CA2394803A1 - Novel human protein kinases and protein kinase-like enzymes - Google Patents

Abstract

The present invention relates to kinase polypeptides, nucleotide sequences encoding the kinase polypeptides, as well as various products and methods useful for the diagnosis and treatment of various kinase-related diseases and conditions. Through the use of a bioinformatics strategy, mammalian members of the PTK's and STK's have been identified and their protein structure predicted.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

~~ TTENANT LES PAGES 1 A 176 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:

PROTEIN KINASE-LIKE ENZYMES
The present invention claims priority on provisional application serial nos.
60/190,162;
60/174,185; 60/168,997; 60/179,364; 60/183,173; 60/178,078; 60/193,404;
60/195,953; and 1 S 60/187,150, all of which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates to kinase polypeptides, nucleotide sequences encoding the kinase polypeptides, as well as various products and methods useful for the diagnosis and 20 treatment of various kinase-related diseases and conditions.
BACKGROUND OF THE INVENTION
The following description of the background of the invention is provided to aid in 25 understanding the invention, but is not admitted to be or to describe prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure 30 and function.
Protein phosphorylation plays a pivotal role in cellular signal transduction.
Among the biological functions controlled by this type of postranslational modification are: cell division, differentiation and death (apoptosis); cell motility and cytoskeletal structure; control of DNA

replication, transcription, splicing and translation; protein translocation events from the endoplasmic reticulum and Golgi apparatus to the membrane and extracellular space; protein nuclear import and export; regulation of metabolic reactions, etc. Abnormal protein phosphorylation is widely recognized to be causally linked to the etiology of many diseases including cancer as well as immunologic, neuronal and metabolic disorders.
The following abbreviations are used for kinases throught this application:
ASK Apoptosis signal-regulating kinase CaMK Ca2+/calinodulin-dependent protein kinase CCRK Cell cycle-related kinase CDK Cyclin-dependent kinase CK Casein kinase DAPK Death-associated protein kinase DM myotonic dystrophy kinase Dyrk dual-specificity-tyrosine phosphorylating-regulated kinase GAK Cyclin G-associated kinase GRK G-protein coupled receptor GuC Guanylate cyclase HIPK Homeodomain-interacting protein kinase IRAK Interleukin-1 receptor-associated kinase MAPKMitogen activated protein kinase MAST Microtubule-associated STK
MLCKMyosin-light'chain kinase MLK Mixed lineage kinase NIMA NimA-related protein kinase PKA cAMP-dependent protein kinase RSK Ribosomal protein S6 kinase RTK Receptor tyrosine kinase SGK Serum and glucocorticoid-regulated kinase STK serine threonine kinase ULK UNC-51-like kinase The best-characterized protein kinases in eukaryotes phosphorylate proteins on the hydroxyl substituent of serine, threonine and tyrosine residues, which are the most common phospho-acceptor amino acid residues. However, phosphorylation on histidine has also been observed in bacteria.
The presence of a phosphate moiety modulates protein function in multiple ways. A
common mechanism includes changes in the catalytic properties (Vmax and Km) of an enzyme, leading to its activation or inactivation.
A second widely recognized mechanism involves promoting protein-protein interactions.
An example of this is the tyrosine autophosphorylation of the ligand-activated EGF receptor tyrosine kinase. This event triggers the high-affinity binding to the phosphotyrosine residue on the receptor's C-terminal intracellular domain to the SH2 motif of the adaptor molecule Grb2.
Grb2, in turn, binds through its SH3 motif to a second adaptor molecule, such as SHC. The formation of this ternary complex activates the signaling events that are responsible for the biological effects of EGF. Serine and threonine phosphorylation events also have been recently recognized to exert their biological function through protein-protein interaction events that are mediated by the high-affinity binding of phosphoserine and phosphothreonine to WW motifs present in a large variety of proteins (Lu, P.J. et al (1999) Science 283:1325-1328).
A third important outcome of protein phosphorylation is changes in the subcellular localization of the substrate. As an example, nuclear import and export events in a large diversity of proteins are regulated by protein phosphorylation (Drier E.A. et al (1999) Genes Dev 13: 556-568).
Protein kinases are one of the largest families of eukaryotic proteins with several hundred known members. These proteins share a 250-300 amino acid domain that can be subdivided into 12 distinct subdomains that comprise the common catalytic core structure.
These conserved protein motifs have recently been exploited using PCR-based and bioinformatic strategies leading to a significant expansion of the known kinases. Multiple alignment of the sequences in the catalytic domain of protein kinases and subsequent parsimony analysis permits their segregation into sub-families of related kinases.

