80% identical with the sequence contained in FIGS. 1 or 2 (SEQ ID 
NO: 1 or 3) encoding hYAK3 polypeptide, or at least 80% identical to a 
nucleotide sequence encoding the polypeptide of SEQ ID NO:2 or 4. 
When the polynucleotides of the invention are used for the recombinant 
production of hYAK3 polypeptide, the polynucleotide may include the coding 
sequence for the mature polypeptide or a fragment thereof, by itself; the 
coding sequence for the mature polypeptide or fragment in reading frame 
with other coding sequences, such as those encoding a leader or secretory 
sequence, a pre-, or pro- or prepro- protein sequence, or other fusion 
peptide portions. For example, a marker sequence which facilitates 
purification of the fused polypeptide can be encoded. In certain preferred 
embodiments of this aspect of the invention, the marker sequence is a 
hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and 
described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is 
an HA tag. The polynucleotide may also contain non-coding 5' and 3' 
sequences, such as transcribed, non-translated sequences, splicing and 
polyadenylation signals, ribosome binding sites and sequences that 
stabilize mRNA. 
Further preferred embodiments are polynucleotides encoding hYAK3 variants 
comprise the amino acid sequence hYAK3 polypeptide of FIGS. 1 or 2 (SEQ ID 
NO:2 or 4) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues 
are substituted, deleted or added, in any combination. 
The present invention further relates to polynucleotides that hybridize to 
the herein above-described sequences. In this regard, the present 
invention especially relates to polynucleotides which hybridize under 
stringent conditions to the herein abovedescribed polynucleotides. As 
herein used, the term "stringent conditions" means hybridization will 
occur only if there is at least 95% and preferably at least 97% identity 
between the sequences. 
Polynucleotides of the invention, which are identical or sufficiently 
identical to a nucleotide sequence contained in SEQ ID NO:1 or 3, may be 
used as hybridization probes for cDNA and genomic DNA, to isolate 
fulfill-length cDNAs and genomic clones encoding hYAK3 polypeptide and to 
isolate cDNA and genomic clones of other genes that have a high sequence 
sinilarity to the hYAK3 gene. Such hybridization techniques are known to 
those of skill in the art. Typically these nucleotide sequences are 70% 
identical preferably 80% identical more preferably 90% identical to that 
of the referent. The probes generally will comprise at least 15 
nucleotides. Preferably, such probes will have at least 30 nucleotides and 
may have at least 50 nucleotides. Particularly preferred probes will range 
between 30 and 50 nucleotides. 
In one embodiment, to obtain a polynucleotide encoding hYAK3 comprises the 
steps of screening an appropriate library under stingent hybridization 
conditions with a labeled probe having the SEQ ID NO:1 or 3 or a fragment 
thereof; and isolating full-length cDNA and genomic clones containing said 
polynucleotide sequence. Such hybridization techniques are well known to 
those of skill in the art. Stringent hybridization conditions are as 
defined above or alternatively conditions under overnight incubation at 
42.degree. C. in a solution comprising 50% formamide, 5.times. SSC (150 mM 
NaCl, 15 mM trisodiurn citrate), 50 mM sodium phosphate (pH 7.6), 5.times. 
Denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, 
sheared salmon sperm DNA, followed by washing the filters in 0.1.times. 
SSC at about 65.degree. C. 
The polynucleotides and polypeptides of the present invention may be 
employed as research reagents and materials for discovery of treatments 
and diagnostics to animal and human disease. 
Vectors, Host Cells, Expression 
The present invention also relates to vectors which comprise a 
polynucleotide or polynucleotides of the present invention, and host cells 
which are genetically engineered with vectors of the invention and to the 
production of polypeptides of the invention by recombinant techniques. 
Cell-free translation systems can also be employed to produce such 
proteins using RNAs derived from the DNA constructs of the present 
invention. 
For recombinant production, host cells can be genetically engineered to 
incorporate expression systems or portions thereof for polynucleotides of 
the present invention. Introduction of polynucleotides into host cells can 
be effected by methods described in many standard laboratory manuals, such 
as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et 
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium 
phosphate transfection, DEAE-dextran mediated transfection, transvection, 
microinjection, cationic lipid-mediated transfection, electroporation, 
transduction, scrape loading, ballistic introduction or infection. 
Representative examples of appropriate hosts include bacterial cells, such 
as streptococci staphylococci, E. coli, Streptomyces and Bacillus subtilis 
cells; fungal cells, such as yeast cells and Aspergillus cells; insect 
cels such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as 
CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant 
cells. 
A great variety of expression systems can be used. Such systems include, 
among others, chromosomal, episomal and virus-derived systems, e.g., 
vectors derived from bacterial plasmids, from bacteriophage, from 
transposons, from yeast episomes, from insertion elements, from yeast 
chromosomal elements, from viruses such as baculoviruses, papova viruses, 
such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, 
pseudorabies viruses and retroviruses, and vectors derived from 
combinations thereof, such as those derived from plasmid and bacteriophage 
genetic elements, such as cosmids and phagemids. The expression systems 
may contain control regions that regulate as well as engender expression. 
Generally, any system or vector suitable to maintain, propagate or express 
polynucleotides to produce a polypeptide in a host may be used. The 
appropriate nucleotide sequence may be inserted into an expression system 
by any of a variety of well-known and routine techniques, such as, for 
example, those set forth in Sambrook et al., MOLECULAR CLONING, A 
LABORATORY MANUAL (supra). 
For secretion of the translated protein into the lumen of the endoplasmic 
reticulum, into the periplasmic space or into the extracellular 
environment, appropriate secretion signals may be incorporated into the 
desired polypeptide. These signals may be endogenous to the polypeptide or 
they may be heterologous signals. 
If the hYAK3 polypeptide is to be expressed for use in screening assays, 
generally, it is preferred that the polypeptide be produced at the surface 
of the cell. In this event, the cells may be harvested prior to use in the 
screening assay. If hYAK3 polypeptide is secreted into the medium, the 
medium can be recovered in order to recover and purify the polypeptide; if 
produced intracellularly, the cells must first be lysed before the 
polypeptide is recovered. hYAK3 polypeptides can be recovered and purified 
from recombinant cell cultures by well-known methods including ammonium 
sulfate or ethanol precipitation, acid extraction, anion or cation 
exchange chromatography, phosphocellulose chromatography, hydrophobic 
interaction chromatography, affinity chromatography, hydroxylapatite 
chromatography and lectin chromatography. Most preferably, high 
performance liquid chromatography is employed for purification. Well known 
techniques for refolding proteins may be employed to regenerate active 
conformation when the polypeptide is denatured during isolation and or 
purification. 
Diagnostic Assays 
This invention also relates to the use of hYAK3 polynucleotides for use as 
diagnostic reagents. Detection of a mutated form of hYAK3 gene associated 
with a dysfunction will provide a diagnostic tool that can add to or 
define a diagnosis of a disease or susceptibility to a disease which 
results from under-expression, over-expression or altered expression of 
hYAK3. Individuals carrying mutations in the hYAK3 gene may be detected at 
the DNA level by a variety of techniques. 
Nucleic acids for diagnosis may be obtained from a subject's cells, such as 
from blood, urine, saliva, tissue biopsy or autopsy material. The genomic 
DNA may be used directly for detection or may be amplified enzymatically 
by using PCR or other amplification techniques prior to analysis. RNA or 
cDNA may also be used in similar fashion. Deletions and insertions can be 
detected by a change in size of the amplified product in comparison to the 
normal genotype. Point mutations can be identified by hybridizing 
amplified DNA to labeled hYAK3 nucleotide sequences. Perfectly matched 
sequences can be distinguished from mismatched duplexes by RNase digestion 
or by differences in melting temperatures. DNA sequence differences may 
also be detected by alterations in electrophoretic mobility of DNA 
fragments in gels, with or without denaturing agents, or by direct DNA 
sequencing. See, e.g., Myers et al., Science (1985) 230:1242. Sequence 
changes at specific locations may also be revealed by nuclease protection 
assays, such as RNase and S1 protection or the chemical cleavage rnethod. 
See Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another 
embodiment, an array of oligonucleotides probes comprising hYAK3 
nucleotide sequence or fragments thereof can be constructed to conduct 
efficient screening of e.g., genetic mutations. Array technology methods 
are well known and have general applicability and can be used to address a 
variety of questions in molecular genetics including gene expression, 
genetic linkage, and genetic variability. (See for example: M.Chee et al., 
Science, Vol 274, pp 610-613 (1996)). 
The diagnostic assays offer a process for diagnosing or determining a 
susceptibility to bone loss including osteoporosis; inflammatory diseases 
such as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthritis, 
Osteoarthritis, Inflammnatory 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 including testicular cancer; 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 dela 
Tourett's syndrome. through detection of mutation in the hYAK3 gene by the 
methods described. 
In addition, bone loss including osteoporosis; inflammatory diseases such 
as Adult Respiratory Disease Syndrome (ARDS), Rheumatoid arthritis, 
Osteoarthritis, Inflammatory Bowel Disease (IBD), psoriasis, derratitis, 
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 including testicular cancer; 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 dela 
Tourett's syndrome., can be diagnosed by methods comprising determining 
from a sample derived from a subject an abnormally decreased or increased 
level of hYAK3 polypeptide or hYAK3 mRNA. Decreased or increased 
expression can be measured at the RNA level using any of the methods well 
known in the art for the quantitation of polynucleotides, such as, for 
example, PCR, RT-PCR, RNase protection, Northern blotting and other 
hybridization methods. Assay techniques that can be used to determine 
levels of a protein, such as an hYAK3 polypeptide, in a sample derived 
from a host are well-known to those of skill in the art. Such assay 
methods include radioimmunoassays, competitive-binding assays, Western 
Blot analysis and ELISA assays. 
Chromosome Assays 
The nucleotide sequences of the present invention are also valuable for 
chromosome identification. The sequence is specifically targeted to and 
can hybridize with a particular location on an individual human 
chromosome. The mapping of relevant sequences to chromosomes according to 
the present invention is an important first step in correlating those 
sequences with gene associated disease. Once a sequence has been mapped to 
a precise chromosomal location, the physical position of the sequence on 
the chromosome can be correlated with genetic map data. Such data are 
found, for example, in V. McKusick, Mendelian Inheritance in Man 
(available on line through Johns Hopkins University Welch Medical 
Library). The relationship between genes and diseases that have been 
mapped to the same chromosomal region are then identified through linkage 
analysis (coinheritance of physically adjacent genes). 
The differences in the cDNA or genomic sequence between affected and 
unaffected individuals can also be determined. If a mutation is observed 
in some or all of the affected individuals but not in any normal 
individuals, then the mutation is likely to be the causative agent of the 
disease. 
Antibodies 
The polypeptides of the invention or their fragments or analogs thereof, or 
cells expressing them can also be used as immunogens to produce antibodies 
immunospecific for the hYAK3 polypeptides. The term "immunospecific" means 
that the antibodies have substantial greater affinity for the polypeptides 
of the invention than their affnity for other related polypeptides in the 
prior art. 
Antibodies generated against the hYAK3 polypeptides can be obtained by 
administering the polypeptides or epitopebea- g fragments, analogs or 
cells to an animal, preferably a nonhuman, using routine protocols. For 
preparation of monoclonal antibodies, any technique which provides 
antibodies produced by continuous cell line cultures can be used. Examples 
include the hybridorna technique (Kohler, G. and Milstein, C., Nature 
(1975) 256:495-497), the trioma technique, the human B-cell hybridorna 
technique (Kozbor et al., Immunology Today (1983) 4:72) and the 
EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER 
THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985). 