Kinases largely fall into two groups: those specific for phosphorylating seriries and threonines, and those specific for phosphorylating tyrosines. Some kinases, referred to as "dual specificity" kinases, are able to phosphorylate on tyrosine as well as serine/threonine residues.
Protein kinases can also be characterized by their location within the cell.
Some kinases are transmembrane receptor-type proteins capable of directly altering their catalytic activity in response to the external environment such as the binding of a ligand. Others are non-receptor-type proteins lacking any transmembrane domain. They can be found in a variety of cellular compartments from the inner surface of the cell membrane to the nucleus.
Many kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state.
Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway. The conserved protein motifs of these kinases have recently been exploited using PCR-based cloning strategies leading to a significant expansion of the known kinases.
Multiple alignment of the sequences in the catalytic domain of protein kinases and subsequent parsimony analysis permits. the segregation of related kinases into distinct branches of subfamilies including: tyrosine kinases (PTK's), dual-specificity kinases, and serine/threonine kinases (STK's). The latter subfamily includes cyclic-nucleotide-dependent kinases, calcium/calmodulin kinases, cyclin-dependent kinases (CDK's), MAP-kinases, serine-threonine kinase receptors, and several other less defined subfamilies.
The protein kinases may be classified into several major groups including AGC, CAMK, Casein kinase 1, CMGC, STE, tyrosine kinases, and atypical kinases (Plowman, GD et al., Proceedings of the National Academy of Sciences, USA, Vol. 96, Issue 24, 13603-13610, November 23, 1999; see also www.kinase.com). In addition, there are a number of minor yet distinct families, including families related to worm- or fungal-specific kinases, and a family designated "other" to represent several smaller families. Within each group are several distinct families of more closely related kinases. In addition, an "atypical" family represents those protein kinases whose catalytic domain has little or no primary sequence homology to conventional kinases, including the A6 kinases and PI3 kinases.
AGC~roun The AGC kinases are basic amino acid-directed enzymes that phosphorylate residues found proximal to Arg and Lys. Examples of this group are the G protein-coupled receptor kinases (GRKs), the cyclic nucleotide-dependent kinases (PKA, PKC, PKG), NDR
or DBF2 kinases, ribosomal S6 kinases, AKT kinases, myotonic dystrophy kinases (DMPKs), MAPK
interacting kinases (MNKs), MAST kinases, and Mo3C11.1 ce family originally identified only in nematodes.
GRKs regulate signaling from heterotrimeric guanine protein coupled receptors (GPCRs).
Mutations in GPCRs cause a number of human diseases, including retinitis pigmentosa, stationary night blindness, color blindness , hyperfunctioning thyroid adenomas, familial precocious puberty , familial hypocalciuric hypercalcemia and neonatal severe hyperparathroidism (OMIM, htt~://www.ncbi.nhn.nih.~ov/Omim~. The regulation of GPCRs by GRKs indirectly implicates GRKs in these diseases.
The cAMP-dependent protein kinases (PKA) consist of heterotetramers comprised of 2 catalytic (C) and 2 regulatory (R) subunits, in which the R subunits bind to the second messenger cAMP, leading to dissociation of the active C subunits from the complex. Many of these kinases respond to second messengers such as cAMP resulting in a wide range of cellular responses to hormones and neurotransmitters.
AKT is a mammalian proto-oncoprotein regulated by phosphatidylinositol 3-kinase (PI3-K), which appears to function as a cell survival signal to protect cells from apoptosis. Insulin receptor, RAS, PI3-K, and PDKl all act as upstream activators of AKT, whereas the lipid phosphatase PTEN functions as a negative regulator of the PI3-K/AKT pathway.
Downstream targets for AKT-mediated cell survival include the pro-apoptotic factors BAD
and Caspase9, and transcription factors in the forkhead family, such as DAF-16 in the worm. AKT
is also an essential mediator in insulin signaling, in part due to its use of GSK-3 as another downstream taxget.
The S6 kinases regulate a wide array of cellular processes involved in mitogenic response including protein synthesis, translation of specific mRNA species, and cell cycle progression from G1 to S phase. The gene has been localized to chromosomal region 17q23 and is amplified in breast cancer (Couch, et al., Cancer Res. 1999 Apr 1;59(7):1408-11).
-S-CAMK Group The CAMK kinases are also basic amino acid-directed kinases. They include the Ca2+/calmodulin-regulated and AMP-dependent protein kinases (AMPK), myosin light chain kinases (MLCK), MAP kinase activating protein kinases (MAPKAPKs) checkpoint 2 kinases (CHK2), death-associated protein kinases (DAPKs), phosphorylase kinase (PHK), Rac and Rho-binding Trio kinases, a "unique" family of CAMKs, and the EMK-related protein kinases.
The EMK family of STKs are involved in the control of cell polarity, microtubule stability and cancer. One member of the EMK family, C-TAK1, has been reported to control entry into mitosis by activating Cdc25C which in turn dephosphorylates Cdc2.
Also included in the EMK family is MAKV, which has been shown to be overexpressed in metastatic tumors (Dokl. Akad. Nauk 354 (4), 554-556 (1997)).
CMGC Group The CMGC kinases are "proline-directed" enzymes phosphorylating residues that exist in a proline-rich context. They include the cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), GSK3s, RCKs, and CLKs. Most CMGC kinases have larger-than-average kinase domains owing to the presence of insertions within subdomains X
and XI.
CDK's play a pivotal role iri the regulation of mitosis during cell division.
The process of cell division occurs in four stages: S phase, the period during which chromosomes duplicate, G2, mitosis and G1 or interphase. During mitosis the duplicated chromosomes are evenly segregated allowing each daughter cell to receive a complete copy of the genome. A key mitotic regulator in all eukaryotic cells is the STK cdc2, a CDK regulated by cyclin B. However some CDK-like kinases, such as CDKS are not cyclin associated nor are they cell cycle regulated.
MAPKs play a pivotal role in many cellular signaling pathways, including stress response and mitogenesis (Lewis, T. S., Shapiro, P. S., and Ahn, N. G. (1998) Adv.
Cancer Res. 74, 49-139). MAP kinases can be activated by growth factors such as EGF, and cytokines such as TNF-alpha. In response to EGF, Ras becomes activated and recruits Rafl to the membrane where Rafl is activated by mechanisms that may involve phosphorylation and conformational changes (Mornson, D. K., and Cutler, R. E. (1997) Curr. Opin. Cell Biol. 9, 174-179).
Active Rafl phosphorylates MEK1 which in turn phosphorylates and activates the ERKs.
S Tyrosine Protein Kinase Group The tyrosine kinase group encompass both cytoplasmic (e.g. src) as well as transmembrane receptor tyrosine kinases (e.g. EGF receptor). These kinases play a pivotal role in the signal transduction processes that mediate cell proliferation, differentiation and apoptosis.
One of the sequences, 17000030181412, is related to the human RET kinase.
Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullaiy thyroid carcinomas. The kinase activity can be aberrantly activated by missense mutations affecting cysteine residues within the extracellular domain, leading to potent oncogenicity (Oncogene 1999 Aug 26;18(34):4833-8).
STE Group The STE family refers to the 3 classes of protein kinases that lie sequentially upstream of the MAPKs. This group includes STE7 (MEK or MAPKK) kinases, STE11 (MEKK or MAPKKK) kinases and STE20 (MEKKK) kinases. In humans, several protein kinase families that bear only distant homology with the STE11 family also operate at the level of MAPKKKs including RAF, MLK, TAK1, and COT. Since crosstalk takes place between protein kinases functioning at different levels of the MAPK cascade, the large number of STE
family kinases could translate into an enormous potential for upstream signal specificity.
The prototype STE20 from baker's yeast is regulated by a hormone receptor, signaling to directly affect cell cycle progression through modulation of CDK activity. It also coordinately regulates changes in the cytoskeleton and in transcriptional programs in a bifurcating pathway. In a similar way, the homologous kinases in humans are likely to play a role in extracellular regulation of growth, cell adhesion and migration, and changes in transcriptional programs, all _7_ three of which have critical roles in tumorigenesis. Mammalian S'TE20-related protein kinases have been implicated in response to growth factors or cytokines, oxidative-, UV-, or irradiation-related stress pathways, inflammatory signals (e.g. TNFa), apoptotic stimuli (e.g. Fas), T and B
cell costimulation, the control of cytoskeletal architecture, and cellular transformation.
Typically the STE20-related kinases serve as upstream regulators of MAPK
cascades. Examples include: HPK1, a protein-serine/threonine kinase (STK) that possesses a STE20-like kinase domain that activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK; PAKl, an STK with an upstream CDC42-binding domain that interacts with Rac and plays a role in cellular transformation through the Ras-MAPK pathway; and murine NIK, which interacts with upstream receptor tyrosine kinases and connects with downstream STE11-family kinases.
NEK kinases are related to NIMA, which is required for entry into mitosis in the filamentous fungus A. nidulans. Mutations in the nimA gene cause the nim (never in mitosis) G2 arrest phenotype in this fungus (Fry, A.M. and Nigg, E.A. (1995) Current Biology 5: 1122-1 S 1125). Several observations suggest that higher eukaryotes may have a NIMA
functional counterpart(s): (1) expression of a dominant-negative form of NIMA in HeLa cells causes a G2 arrest; (2)overexpression of NIMA causes chromatin condensation, not only in A. nidulans, but also in yeast, Xenopus oocytes and HeLa cells (Lu, K.P. and Hunter, T. (1995) Prog. Cell Cycle Res. 1, 187-205); (3) NIMA when expressed in mammalian cells interacts with pinl, a prolyl-prolyl isomerase that functions in cell cycle regulation (Lu, K.P. et al.
(1996) Nature 380, 544-547); (4) okadaic acid inhibitor studies suggests the presence of cdc2-independent mechanism to induce mitosis (Ghosh, S. et a1.(1998) Exp. Cell Res. 242, 1-9) and (5) a NIMA-like kinase (finl) exists in another eukaryote besides Aspergillus, Saccharomyces pombe (Krien, M.J.E. et a1.(1998) J. Cell Sci. 111, 967-976). Four mammalian NIMA-like kinases have been identified.
NEKl, NEK2, NEK3 and NRK2. Despite the similarity of the NIMA-related kinases to NIMA
over the catalytic region, the mammalian kinases are structurally different to NIMA over the extracatalytic regions. In addition the mammalian kinases are unable to complement the nim phenotype in Aspergillus nimA mutants. These observations lead to the following three possibilities: 1) the mammalian NIMA homologue remains unidentified; 2) there is no NIMA
homologue in higher eukaryotes; 3) the biological function of NIMA is carried out by multiple, _g_ related kinases in higher eukaryotes. The elucidation and biological characterization of additional mammalian NIMA- and NEK-related kinases should assist in elucidating this question.
Casein Kinase 1 Group The CKl family represents a distant branch of the protein kinase family. The hallmarks of protein kinase subdomains VIII and IX are difficult to identify. One or more forms are ubiquitously distributed in mammalian tissues and cell lines. CKl kinases are found in cytoplasm, in nuclei, membrane-bound, and associated with the cytoskeleton.
Splice variants differ in their subcellular distribution.
"Other" Group Several families cluster within a group of unrelated kinases termed "Other".
Included are: CHK1; Elongation 2 factor kinases (EIFK); homologues of the yeast sterile family kinases (STE), which refers to 3 classes of kinases which lie sequentially upstream of the MAPKs;
Calcium-calmodulin kinase kinases (CAMKK); dual-specific tyrosine kinases (DYRK); IkB
kinases (IKK); Integrin receptor kinase (IRAK); endoribonuclease-associated kinases (IRE);
Mixed lineage kinase (MLK); LIM-domain containing kinase (LIMK); MOS; PIM;
Receptor interacting kinase (RIP); SR-protein specific kinase (SRPK); RAF; Serine-threonine kinase receptors (STKR); TAK1; Testis specific kinase (TSK); tousled-related kinase (TSL); UNC51-related kinase (UNC); VRK; WEE; mitotic kinases (BUB1, AURORA, PLK, and NIMA/NEK);
several families that are close homologues to worm (C26C2.1, YQ09, ZC581.9, YFL033c, C24A1.3); Drosophila (SLOB), or yeast (YDOD sp, YGR262 sc) kinases; and others that are "unique," that is, those which do not cluster into any obvious family.
Additional families are even less well defined and first were identified in lower eukaryotes such as yeast or worms (YNL020, YPL236, YQ09, YWY3, SCY1, CO1H6.9, C26C2.1) RIP2 is a serine-threonine kinase associated with the tumor necrosis factor (TNF) receptor complex and is implicated in the activation of NF-kappa B and cell death in mammalian cells. It has recently been demonstrated that RIP2 activates the MAPK pathway (Navas, et al., J