Techniques for the production of single chain antibodies (U.S. Pat. No. 
4,946,778) can also be adapted to produce single chain antibodies to 
polypeptides of this invention. Also, transgenic mice, or other organisms 
including other mamnals, may be used to express humanized antibodies. 
The above-described antibodies may be employed to isolate or to identify 
clones expressing the polypeptide or to purify the polypeptides by 
affinity chromatography. 
Antibodies against hYAK3 polypeptides may also be employed to treat bone 
loss including osteoporosis; inflannmatory 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 including testicular cancer; anorexia; 
bulnnia; 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 dela 
Tourett's syndrome., among others. 
Vaccines 
Another aspect of the invention relates to a method for inducing an 
immunological response in a mammal which comprises inoculating the mammal 
with hYAK3 polypeptide, or a fragment thereof, adequate to produce 
antibody and/or T cell immune response to protect said animal from bone 
loss including osteoporosis; inflammatory diseases such as Adult 
Respiratory Disease Syndrome (ARDS), Rheumatoid arthritis, Osteoarthritis, 
Inflammnatory 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 including testicular cancer; 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 dela 
Tourett's syndrome., among others. Yet another aspect of the invention 
relates to a method of inducing immunological response in a mammal which 
comprises, delivering hYAK3 polypeptide via a vector directing expression 
of hYAK3 polynucleotide in vivo in order to induce such an immunological 
response to produce antibody to protect said animal from diseases. 
Further aspect of the invention relates to an immunological/vaccine 
formulation (composition) which, when introduced into a mammalian host, 
induces an immunological response in that mammal to a hYAK3 polypeptide 
wherein the composition comprises a hYAK3 polypeptide or hYAK3 gene. The 
vaccine formulation may further comprise a suitable carrier. Since hYAK3 
polypeptide may be broken down in the stomach, it is preferably 
administered parenterally (including subcutaneous, intramuscular, 
intravenous, intradermal etc. injection). Formulations suitable for 
parenteral administration include aqueous and non-aqueous sterile 
injection solutions which may contain anti-oxidants, buffers, 
bacteriostats and solutes which render the formulation instonic with the 
blood of the recipient; and aqueous and non-aqueous sterile suspensions 
which may include suspending agents or thickening agents. The formulations 
may be presented in unit-dose or multi-dose containers, for example, 
sealed ampoules and vials and may be stored in a freeze-dried condition 
requiring only the addition of the sterile liquid carrier immediately 
prior to use. The vaccine formulation may also include adjuvant systems 
for enhancing the immunogenicity of the formulation, such as oil-in water 
systems and other systems known in the art. The dosage will depend on the 
specific activity of the vaccine and can be readily determined by routine 
experimentation. 
Screening Assays 
The hYAK3 polypeptide 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 hYAK3 
polypeptide of the present invention. Thus, polypeptides of the invention 
may also be used to assess identify agonist or antagonists from, for 
example, cells, cell-free preparations, chemical libraries, and natural 
product mixtures. These agonists or antagonists may be natural substrates, 
ligands, receptors, etc., as the case may be, of the polypeptide of the 
present invention; or may be structural or functional mnimetics of the 
polypeptide of the present invention. See Coligan et al., Current 
Protocols in Imnumology 1(2):Chapter 5 (1991). 
hYAK3 polypeptides are ubiquitous in the mammalian host and are responsible 
for rany biological functions, including many pathologies. Accordingly, it 
is desirous to find compounds and drugs which stimulate hYAK3 polypeptide 
on the one hand and which can inhibit the function of hYAK3 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 arthritis, Osteoarthritis, hfflammatory Bowel 
Disease (IBD), psoriasis, dernatitis, 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 including 
testicular cancer; 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 dela Tourett's syndrome. 
Antagonists may be employed for a variety of therapeutic and prophylactic 
purposes for such conditions as bone loss including osteoporosis; 
inflammatory diseases such as Adult Respiratory Disease Syndrome (ARDS), 
Rheumatoid arthritis, Osteoarthritis, fIflammatory Bowel Disease (IBD), 
psoriasis, dermatitis, asthma, allergies; infections such as bacterial, 
flngal, 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 including testicular 
cancer; anorexia; bulnima; 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 dela Tourett's syndrome. 
In general, such screening procedures ray involve using appropriate cells 
which express the hYAK3 polypeptide or respond to hYAK3 polypeptide of the 
present invention. Such cels include cells from mammals, yeast, Drosophila 
or E coli. Cells which express the hYAK3 polypeptide (or cell membrane 
containing the expressed polypeptide) or respond to hYAK3 polypeptide 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 hYAK3 activity. 
The knowledge that the hYAK3 encodes a protein kinase suggests that 
recombinant forms can be used to establish a protein kdnase activity. 
Typically this would involve the direct incubation of hYAK3 with a protein 
or peptide substrate in the presence of .gamma.-32P- ATP, followed by the 
measurement of radioactivity incorporated into the substrate by separation 
and counting. Separation methods include immunoprecipitation, conjugation 
of substrate to a bead allowing separation by centrifugation 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 hYAK3 itself 
(autophosphorylation), myelin basic protein, casein, histone and HSP27. 
Other substances might be discovered by incubating hYAK3 with random 
peptides conjugated to solid supports or displayed on the surface of phage 
or by incubation of hYAK3 with mammalian cell lysates and .gamma.-32P- 
ATP, followed by separation of the labelled target proteins, and 
sequencing. The protein kinase activity of hYAK3 may require incubation 
with a specific upstream effector. This may be achieved by preincubating 
HYAK3 with lysates from a variety of stimulated eukaryotic cells and ATP. 
These assays permit the discovery and modification of compounds which 
inhibit hYAK3 kinase activity in vitro and would be expected to have 
effects on proliferation of osteoblasts, chondorcytes, 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, 
osteoarthritis, cardiomyopathy and chachexia. 
This invention contemplates the treatment and/or amelioration of such 
diseases by administering an hYAK3 inhibiting amount of a compound. 
Without wishing to be bound by any particular theory of the functioning of 
the hYAK3 of this invention, it is believed that among the useful 
inhibitors of hYAK3 function are those compounds which inhibit the kinase 
activity of the hYAK3. Other sites of inhibition are, of course, possible 
owing to its position in a signal transduction cascade. Therefore, 
inhibiting the interaction of hYAK3 with one or more of its upstream or 
downstream modulators/substrates is also contemplated by this invention. 
Inhibitors of protein-protein interactions between hYAK3 and other factors 
could lead to the development of pharmaceutical agents for the modulation 
of hYAK3 activity. 
The assays may simply test binding of a candidate compound wherein 
adherence to the cells bearing the hYAK3 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 hYAK3 polypeptide, using detection systems 
appropriate to the cells bearing the hYAK3 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 hYAK3 polypeptide antagonists include antibodies or, 
in some cases, oligonucleotides or proteins which are closely related to 
the ligands, substrates, receptors, etc., as the case may be, of the hYAK3 
polypeptide, e.g., a fragment of the ligands, substrates, receptors, or 
small molecules which bind to the polypetide 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 conditions related 
to both an excess of and insufficient amounts of hYAK3 polypeptide 
activity. 
If the activity of hYAK3 polypeptide is in excess, several approaches are 
available. One approach comprises administering to a subject an inhibitor 
compound (antagonist) as hereinabove described along with a 
pharmaceutically acceptable carrier in an amount effective to inhibit 
activation by blocldng binding of ligands to the hYAK3 polypeptide, or by 
inhibiting a second signal, and thereby alleviating the abnormal 
condition. 
In another approach, soluble forms of hYAK3 polypeptides still capable of 
binding the ligand in competition with endogenous hYAK3 polypeptide may be 
administered. Typical embodiments of such competitors comprise fragments 
of the hYAK3 polypeptide. 
In still another approach, expression of the gene encoding endogenous hYAK3 
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, Fla. (1988). Alternatively, 
oligonucleotides 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 
oligomers can be administered per se or the relevant oligomers can be 
expressed in vivo. 
For treating abnormal conditions related to an under-expression of hYAK3 
and its activity, several approaches are also available. One approach 
comprises administering to a subject a therapeutically effective amount of 
a compound which activates hYAK3 polypeptide, i.e., an agonist as 
described above, in combination with a pharmaceutically acceptable 
carrier, to thereby alleviate the abnormal condition. Alternatively, gene 
therapy may be employed to effect the endogenous production of hYAK3 by 
the relevant cells in 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 packaging 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 engineering 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 therein) in Human Molecular Genetics, T Strachan and 
A P Read, BIOS Scientific Publishers Ltd (1996). 
Formulation and Administration 
Peptides, such as the soluble form of hYAK3 polypeptides, and agonist and 
antagonist peptides or small molecules, may be formulated in combination 
with a suitable pharmaceutical carrier. Such formulations comprise a 
therapeutically effective amount of the polypeptide or compound, and a 
pharmaceutically acceptable carrier or excipient. Such carriers include 
but are not limited to, saline, buffered saline, dextrose, water, 
glycerol, ethanol, and combinations thereof. Formulation should suit the 
mode of administration, and is well within the skill of the art. The 
invention further relates to pharmaceutical packs and kits comprising 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 in 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 intraperitoneal, 
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 enteric 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 .mu.g/kg of subject. Wide 
variations in the needed dosage, however, are to be expected in view of 
the variety of compounds available and the differing efficiencies of 
various routes of administration. For example, oral administration would 
be expected to require higher dosages than administration by intravenous 
injection. Variations in these dosage levels can be adjusted using 
standard empirical routines for optimization, as is well understood in the 
art. 
Polypeptides used in 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 carried out using standard techniques, which are 
well known and routine to those of skill in the art, except where 
otherwise described in detail. The examples illustrate, but do not limit 
the invention. 
Example 1 
A partial clone was initially identified through random searches of the 
Human Genome Sciences database. This partial clone (.about.1 kb) showed 
significant homology to YAK1 from S. cerevisiae. To obtain the fill length 
cDNA: Using the insert of the above partial clone as a probe, 1M plaques 
were screened from both a human testis and skeletal muscle cDNA library 
(Stratagene, LaJolla Calif.). Library screening procedure is described by 
(Elgin, et al. Stratagies 4: 8-9, 1991). The probes were .alpha.-32P 
labeled, using a Random Primed Labeling Kit (Boheringer Manheim, Germany, 
Cat. # 1585584) and purified by running over Sephadex G-50 columns 
(Pharmacia Biotech. Cat. # 17-0855-02). The hybirdization and washing 
conditions were according to J. Sambrook, E. F. Fritch and T. Maniatis 
(1989) A Laboratory Manaul Second. Ed. Vol. 1 pp. 2.69-2.81 Cold Spring 
Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Several positive 
clones were isolated from each library by plaque purification and 
fragments containing the inserts were excised and sequenced. The longest 
insert obtained from the skeletal muscle and testis libraries was 2.1 kb 
and 2.3 kb, respectively (SEQ ID NOS:1 and 3, respectively). The presence 
of an in-frame stop codon immediately 5' of an initiation codon indicated 
that both cDNA's were full length. Comparison of these two cDNAs indicates 
that the 3' most 1844 nucleotides are identical. The skeletal muscle cDNA 
(hYAK3.alpha.) is 266 nucleotides shorter than the testis cDNA 
(hYAK3.beta.); however, the skeletal muscle cDNA is 20 amino acids longer 
at the amino terminus than that obtained from the testis library. The 
presence of the splice site consensus sequence, AGG, suggests that the 
differences between these two cDNAs rnay be due to alternative splicing. 