Biol. Chem. 1999 Nov 19;274(47):33684-33690). RIP2 activates AP-1 and serum response element regulated expression by inducing the activation of the Elkl transcription factor. RIP2 directly phosphorylates and activates ERK2 in vivo and in vitro. RIP2 in turn is activated through its interaction with Ras-activated Rafl. These results highlight the integrated nature of kinase signaling pathway.
The tousled (TSL) kinase was first identified in the plant Arabidopsis thaliana. TSL
encodes a serine/threonine kinase that is essential for proper flower development. Human tousled-like kinases (Tlks) are cell-cycle-regulated enzymes, displaying maximal activities during S phase. This regulated activity suggests that Tlk function is linked to ongoing DNA
replication (Sillje, et al., EMBO J 1999 Oct 15;18(20):5691-5702).
Atypical Protein Kinase Group There are several proteins with protein kinase activity that appear structurally unrelated to the eukaryotic protein kinases. These include; Dictyostelium myosin heavy chain kinase A
(MHCKA), Physarum polycephalum actin-fragmin kinase, the human A6 PTK, human BCR, mitochondrial pyruvate dehydrogenase and branched chain fatty acid dehydrogenase kinase, and the prokaryotic "histidine" protein kinase family. The slime mold, worm, and human eEF-2 kinase homologues have all been demonstrated to have protein kinase activity, yet they bear little resemblance to conventional protein kinases except for the presence of a putative GxGxxG ATP-binding motif.
The so-called histidine kinases are abundant in prokaryotes, with more than 20 representatives in E. coli, and have also been identified in yeast, molds, and plants. In response to external stimuli, these kinases act as part of two-component systems to regulate DNA
replication, cell division, and differentiation through phosphorylation of an aspartate in the target protein. To date, no "histidine" kinases have been identified in metazoans, although mitochondrial pyruvate dehydrogenase (PDK) and branched chain alpha-ketoacid dehydrogenase kinase (BCKD kinase), are related in sequence. PDK and BCKD kinase represent a unique family of atypical protein kinases involved in regulation of glycolysis, the citric acid cycle, and protein synthesis during protein malnutrition. Structurally they conserve only the C-terminal portion of "histidine" kinases including the G box regions. BCKD kinase phosphorylates the Ela subunit of the BCKD complex on Ser-293, proving it to be a functional protein kinase. Although no bona fide "histidine" kinase has yet been identified in humans, they do contain PDK.
Several other proteins contain protein kinase-like homology including:
receptor guanylyl cyclases, diacylglycerol kinases, choline/ethanolamine kinases, and YLKl-related antibiotic resistance kinases. Each of these families contain short motifs that were recognized by our profile searches with low scoring E-values, but a priori would not be expected to function as . protein kinases. Instead, the similarity could simply reflect the modular nature of protein evolution and the primal role of ATP binding in diverse phosphotransfer enzymes. However, two recent papers on a bacterial homologue of the YLK1 family suggests that the aminoglycoside phosphotransferases (APHs) are structurally and functionally related to protein kinases. There are over 40 APHs identified from bacteria that are resistant to aminoglycosides such as kanamycin, gentamycin, or amikacin. The crystal structure of one well characterized APH
reveals that it shares greater than 40% structural identity with the 2 lobed structure of the catalytic domain of cAMP-dependent protein kinase (PKA), including an N-terminal lobe composed of a 5-stranded antiparallel beta sheet and the core of the C-terminal lobe including several invariant segments found in all protein kinases. APHs lack the GxGxxG
normally present in the loop between beta strands 1 and 2 but contain 7 of the 12 strictly conserved residues present in most protein kinases, including the HGDxxxN signature sequence in kinase subdomain VIB. Furthermore, APH also has been shown to exhibit protein-serine/threonine kinase activity, suggesting that other YLK-related molecules may indeed be functional protein kinases.
The eukaryotic lipid kinases (PI3Ks, PI4Ks, and PIPKs) also contain several short motifs similar to protein kinases, but otherwise share minimal primary sequence similarity. However, once again structural analysis of PIPKII-beta defines a conserved ATP-binding core that is strikingly similar to conventional protein kinases. Three residues are conserved among all of these enzymes including (relative to the PKA sequence) Lys-72 which binds the gamma-phosphate of ATP, Asp-166 which is part of the HRDLK motif and Asp-184 from the conserved Mgr or Mn~ binding DFG motif. The worm genome contains 12 phosphatidylinositol kinases, including 3 PI3-kinases, 2 PI4-kinases, 3 PIPS-kinases, and 4 PI3-kinase-related kinases. The latter group has 4 mammalian members (DNA-PK, FRAP/TOR, ATM, and ATR), which have been shown to participate in the maintenance of genomic integrity in response to DNA damage, and exhibit true protein kinase activity, raising the possibility that other PI-kinases may also 'act as protein kinases. Regardless of whether they have true protein kinase activity, PI3-kinases are tightly linked to protein kinase signaling, as evidenced by their involvement downstream of many growth factor receptors and as upstream activators of the cell survival response mediated by the AKT protein kinase.