Fasta analysis show both peptides to have high homology to a putative 
serine/threonine kinase of unknown function from C. elegans (F49E11.1). 
Example 2 
Northern analysis was carried out to determine the distribution of hYAK3 
mRNA in human tissues. The original partial clone was radiolabelled with 
[32P]-dATP using a randomly primed labelling kit. Membranes containing 
mRNA from multiple human tissues (Clontech #7760-1 and #7759-1)) were 
hybridized with the probe and washed under high stringency conditions as 
directed. Hybridized mRNA was visualized by exposing the membranes to 
X-ray film One major transcript at .about.2.3 kb was present in testis. 
The transcript was not visible in any other tissues; however, dot blot 
analysis using a Human Master blot (Clontech #7770-1) indicated that hYAK3 
is expressed in other tissues including skeletal muscle. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 4 
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- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2061 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- GGAGCGAAAT GCGCTGAGCT GCAGTGTCTG GTCGAGAGTA CCCGTGGGAG CG - #TCGCGCCG 
60 
- CGGAGGCAGC CGTCCCGGCG TAGGTGGCGT GGCCGACCGG ACCCCCAACT GG - #CGCCTCTC 
120 
- CCCGCGCGGG GTCCCGAGCT AGGAGATGGG AGGCACAGCT CGTGGGCCTG GG - #CGGAAGGA 
180 
- TGCGGGGCCG CCTGGGGCCG GGCTCCCGCC CCAGCAGCGG AGGTTGGGGG AT - #GGTGTCTA 
240 
- TGACACCTTC ATGATGATAG ATGAAACCAA ATGTCCCCCC TGTTCAAATG TA - #CTCTGCAA 
300 
- TCCTTCTGAA CCACCTCCAC CCAGAAGACT AAATATGACC ACTGAGCAGT TT - #ACAGGAGA 
360 
- TCATACTCAG CACTTTTTGG ATGGAGGTGA GATGAAGGTA GAACAGCTGT TT - #CAAGAATT 
420 
- TGGCAACAGA AAATCCAATA CTATTCAGTC AGATGGCATC AGTGACTCTG AA - #AAATGCTC 
480 
- TCCTACTGTT TCTCAGGGTA AAAGTTCAGA TTGCTTGAAT ACAGTAAAAT CC - #AACAGTTC 
540 
- ATCCAAGGCA CCCAAAGTGG TGCCTCTGAC TCCAGAACAA GCCCTGAAGC AA - #TATAAACA 
600 
- CCACCTCACT GCCTATGAGA AACTGGAAAT AATTAATTAT CCAGAAATTT AC - #TTTGTAGG 
660 
- TCCAAATGCC AAGAAAAGAC ATGGAGTTAT TGGTGGTCCC AATAATGGAG GG - #TATGATGA 
720 
- TGCAGATGGG GCCTATATTC ATGTACCTCG AGACCATCTA GCTTATCGAT AT - #GAGGTGCT 
780 
- GAAAATTATT GGCAAGGGGA GTTTTGGGCA GGTGGCCAGG GTCTATGATC AC - #AAACTTCG 
840 
- ACAGTACGTG GCCCTAAAAA TGGTGCGCAA TGAGAAGCGC TTTCATCGTC AA - #GCAGCTGA 
900 
- GGAGATCCGG ATTTTGGAGC ATCTTAAGAA ACAGGATAAA ACTGGTAGTA TG - #AACGTTAT 
960 
- CCACATGCTG GAAAGTTTCA CATTCCGGAA CCATGTTTGC ATGGCCTTTG AA - #TTGCTGAG 
1020 
- CATAGACCTT TATGAGCTGA TTAAAAAAAA TAAGTTTCAG GGTTTTAGCG TC - #CAGTTGGT 
1080 
- ACGCAAGTTT GCCCAGTCCA TCTTGCAATC TTTGGATGCC CTCCACAAAA AT - #AAGATTAT 
1140 
- TCACTGCGAT CTGAAGCCAG AAAACATTCT CCTGAAACAC CACGGGCGCA GT - #TCAACCAA 
1200 
- GGTCATTGAC TTTGGGTCCA GCTGTTTCGA GTACCAGAAG CTCTACACAT AT - #ATCCAGTC 
1260 
- TCGGTTCTAC AGAGCTCCAG AAATCATCTT AGGAAGCCGC TACAGCACAC CA - #ATTGACAT 
1320 
- ATGGAGTTTT GGCTGCATCC TTGCAGAACT TTTAACAGGA CAGCCTCTCT TC - #CCTGGAGA 
1380 
- GGATGAAGGA GACCAGTTGG CCTCCATGAT GGAGCTTCTA GGGATGCCAC CA - #CCAAAACT 
1440 
- TCTGGAGCAA TCCAAACGTG CCAAGTACTT TATTAATTCC AAGGGCATAC CC - #CGCTACTG 
1500 
- CTCTGTGACT ACCCAGGCAG ATGGGAGGGT TGTGCTTGTG GGGGGTCGCT CA - #CGTAGGGG 
1560 
- TAAAAAGCGG GGTCCCCCAG GCAGCAAAGA CTGGGGGACA GCACTGAAAG GG - #TGTGATGA 
1620 
- CTACTTGTTT ATAGAGTTCT TGAAAAGGTG TCTTCACTGG GACCCCTCTG CC - #CGCTTGAC 
1680 
- CCCAGCTCAA GCATTAAGAC ACCCTTGGAT TAGCAAGTCT GTCCCCAGAC CT - #CTCACCAC 
1740 
- CATAGACAAG GTGTCAGGGA AACGGGTAGT TAATCCTGCA AGTGCTTTCC AG - #GGATTGGG 
1800 
- TTCTAAGCTG CCTCCAGTTG TTGGAATAGC CAATAAGCTT AAAGCTAACT TA - #ATGTCAGA 
1860 
- AACCAATGGT AGTATACCCC TATGCAGTGT ATTGCCAAAA CTGATTAGCT AG - #TGGACAGA 
1920 
- GATATGCCCA GAGATGCATA TGTGTATATT TTTATGATCT TACAAACCTG CA - #AATGGAAA 
1980 
- AAATGCAAGC CCATTGGTGG ATGTTTTTGT TAGAGTAGAC TTTTTTTAAA CA - #AGACAAAA 
2040 
# 2061AA A 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 588 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Met Gly Gly Thr Ala Arg Gly Pro Gly Arg Ly - #s Asp Ala Gly Pro Pro 
# 15 
- Gly Ala Gly Leu Pro Pro Gln Gln Arg Arg Le - #u Gly Asp Gly Val Tyr 
# 30 
- Asp Thr Phe Met Met Ile Asp Glu Thr Lys Cy - #s Pro Pro Cys Ser Asn 
# 45 
- Val Leu Cys Asn Pro Ser Glu Pro Pro Pro Pr - #o Arg Arg Leu Asn Met 
# 60 
- Thr Thr Glu Gln Phe Thr Gly Asp His Thr Gl - #n His Phe Leu Asp Gly 
#80 
- Gly Glu Met Lys Val Glu Gln Leu Phe Gln Gl - #u Phe Gly Asn Arg Lys 
# 95 
- Ser Asn Thr Ile Gln Ser Asp Gly Ile Ser As - #p Ser Glu Lys Cys Ser 
# 110 
- Pro Thr Val Ser Gln Gly Lys Ser Ser Asp Cy - #s Leu Asn Thr Val Lys 
# 125 
- Ser Asn Ser Ser Ser Lys Ala Pro Lys Val Va - #l Pro Leu Thr Pro Glu 
# 140 
- Gln Ala Leu Lys Gln Tyr Lys His His Leu Th - #r Ala Tyr Glu Lys Leu 
145 1 - #50 1 - #55 1 - 
#60 
- Glu Ile Ile Asn Tyr Pro Glu Ile Tyr Phe Va - #l Gly Pro Asn Ala Lys 
# 175 
- Lys Arg His Gly Val Ile Gly Gly Pro Asn As - #n Gly Gly Tyr Asp Asp 
# 190 
- Ala Asp Gly Ala Tyr Ile His Val Pro Arg As - #p His Leu Ala Tyr Arg 
# 205 
- Tyr Glu Val Leu Lys Ile Ile Gly Lys Gly Se - #r Phe Gly Gln Val Ala 
# 220 
- Arg Val Tyr Asp His Lys Leu Arg Gln Tyr Va - #l Ala Leu Lys Met Val 
225 2 - #30 2 - #35 2 - 
#40 
- Arg Asn Glu Lys Arg Phe His Arg Gln Ala Al - #a Glu Glu Ile Arg Ile 
# 255 
- Leu Glu His Leu Lys Lys Gln Asp Lys Thr Gl - #y Ser Met Asn Val Ile 
# 270 
- His Met Leu Glu Ser Phe Thr Phe Arg Asn Hi - #s Val Cys Met Ala Phe 
# 285 
- Glu Leu Leu Ser Ile Asp Leu Tyr Glu Leu Il - #e Lys Lys Asn Lys Phe 
# 300 
- Gln Gly Phe Ser Val Gln Leu Val Arg Lys Ph - #e Ala Gln Ser Ile Leu 
305 3 - #10 3 - #15 3 - 
#20 
- Gln Ser Leu Asp Ala Leu His Lys Asn Lys Il - #e Ile His Cys Asp Leu 
# 335 
- Lys Pro Glu Asn Ile Leu Leu Lys His His Gl - #y Arg Ser Ser Thr Lys 
# 350 
- Val Ile Asp Phe Gly Ser Ser Cys Phe Glu Ty - #r Gln Lys Leu Tyr Thr 
# 365 
- Tyr Ile Gln Ser Arg Phe Tyr Arg Ala Pro Gl - #u Ile Ile Leu Gly Ser 
# 380 
- Arg Tyr Ser Thr Pro Ile Asp Ile Trp Ser Ph - #e Gly Cys Ile Leu Ala 
385 3 - #90 3 - #95 4 - 
#00 
- Glu Leu Leu Thr Gly Gln Pro Leu Phe Pro Gl - #y Glu Asp Glu Gly Asp 
# 415 
- Gln Leu Ala Ser Met Met Glu Leu Leu Gly Me - #t Pro Pro Pro Lys Leu 
# 430 
- Leu Glu Gln Ser Lys Arg Ala Lys Tyr Phe Il - #e Asn Ser Lys Gly Ile 
# 445 
- Pro Arg Tyr Cys Ser Val Thr Thr Gln Ala As - #p Gly Arg Val Val Leu 
# 460 
- Val Gly Gly Arg Ser Arg Arg Gly Lys Lys Ar - #g Gly Pro Pro Gly Ser 
465 4 - #70 4 - #75 4 - 
#80 
- Lys Asp Trp Gly Thr Ala Leu Lys Gly Cys As - #p Asp Tyr Leu Phe Ile 
# 495 
- Glu Phe Leu Lys Arg Cys Leu His Trp Asp Pr - #o Ser Ala Arg Leu Thr 
# 510 
- Pro Ala Gln Ala Leu Arg His Pro Trp Ile Se - #r Lys Ser Val Pro Arg 