SUMMARY OF THE INVENTION
The present invention relates, in part, to human protein kinases and protein kinase-like enzymes identified from genomic sequencing.
Tyrosine and serine/threonine kinases (PTK's and STK's) have been identified and their protein sequence predicted as part of the instant invention. Mammalian members of these families were identified through the use of a bioinformatics strategy. The partial or complete sequences of these kinases are presented here, together with their classification, predicted or deduced protein structure.
One aspect of the invention features an identified, isolated, enriched, or purified nucleic acid molecule encoding a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ LD N0:58, SEQ ID N0:59, SEQ ID
N0:60, SEQ ID
N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:
66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID
N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ
ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID
N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID
N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ
ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ
117 N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID
NO:11 l, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID N0:114.
The term "identified" in reference to a nucleic acid is meant that a sequence was selected from a genomic, EST, or cDNA sequence database based on it being predicted to encode a portion of a previously unknown or novel protein kinase.
By "isolated," in reference to nucleic acid, is meant a polymer of 10 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA
and RNA that is isolated from a natural source or that is synthesized as the sense or complementary antisense strand. In certain embodiments of the invention, longer nucleic acids are preferred, for example those of 300, 600, 900, 1200, 1500, or more nucleotides and/or those having at least 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% identity to a sequence selected from the group consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ
ID N0:3, SEQ
ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ
ID
NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ
ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID
N0:27,,SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ
ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID
N0:44, SEQ ID N0:45, SEQ 117 N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ
ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID
NO:55, SEQ ID N0:56, and SEQ ID N0:57.
The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term "isolated"
indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment.
Thus, the sequence may be in a cell-free solution or placed in a different cellular environment.
The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90 - 95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
By the use of the term "enriched" in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2- to 5-fold) of the total DNA
or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
The term "significant" is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2-fold, more preferably at least 5- to 10-fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The DNA from other sources may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUCl9. This term distinguishes from naturally occurring events, such as viral infection, or tumor-type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA.
That is, the term is meant to cover only those situations in which a person has intervened to S elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form.
The term "purified" in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2- to 5-fold greater, e.g., in terms of mg/mL). Individual clones isolated from a cDNA
library may be purified to electrophoretic homogeneity. The claimed DNA
molecules obtained from these clones could be obtained directly from total DNA or from total RNA.
The cDNA
clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurnng substance (messenger RNA). The construction of a cDNA
library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA
library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
By a "kinase polypeptide" is meant 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids in a polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID
N0:59, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ
ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID
N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID
N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ
ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID
N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID
N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ

ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID
NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID
NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ 117 N0:113, and SEQ ID NO:l 14. In certain aspects, polypeptides of 100, 200, 300, 400, 450, 500, 550, 600, 700, 800, 900 or more amino acids are preferred. The kinase polypeptide can be encoded by a full-length nucleic acid sequence or any portion (e.g., a "fragment" as defined herein) of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained, including, for example, a catalytic domain, as defined herein, or a portion thereof. One of skill in the art would be able to select those catalytic domains, or portions thereof, which exhibit a kinase or kinase-like activity, e.g., catalytic activity, as defined herein. It is well known in the art that due to the degeneracy of the genetic code numerous different nucleic acid sequences can code for the same amino acid sequence. Equally, it is also well known in the art that conservative changes in amino acid can be made to arrive at a protein or polypeptide which retains the functionality of the original. Such substitutions may include the replacement of an amino acid by a residue having similar physicochemical properties, such as substituting one aliphatic residue (Ile, Val, Leu or Ala) for another, or substitution between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Further information regarding making amino acid exchanges which have only slight, if any, effects on the overall protein can be found in Bowie et al., Science, 1990, 247, 1306-1310, which is incorporated herein by reference in its entirety including any figures, tables, or drawings. In all cases, all permutations are intended to be covered by this disclosure.
The amino acid sequence of a kinase peptide of the invention will be substantially similar to a sequence having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ
ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID
NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ ID N0:75, SEQ II7 N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ
ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID
N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ

ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:l 11, SEQ ID
N0:112, SEQ ID N0:113, and SEQ ID NO:l 14, or the corresponding full-length amino acid S sequence, or fragments thereof.
A sequence that is substantially similar to a sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
67, SEQ ID NO: 68, SEQ ID N0:69, SEQ 117 N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID
N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ
ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID
N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID
N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ
ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID
NO:101, 1 S SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ
ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:l 10, SEQ ID NO:111, SEQ ID
N0:112, SEQ ID N0:113, and SEQ ID N0:114, will preferably have at least 90%
identity (more preferably at least 95% and most preferably 99-100%) to the sequence.
By "identity" is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. "Gaps" are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Gapped BLAST or PSI-BLAST
(Altschul, et al.
(1997) Nucleic Acids Res. 25:3389-3402), BLAST (Altschul, et al. (1990) J.
Mol. Biol. 215:403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol. 147:195-197).
Preferably, the default settings of these programs will be employed, but those skilled in the art recognize whether these settings need to be changed and know how to make the changes.