# 525 
- Pro Leu Thr Thr Ile Asp Lys Val Ser Gly Ly - #s Arg Val Val Asn Pro 
# 540 
- Ala Ser Ala Phe Gln Gly Leu Gly Ser Lys Le - #u Pro Pro Val Val Gly 
545 5 - #50 5 - #55 5 - 
#60 
- Ile Ala Asn Lys Leu Lys Ala Asn Leu Met Se - #r Glu Thr Asn Gly Ser 
# 575 
- Ile Pro Leu Cys Ser Val Leu Pro Lys Leu Il - #e Ser 
# 585 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2327 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: Other 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- CGGCGCTGGC AAGCGAAGCT TGGGGGTGGG GAGGTAGAGT GAGCCCTCAG TA - #GGAGGGAC 
60 
- GAGGGCAGGG GTCTGACTGC CTCCCCGGGA CCGCCCCCAC CTCCTCTCTA TC - #AGGGCCCC 
120 
- CTCCCCCCAT CCCTGTCTCA CCGGGCGCGG GGGACGGGGC TAGAGCGGAG TT - #AGAGCAAG 
180 
- AAGAATTTCC ACCCCTGGAT TCCCTCTGAA ACCCTAGATC GGGGTATATG TT - #AAGGGATT 
240 
- ACGAAAATCT AGGACTTTTT GTGGGGCTTT TTATTAAAGG GGGGGAGCCC GG - #GAGCAATA 
300 
- CCTTGGAAAG AAGCCCTGTT GCTTAGAGCG GATAACCAAC GGCTGAACTC TT - #GGGGTTTG 
360 
- CTGTGAGGGG TGCGGTCTAG CTTCGAATGT ACAGTGGTGG AGCCACAGTG TT - #AAAGAACA 
420 
- GAGAAGTGAT CCTTAATCAT TTAGAATTTT GCCTCCACCA TCCACCAGAA AA - #TGAAGTGG 
480 
- AAAGAGAAGT TGGGGGATGG TGTCTATGAC ACCTTCATGA TGATAGATGA AA - #CCAAATGT 
540 
- CCCCCCTGTT CAAATGTACT CTGCAATCCT TCTGAACCAC CTCCACCCAG AA - #GACTAAAT 
600 
- ATGACCACTG AGCAGTTTAC AGGAGATCAT ACTCAGCACT TTTTGGATGG AG - #GTGAGATG 
660 
- AAGGTAGAAC AGCTGTTTCA AGAATTTGGC AACAGAAAAT CCAATACTAT TC - #AGTCAGAT 
720 
- GGCATCAGTG ACTCTGAAAA ATGCTCTCCT ACTGTTTCTC AGGGTAAAAG TT - #CAGATTGC 
780 
- TTGAATACAG TAAAATCCAA CAGTTCATCC AAGGCACCCA AAGTGGTGCC TC - #TGACTCCA 
840 
- GAACAAGCCC TGAAGCAATA TAAACACCAC CTCACTGCCT ATGAGAAACT GG - #AAATAATT 
900 
- AATTATCCAG AAATTTACTT TGTAGGTCCA AATGCCAAGA AAAGACATGG AG - #TTATTGGT 
960 
- GGTCCCAATA ATGGAGGGTA TGATGATGCA GATGGGGCCT ATATTCATGT AC - #CTCGAGAC 
1020 
- CATCTAGCTT ATCGATATGA GGTGCTGAAA ATTATTGGCA AGGGGAGTTT TG - #GGCAGGTG 
1080 
- GCCAGGGTCT ATGATCACAA ACTTCGACAG TACGTGGCCC TAAAAATGGT GC - #GCAATGAG 
1140 
- AAGCGCTTTC ATCGTCAAGC AGCTGAGGAG ATCCGGATTT TGGAGCATCT TA - #AGAAACAG 
1200 
- GATAAAACTG GTAGTATGAA CGTTATCCAC ATGCTGGAAA GTTTCACATT CC - #GGAACCAT 
1260 
- GTTTGCATGG CCTTTGAATT GCTGAGCATA GACCTTTATG AGCTGATTAA AA - #AAAATAAG 
1320 
- TTTCAGGGTT TTAGCGTCCA GTTGGTACGC AAGTTTGCCC AGTCCATCTT GC - #AATCTTTG 
1380 
- GATGCCCTCC ACAAAAATAA GATTATTCAC TGCGATCTGA AGCCAGAAAA CA - #TTCTCCTG 
1440 
- AAACACCACG GGCGCAGTTC AACCAAGGTC ATTGACTTTG GGTCCAGCTG TT - #TCGAGTAC 
1500 
- CAGAAGCTCT ACACATATAT CCAGTCTCGG TTCTACAGAG CTCCAGAAAT CA - #TCTTAGGA 
1560 
- AGCCGCTACA GCACACCAAT TGACATATGG AGTTTTGGCT GCATCCTTGC AG - #AACTTTTA 
1620 
- ACAGGACAGC CTCTCTTCCC TGGAGAGGAT GAAGGAGACC AGTTGGCCTC CA - #TGATGGAG 
1680 
- CTTCTAGGGA TGCCACCACC AAAACTTCTG GAGCAATCCA AACGTGCCAA GT - #ACTTTATT 
1740 
- AATTCCAAGG GCATACCCCG CTACTGCTCT GTGACTACCC AGGCAGATGG GA - #GGGTTGTG 
1800 
- CTTGTGGGGG GTCGCTCACG TAGGGGTAAA AAGCGGGGTC CCCCAGGCAG CA - #AAGACTGG 
1860 
- GGGACAGCAC TGAAAGGGTG TGATGACTAC TTGTTTATAG AGTTCTTGAA AA - #GGTGTCTT 
1920 
- CACTGGGACC CCTCTGCCCG CTTGACCCCA GCTCAAGCAT TAAGACACCC TT - #GGATTAGC 
1980 
- AAGTCTGTCC CCAGACCTCT CACCACCATA GACAAGGTGT CAGGGAAACG GG - #TAGTTAAT 
2040 
- CCTGCAAGTG CTTTCCAGGG ATTGGGTTCT AAGCTGCCTC CAGTTGTTGG AA - #TAGCCAAT 
2100 
- AAGCTTAAAG CTAACTTAAT GTCAGAAACC AATGGTAGTA TACCCCTATG CA - #GTGTATTG 
2160 
- CCAAAACTGA TTAGCTAGTG GACAGAGATA TGCCCAGAGA TGCATATGTG TA - #TATTTTTA 
2220 
- TGATCTTACA AACCTGCAAA TGGAAAAAAT GCAAGCCCAT TGGTGGATGT TT - #TTGTTAGA 
2280 
# 2327CAAG ACAAAACATT TTTATATGAT TATAAAA 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 568 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- Met Lys Trp Lys Glu Lys Leu Gly Asp Gly Va - #l Tyr Asp Thr Phe Met 
# 15 
- Met Ile Asp Glu Thr Lys Cys Pro Pro Cys Se - #r Asn Val Leu Cys Asn 
# 30 
- Pro Ser Glu Pro Pro Pro Pro Arg Arg Leu As - #n Met Thr Thr Glu Gln 
# 45 
- Phe Thr Gly Asp His Thr Gln His Phe Leu As - #p Gly Gly Glu Met Lys 
# 60 
- Val Glu Gln Leu Phe Gln Glu Phe Gly Asn Ar - #g Lys Ser Asn Thr Ile 
#80 
- Gln Ser Asp Gly Ile Ser Asp Ser Glu Lys Cy - #s Ser Pro Thr Val Ser 
# 95 
- Gln Gly Lys Ser Ser Asp Cys Leu Asn Thr Va - #l Lys Ser Asn Ser Ser 
# 110 
- Ser Lys Ala Pro Lys Val Val Pro Leu Thr Pr - #o Glu Gln Ala Leu Lys 
# 125 
- Gln Tyr Lys His His Leu Thr Ala Tyr Glu Ly - #s Leu Glu Ile Ile Asn 
# 140 
- Tyr Pro Glu Ile Tyr Phe Val Gly Pro Asn Al - #a Lys Lys Arg His Gly 
145 1 - #50 1 - #55 1 - 
#60 
- Val Ile Gly Gly Pro Asn Asn Gly Gly Tyr As - #p Asp Ala Asp Gly Ala 
# 175 
- Tyr Ile His Val Pro Arg Asp His Leu Ala Ty - #r Arg Tyr Glu Val Leu 
# 190 
- Lys Ile Ile Gly Lys Gly Ser Phe Gly Gln Va - #l Ala Arg Val Tyr Asp 
# 205 
- His Lys Leu Arg Gln Tyr Val Ala Leu Lys Me - #t Val Arg Asn Glu Lys 
# 220 
- Arg Phe His Arg Gln Ala Ala Glu Glu Ile Ar - #g Ile Leu Glu His Leu 
225 2 - #30 2 - #35 2 - 
#40 
- Lys Lys Gln Asp Lys Thr Gly Ser Met Asn Va - #l Ile His Met Leu Glu 
# 255 
- Ser Phe Thr Phe Arg Asn His Val Cys Met Al - #a Phe Glu Leu Leu Ser 
# 270 
- Ile Asp Leu Tyr Glu Leu Ile Lys Lys Asn Ly - #s Phe Gln Gly Phe Ser 
# 285 
- Val Gln Leu Val Arg Lys Phe Ala Gln Ser Il - #e Leu Gln Ser Leu Asp 
# 300 
- Ala Leu His Lys Asn Lys Ile Ile His Cys As - #p Leu Lys Pro Glu Asn 
305 3 - #10 3 - #15 3 - 
#20 
- Ile Leu Leu Lys His His Gly Arg Ser Ser Th - #r Lys Val Ile Asp Phe 
# 335 
- Gly Ser Ser Cys Phe Glu Tyr Gln Lys Leu Ty - #r Thr Tyr Ile Gln Ser 
# 350 
- Arg Phe Tyr Arg Ala Pro Glu Ile Ile Leu Gl - #y Ser Arg Tyr Ser Thr 
# 365 
- Pro Ile Asp Ile Trp Ser Phe Gly Cys Ile Le - #u Ala Glu Leu Leu Thr 
# 380 
- Gly Gln Pro Leu Phe Pro Gly Glu Asp Glu Gl - #y Asp Gln Leu Ala Ser 
385 3 - #90 3 - #95 4 - 
#00 
- Met Met Glu Leu Leu Gly Met Pro Pro Pro Ly - #s Leu Leu Glu Gln Ser 
# 415 
- Lys Arg Ala Lys Tyr Phe Ile Asn Ser Lys Gl - #y Ile Pro Arg Tyr Cys 
# 430 
- Ser Val Thr Thr Gln Ala Asp Gly Arg Val Va - #l Leu Val Gly Gly Arg 
# 445 
- Ser Arg Arg Gly Lys Lys Arg Gly Pro Pro Gl - #y Ser Lys Asp Trp Gly 
# 460 
- Thr Ala Leu Lys Gly Cys Asp Asp Tyr Leu Ph - #e Ile Glu Phe Leu Lys 
465 4 - #70 4 - #75 4 - 
#80 
- Arg Cys Leu His Trp Asp Pro Ser Ala Arg Le - #u Thr Pro Ala Gln Ala 
# 495 
- Leu Arg His Pro Trp Ile Ser Lys Ser Val Pr - #o Arg Pro Leu Thr Thr 
# 510 
- Ile Asp Lys Val Ser Gly Lys Arg Val Val As - #n Pro Ala Ser Ala Phe 
# 525 
- Gln Gly Leu Gly Ser Lys Leu Pro Pro Val Va - #l Gly Ile Ala Asn Lys 
# 540 
- Leu Lys Ala Asn Leu Met Ser Glu Thr Asn Gl - #y Ser Ile Pro Leu Cys 
545 5 - #50 5 - #55 5 - 
#60 
- Ser Val Leu Pro Lys Leu Ile Ser 
565 
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