"Similarity" is measured by dividing the number of identical residues plus the number of conservatively substituted residues (see Bowie, et al. Science, 1999), 247, 1306-1310, which is incorporated herein by reference in its entirety, including any drawings, figures, or tables) by the total number of residues and gaps and multiplying the product by 100.
In preferred embodiments, the invention features isolated, enriched, or purified nucleic acid molecules encoding a kinase polypeptide comprising a nucleotide sequence that: (a) encodes a polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ
ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID
NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ ID N0:75, SEQ 117 N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ
ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID
N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ
ID N0:97, SEQ ID N0:98, SEQ 117 N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:l 10, SEQ ID NO:111, SEQ ID
N0:112, SEQ ID N0:113, and SEQ ID N0:114; (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecule of (a) and encodes a naturally occurnng kinase polypeptide; (d) encodes a polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID
NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ
ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID
N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID
N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ
ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID
N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID
N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID
N0:103, SEQ ID N0:104, SEQ 117 N0:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ

ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ
ID
N0:114, except that it lacks one or more, but not all, of the domains selected from the group consisting of an N-terminal domain, a catalytic domain, a C-terminal catalytic domain, a C-terminal domain, a coiled-coil structure region, a proline-rich region, a spacer region, and a C-terminal tail; and (e) is the complement of the nucleotide sequence of (d).
The term "complement" refers to two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. A nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence.
Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. These conditions are well known to those skilled in the art.
Under stringent hybridization conditions only highly complementary nucleic acid sequences 1 S hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides, more preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 50 contiguous nucleotides, most preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 100 contiguous nucleotides. In some instances, the conditions may prevent hybridization of nucleic acids having more than 5 mismatches in the full-length sequence.
By stringent hybridization assay conditions is meant hybridization assay conditions at least as stringent as the following: hybridization in 50% formamide, SX SSC, 50 mM NaH2P04, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and SX Denhardt's solution at 42 °C overnight; washing with 2X SSC, 0.1% SDS at 45 °C; and washing with 0.2X SSC, 0.1%
SDS at 45 °C. Under some of the most stringent hybridization assay conditions, the second wash can be done with O.1X SSC at a temperature up to 70 °C (Berger et al.
(1987) Guide to Molecular Cloning TechnicLues pg 421, hereby incorporated by reference herein in its entirety including any figures, tables, or drawings.). However, other applications may require the use of conditions falling between these sets of conditions. Methods of determining the conditions required to achieve desired hybridizations are well known to those with ordinary skill in the art, and are based on several factors, including but not limited to, the sequences to be hybridized and the samples to be tested. Washing conditions of lower stringency frequently utilize a lower temperature during the washing steps, such as 65 °C, 60 °C, 55 °C, 50 °C, or 42 °C.
The term "domain" refers to a region of a polypeptide which serves a particular function.
For instance, N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal. Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
The term "N-terminal domain" refers to the extracatalytic region located between the initiator methionine and the catalytic domain of the protein kinase. The N-terminal domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the N-terminal boundary of the catalytic domain.
Depending on its length, the N-terminal domain may or may not play a regulatory role in kinase function. An example of a protein kinase whose N-terminal domain has been shown to play a regulatory role is PAK65, which contains a CRIB motif used for Cdc42 and rac binding (Burbelo, P.D. et al. (1995) J. Biol. Chem. 270, 29071-29074).
The term "catalytic domain" refers to a region of the protein kinase that is typically 25-300 amino acids long and is responsible for carrying out the phosphate transfer reaction from a high-energy phosphate donor molecule such as ATP or GTP to itself (autophosphorylation) or to other proteins (exogenous phosphorylation). The catalytic domain of protein kinases is made up of ~12 subdomains that contain highly conserved amino acid residues, and are responsible for proper polypeptide folding and for catalysis. The catalytic domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database.
The term "catalytic activity", as used herein, defines the rate at which a kinase catalytic domain phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a phosphorylated product as a function of time. Catalytic activity can be measured by methods of the invention by holding time constant and determining the concentration of a phosphorylated substrate after a fixed period of time.
Phosphorylation of a substrate occurs at the active site of a protein kinase.
The active site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
The term "substrate" as used herein refers to a molecule phosphorylated by a kinase of the invention. Kinases phosphorylate substrates on serine/threonine or tyrosine amino acids.
The molecule may be another protein or a polypeptide.
The term "C-terminal domain" refers to the region located between the catalytic domain or the last (located closest to the C-terminus) functional domain and the carboxy-terminal amino acid residue of the protein kinase. By "functional" domain is meant any region of the polypeptide that may play a regulatory or catalytic role as predicted from amino acid sequence homology to other proteins or by the presence of amino acid sequences that may give rise to specific structural conformations (e.g. N-terminal domain). The C-terminal domain can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C-terminal boundary of the catalytic domain or of any functional C-terminal extracatalytic domain. Depending on its length and amino acid composition, the C-terminal domain may or may not play a regulatory role in kinase function.
An example of a protein kinase whose C-terminal domain may play a regulatory role is PAK3 which contains a heterotrimeric Gb subunit-binding site near its C-terminus (Leeuw, T. et al.
(1998) Nature, 391, 191-195). For the some of the kinases of the instant invention, the C-terminal domain may also comprise the catalytic domain (above).
The term "C-terminal tail" as used herein, refers to a C-terminal domain of a protein kinase, that by homology extends or protrudes past the C-terminal amino acid of its closest homolog. C-terminal tails can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign.
Depending on its length, a C-terminal tail may or may not play a regulatory role in kinase function.
The term "coiled-coil structure region" as used herein, refers to a polypeptide sequence that has a high probability of adopting a coiled-coil structure as predicted by computer algorithms such as COILS (Lupas, A. (1996) Meth. Enzymology 266:513-525).
Coiled-coils are formed by two or three amphipathic a-helices in parallel. Coiled-coils can bind to coiled-coil domains of other polypeptides resulting in homo- or heterodimers (Lupas, A.
(1991) Science 252:1162-1164). Coiled-coil-dependent oligomerization has been shown to be necessary for protein function including catalytic activity of serine/threonine kinases (Roe, J. et al. (1997) J.
Biol. Chem. 272:5838-5845).
The term "proline-rich region" as used herein, refers to a region of a protein kinase whose proline content over a given amino acid length is higher than the average content of this amino acid found in proteins(i.e., >10%). Proline-rich regions are easily discernable by visual inspection of amino acid sequences and quantitated by standard computer sequence analysis programs such as the DNAStar program EditSeq. Proline-rich regions have been demonstrated to participate in regulatory protein -protein interactions: Among these interactions, those that are most relevant to this invention involve the "PxxP" proline rich motif found in certain protein kinases (i.e., human PAKI) and the SH3 domain of the adaptor molecule Nck (Galisteo, M.L. et al. (1996) J. Biol. Chem. 271:20997-21000). Other regulatory interactions involving "PxxP"
proline-rich motifs include the WW domain (Sudol, M. (1996) Prog. Biochys.
Mol. Bio. 65:113-132).
The term "spacer region" as used herein, refers to a region of the protein kinase located between predicted functional domains. The spacer region has no detectable homology to any amino acid sequence in the database, and can be identified by using a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the C- .
and N-terminal boundaries of the flanking functional domains. Spacer regions may or may not play a fundamental role in protein kinase function. Precedence for the regulatory role of spacer regions in kinase function is provided by the role of the src kinase spacer in inter-domain interactions (Xu, W. et al. (1997) Nature 385:595-602).
The term "insert" as used herein refers to a portion of a protein kinase that is absent from a close homolog. Inserts may or may not by the product alternative splicing of exons. Inserts can be identified by using a Smith-Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Inserts may play a functional role by presenting a new interface for protein-protein interactions, or by interfering with such interactions.
The term "signal transduction pathway" refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response. The polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, polypeptides containing SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine binding (PTB
and PH) domain containing proteins), proline-rich binding proteins (SH3 domain containing proteins), GTPases, phosphodiesterases, phospholipases, prolyl isomerases, proteases, Ca2+
binding proteins, cAMP binding proteins, guanyl cyclases, adenylyl cyclases, NO generating proteins, nucleotide exchange factors, and transcription factors.
In other preferred embodiments, the invention features isolated, enriched, or purified nucleic acid molecules encoding kinase polypeptides, further comprising a vector or promoter effective to initiate transcription in a host cell. The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence selected from the group consisting of those set forth in SEQ ID NO:1, SEQ ID
N0:2, SEQ ID
N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12; SEQ ID N0:13, SEQ ID N0:14, SEQ
ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID
N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ >D N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID
N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ
ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ 117 N0:36, SEQ ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID
N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ
ID N0:49, SEQ ID N0:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID
N0:54, SEQ ID N0:55, SEQ ID N0:56, and SEQ ID N0:57, or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a kinase polypeptide and a transcriptional termination region functional in a cell. Specific vectors and host cell combinations are discussed herein.
The term "vector" relates to a single or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome. A
circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art. A nucleic acid molecule encoding a kinase can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
The term "transfecting" defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies.
The term "promoter" as used herein, refers to nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to persons skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA
transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT
sequence, and the like.
In preferred embodiments, the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of those set forth in SEQ
ID NO:l, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ
ID
N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ
ID
N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ
ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID
N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID
N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ

ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID
N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID
N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ 117 N0:50, SEQ ID N0:51, SEQ ID N0:52, SEQ
ID N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ 117 N0:56, and SEQ ID N0:57, which encodes an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID NO:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID
NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ
ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID
N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID
N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ
117 N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID
N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID
N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID
N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ
ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ
ID
N0:114, a functional derivative thereof, or at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ 117 N0:63, SEQ ID
NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ
ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID
N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID
N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ
ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID
N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID
N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID
N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ
ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ
ID
N0:114. The nucleic acid may be isolated from a natural source by cDNA cloning or by subtractive hybridization. The natural source may be mammalian, preferably human, preferably blood, semen or tissue, and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer.
The term "mammal" refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes, and most preferably to S humans.
In yet other preferred embodiments, the nucleic acid is a conserved or unique region, for example those useful for: the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, obtaining antibodies to polypeptide regions, and designing antisense oligonucleotides.
By "conserved nucleic acid regions", are meant regions present on two or more nucleic acids encoding a kinase polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding kinase polypeptides are provided in Wahl et al. Meth.
Enzym. 152:399-407 (1987) and in Wahl et al. Meth. Enzym. 152:415-423 (1987), which are hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables.
Preferably, conserved regions differ by no more than 5 out of 20 nucleotides, even more preferably 2 out of 20 nucleotides or most preferably 1 out of 20 nucleotides.
By "unique nucleic acid region" is meant a sequence present in a nucleic acid coding for a kinase polypeptide that is not present in a sequence coding for any other naturally occurnng polypeptide. Such regions preferably encode 32 (preferably 40, more preferably 45, most preferably SS) or more contiguous amino acids, for example, an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID
NO:
66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID
N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ
ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID
N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID
N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ
ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ
ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID N0:112, SEQ 117 N0:113, and SEQ ID N0:114. In particular, a unique nucleic acid region is preferably of mammalian origin.
Another aspect of the invention features a nucleic acid probe for the detection of nucleic acid encoding a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ
ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ
ID
NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ
ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID
N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID
N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ
ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID
N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID
N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID N0:112, SEQ ID NO:I 13, and SEQ ID N0:114. in a sample. The nucleic acid probe contains a nucleotide base sequence that will hybridize to the sequence selected from the group consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ 117 N0:3, SEQ ID
N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ
ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID
N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ
ID N0:33, SEQ TD N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID
N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ
ID NO:SO, SEQ ID NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID
NO:55, SEQ ID N0:56, and SEQ ID N0:57, or a functional derivative thereof.

In preferred embodiments, the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 32, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ
ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ )D N0:19, SEQ ID
N0:20, SEQ ID N0:21, SEQ >D N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ
ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID
N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID
N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ
ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID
N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID
N0:54, SEQ ID N0:55, SEQ ID N0:56, and SEQ ID N0:57, or a functional derivative thereof.
Methods for using the probes include detecting the presence or amount of kinase RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to kinase RNA.
The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a kinase polypeptide may be used in the identification of the sequence of the nucleic acid detected (Nelson et al., in Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Kricka, ed., p. 275, 1992, hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
Methods for using the probes also include using these probes to find, for example, the full-length clone of each of the predicted kinases by techniques known to one skilled in the art.
These clones will be useful for screening for small molecule compounds that inhibit the catalytic activity of the encoded kinase with potential utility in treating cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders. More specifically disorders including cancers of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-1, HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms;
metabolic disorders including Diabetes and obesity and their related syndromes, among others;
cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retinopathy, and macular degeneration; inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplant rejection.
In another aspect, the invention describes a recombinant cell or tissue comprising a nucleic acid molecule encoding a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID
NO:
66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID
N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ 117 N0:76, SEQ ID N0:77, SEQ
ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID
N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID
N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ
ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID
NO:100, SEQ ID NO:141, SEQ 117 N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ
ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ 117 NO:110, SEQ ID
NO:l 11, SEQ ID NO:112, SEQ ID NO:l 13, and SEQ ID N0:114. In such cells, the nucleic acid may be under the control of the genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By "exogenous"
it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the kinase polypeptides.

The polypeptide is preferably a fragment of the protein encoded by an amino acid sequence selected from the group consisting of those set forth in SEQ ID
N0:58, SEQ ID
N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ
117 NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID
N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75., SEQ ID
N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ
ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID
N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID
N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ 117 N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ
ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID
N0:104, SEQ 117 NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID
N0:109, SEQ ID NO:110, SEQ ID NO:I 1 l, SEQ ID N0:112, SEQ ID N0:113, and SEQ
ID
N0:114. By "fragment," is meant an amino acid sequence present in a kinase polypeptide.
Preferably, such a sequence comprises at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of a sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ
ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ
ID
N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ
ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID
N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID
N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ
ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ 117 N0:97, SEQ ID
N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ
ID
N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID
N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID
N0:114.
In another aspect, the invention features an isolated, enriched, or purified kinase polypeptide having the amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ
ID
N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:
68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ 117 N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ
ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ 117 N0:90, SEQ 117 N0:91, SEQ 117 N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ
ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ 117 N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
N0:112, SEQ ID N0:113, and SEQ ID NO:l 14.
By "isolated" in reference to a polypeptide is meant a polymer of 6 (preferably 12, more preferably 18, most preferably 25, 32, 40, or 50) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. In certain aspects longer polypeptides are preferred, such as those comprising 100, 200, 300, 400, 450, 500, 550, 600, 700, 800, 900 or more contiguous amino acids, including an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID
N0:59, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ
ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID
N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID
N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ
ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID
N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID
N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ
ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID
NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID
NO:110, SEQ ID NO:111, SEQ 117 N0:112, SEQ ID NO:l 13, and SEQ ID N0:114.
The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term "isolated"
indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90 - 95% pure at least) of non-amino acid-based material naturally associated with it.
By the use of the term "enriched" in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2- to 5-fold) of the total amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two.
However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term "significantly" here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2-fold, more preferably at least 5- to 10-fold or even more. The term also does not imply that there is no amino acid sequence from other sources.
The other source of amino acid sequences may, for example, comprise amino acid sequence encoded by a yeast or bacterial genome, or a cloning vector such as pUCl9. The term is meant to cover only those situations in which man has intervened to increase the proportion of the desired amino acid sequence.
It is also advantageous for some purposes that an amino acid sequence be in purified form. The term "purified" in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment. Compared to the natural level this level should be at least 2-to 5-fold greater (e.g., in terms of mg/mL). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
In preferred embodiments, the kinase polypeptide is a fragment of the protein encoded by an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID
NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ

117 N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ m N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID
N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ m N0:85, SEQ ID.N0:86, SEQ
ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID
N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID
N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID
N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ
ID N0:109, SEQ ID NO:l 10, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID
N0:114. Preferably, the kinase polypeptide contains at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of a sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID
N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:
68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ m N0:73, SEQ ID
N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ
ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID
N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ )D N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ
ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
N0:112, SEQ ID N0:113, and SEQ ID N0:114, or a functional derivative thereof.
In preferred embodiments, the kinase polypeptide comprises an amino acid sequence having (a) an amino acid sequence selected from the group consisting of those set forth in SEQ
ID N0:58, SEQ 117 N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID
N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ
ID
N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ
ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID
N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID
N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ
ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID
N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ
ID
N0:103, SEQ ID N0:104, SEQ 117 N0:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID
N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID
NO:l 13, and SEQ ID N0:114; and (b) an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ
ID N0:61, SEQ ID N0:62, SEQ B7 N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ
ID
NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ
ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID
N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID
N0:84, SEQ 117 N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ
ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID
N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ 1D N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID
N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID N0:112, SEQ 117 N0:113, and SEQ ID N0:114, except that it lacks one or more of the domains selected from the group consisting of a C-terminal catalytic domain, an N-terminal domain, a catalytic domain, a C-terminal domain, a coiled-coil structure region, a proline-rich region, a spacer region, and a C-terminal tail.
The polypeptide can be isolated from a natural source by methods well-known in the art.
The natural source may be mammalian, preferably human, preferably blood, semen or tissue, and the polypeptide may be synthesized using an automated polypeptide synthesizer.
In some embodiments the invention includes a recombinant kinase polypeptide having (a) an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID
NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ
ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID
N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID
N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ
ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91; SEQ ID
N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID

N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID
N0:103, SEQ ID N0:104, SEQ 117 NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ
ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ
ID
N0:114. By "recombinant kinase polypeptide" is meant a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure.
Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
The polypeptides to be expressed in host cells may also be fusion proteins which include regions from heterologous proteins. Such regions may be included to allow, e.g., secretion, improved stability, or facilitated purification of the polypeptide. For example, a sequence encoding an appropriate signal peptide can be incorporated into expression vectors. A DNA
sequence for a signal peptide (secretory leader) may be fused in-frame to the polynucleotide sequence so that the polypeptide is translated as a fusion protein comprising the signal peptide.
A signal peptide that is functional in the intended host cell promotes extracellular secretion of the polypeptide. Preferably, the signal sequence will be cleaved from the polypeptide upon secretion of the polypeptide from the cell. Thus, preferred fusion proteins can be produced in which the N-terminus of a kinase polypeptide is fused to a carrier peptide.
In one embodiment, the polypeptide comprises a fusion protein which includes a heterologous region used to facilitate purification of the polypeptide. Many of the available peptides used for such a function allow selective binding of the fusion protein to a binding partner. A preferred binding partner includes one or more of the IgG binding domains of protein A are easily purified to homogeneity by affinity chromatography on, for example, IgG-coupled Sepharose. Alternatively, many vectors have the advantage of carrying a stretch of histidine residues that can be expressed at the N-terminal or C-terminal end of the target protein, and thus the protein of interest can be recovered by metal chelation chromatography. A
nucleotide sequence encoding a recognition site for a proteolytic enzyme such as enterokinase, factor X
procollagenase or thrombine may immediately precede the sequence for a kinase polypeptide to permit cleavage of the fusion protein to obtain the mature kinase polypeptide.
Additional examples of fusion-protein binding partners include, but are not limited to, the yeast I-factor, the honeybee melatin leader in sf~3 insect cells, 6-His tag, thioredoxin tag, hemaglutinin tag, GST
tag, and OmpA signal sequence tag. As will be understood by one of skill in the art, the binding partner which recognizes and binds to the peptide may be any ion, molecule or compound including metal ions (e.g., metal affinity columns), antibodies, or fragments thereof, and any protein or peptide which binds the peptide, such as the FLAG tag.
In another aspect, the invention features an antibody r(e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain or fragment where the polypeptide is selected from the group having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID
N0:59, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ
ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID
N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID
N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ
ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID
N0:88, 1 S SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ
ID
N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ
ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID
NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID
NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID N0:114. By "specific binding affinity" is meant that the antibody binds to the target kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions. Antibodies or antibody fragments are polypeptides that contain regions that can bind other polypeptides. The term "specific binding affinity" describes an antibody that binds to a kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions.. Antibodies can be used to identify an endogenous source of kinase polypeptides, to monitor cell cycle regulation, and for immuno-localization of kinase polypeptides within the cell.
The term "polyclonal" refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
"Monoclonal antibodies" are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art (Kohler et al., Nature 256:495-497, 1975, and U.S.
Patent No. 4,37 6,110, both of which are hereby incorporated by reference herein in their entirety including any figures, tables, or drawings).
The term "antibody fragment" refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
Antibodies or antibody fragments having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by probing the sample with the antibody under conditions suitable for kinase-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide. Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for the kinase as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
An antibody or antibody fragment with specific binding affinity to a kinase polypeptide of the invention can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism.
Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above.
Antibodies having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide.
Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
In another aspect, the invention features a hybridoma which produces an antibody having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain, where the polypeptide is selected from the group having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ
ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ
ID
NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ
ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID
N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID
N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ
ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID
N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID
N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID N0:114. By "hybridoma" is meant an immortalized cell line that is capable of secreting an antibody, for example an antibody to a kinase of the invention. In preferred embodiments, the antibody to the kinase comprises a sequence of amino acids that is able to specifically bind a kinase polypeptide of the invention.
In another aspect, the present invention is also directed to kits comprising antibodies that bind to a polypeptide encoded by any of the nucleic acid molecules described above, and a negative control antibody.
The term "negative control antibody" refers to an antibody derived from similar source as the antibody having specific binding affinity, but where it displays no binding affinity to a polypeptide of the invention.
In another aspect, the invention features a kinase polypeptide binding agent able to bind to a kinase polypeptide selected from the group having (a) an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID
N0:60, SEQ
ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:
66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID

N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ
ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ 117 N0:81, SEQ ID N0:82, SEQ ID
N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID
N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ
ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ
ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID N0:114. The binding agent is preferably a purified antibody that recognizes an epitope present on a kinase polypeptide of the invention. Other binding agents include molecules that bind to kinase polypeptides and analogous molecules that bind to a kinase polypeptide. Such binding agents may be identified by using assays that measure kinase binding partner activity, such as those that measure PDGFR
activity.
The invention also features a method for screening for human cells containing a kinase polypeptide of the invention or an equivalent sequence. The method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying the kinases of the invention (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
In another aspect, the invention features methods for identifying a substance that modulates kinase activity comprising the steps of: (a) contacting a kinase polypeptide selected from the group having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ
ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID
NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ
ID N0:80, SEQ ID N0:81, SEQ ID N0:82,.SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID
N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ
ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID

N0:107, SEQ 117 N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:l 12, SEQ ID N0:113, and SEQ ID N0:114 with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide. The skilled artisan will appreciate that the kinase polypeptides of the invention, including, for example, a portion of a full-length sequence such as a catalytic domain or a portion thereof, are useful for the identification of a substance which modulates kinase activity. Those kinase polypeptides having a functional activity (e.g., catalytic activity as defined herein) are useful for identifying a substance that modulates kinase activity.
The term "modulates" refers to the ability of a compound to alter the function of a kinase of the invention. A modulator preferably activates or inhibits the activity of a kinase of the invention depending on the concentration of the compound exposed to the kinase.
The term "modulates" also refers to altering the function of kinases of the invention by increasing or decreasing the probability that a complex forms between the kinase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the kinase and the natural binding partner depending on the concentration of the compound exposed to the kinase, and most preferably decreases the probability that a complex forms between the kinase and the natural binding partner.
The term "activates" refers to increasing the cellular activity of the kinase.
The term inhibit refers to decreasing the cellular activity of the kinase. Kinase activity is preferably the interaction with a natural binding partner.
The term "complex" refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another. For instance, a protein tyrosine receptor protein kinase, GRB2, SOS, R.AF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
The term "natural binding partner" refers to polypeptides, lipids, small molecules, or nucleic acids that bind to kinases in cells. A change in the interaction between a kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of kinase/natural binding partner complex.

The term "contacting" as used herein refers to mixing a solution comprising the test compound with a liquid medium bathing the cells of the methods. The solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the test compound or compounds into the cells of the methods. The solution comprising the test compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipette-based device or syringe-based device.
In another aspect, the invention features methods for identifying a substance that modulates kinase activity in a cell comprising the steps of (a) expressing a kinase polypeptide in a cell, wherein said polypeptide is selected from the group having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID
N0:59, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ
ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ D7 N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID
N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ
ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID
N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID
N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ
ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID
NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID
NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID N0:114; (b) adding a test substance to said cell; and (c) monitoring a change in cell phenotype or the interaction between said polypeptide and a natural binding partner. The skilled artisan will appreciate that the kinase polypeptides of the invention, including, for example, a portion of a full-length sequence such as a catalytic domain or a portion thereof, are useful for the identification of a substance which modulates kinase activity. Those kinase polypeptides having a functional activity (e.g., catalytic activity as defined herein) are useful for identifying a substance that modulates kinase activity.
The term "expressing" as used herein refers to the production of kinases of the invention from a nucleic acid vector containing kinase genes within a cell. The nucleic acid vector is transfected into cells using well known techniques in the art as described herein.

Another aspect of the instant invention is directed to methods of identifying compounds that bind to kinase polypeptides of the present invention, comprising contacting the kinase polypeptides with a compound, and determining whether the compound binds the kinase polypeptides. Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety. The compounds to be screened include, but are not limited to, compounds of extracellular, intracellular, biological or chemical, origin.
The methods of the invention also embrace compounds that are attached to a label, such as a radiolabel (e.g., l2sh 3sS~ 32P~ 33P~ 3H)~ a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label. The kinase polypeptides employed in such a test may 1 S either be free in solution, attached to a solid support, borne on a cell surface, located intracellularly or associated with a portion of a cell. One skilled in the art can, for example, measure the formation of complexes between a kinase polypeptide and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between a kinase polypeptide and its substrate caused by the compound being tested.
Other assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, Enzyme Assays: A Practical Approach, eds. R. Eisenthal and M. J.
Danson, 1992, Oxford University Press, which is incorporated herein by reference in its entirety.
Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of a kinase polypeptide comprising contacting the kinase polypeptide with a compound, and determining whether the compound modifies activity of the kinase polypeptide. As described herein, the kinase polypeptides of the invention include a portion of a full-length sequence, such as a catalytic domain, as defined herein. In some instances, the kinase polypeptides of the invention comprise less than the entire catalytic domain, yet exhibit kinase or kinase-like activity. These compounds are also referred to as "modulators of protein kinases." The activity in the presence of the test compound is measured to the activity in the absence of the test compound. Where the activity of a sample containing the test compound is higher than the activity in a sample lacking the test compound, the compound will have increased the activity. Similarly, where the activity of a sample S containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited the activity.
The present invention is particularly useful for screening compounds by using a kinase polypeptide in any of a variety of drug screening techniques. The compounds to be screened include, but are not limited to, extracellular, intracellular, biological or chemical origin. The kinase polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly. One skilled in the art can, for example, measure the formation of complexes between a kinase polypeptide and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between a kinase polypeptide and its substrate caused by the compound 1 S being tested.
The activity of kinase polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesised peptide ligands.
Alternatively, the activity of the kinase polypeptides can be assayed by examining their ability to bind metal ions such as calcium, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and photons. Thus, modulators of the kinase polypeptide's activity may alter a kinase function, such as a binding property of a kinase or an activity such as signal transduction or membrane localization.
In various embodiments of the method, the assay may take the form of a yeast growth assay, an Aequorin assay, a Luciferase assay, a mitogenesis assay, a MAP
Kinase activity assay, as well as other binding or function-based assays of kinase activity that are generally known in the art. In several of these embodiments, the invention includes any of the receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, polypeptides containing SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC
homology 2 (SH2) and phosphotyrosine binding (PTB and PH) domain containing proteins), proline-rich binding proteins (SH3 domain containing proteins), GTPases, phosphodiesterases, phospholipases, prolyl isomerases, proteases, Ca2+ binding proteins, cAMP
binding proteins, guanyl cyclases, adenylyl cyclases, NO generating proteins, nucleotide exchange factors, and transcription factors. Biological activities of kinases according to the invention include, but are not limited to, the binding of a natural or a synthetic ligand, as well as any one of the functional activities of kinases known in the art. Non-limiting examples of kinase activities include transmembrar<e signaling of various forms, which may involve kinase binding interactions and/or the exertion of an influence over signal transduction.
The modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into mimetics of natural kinase ligands, and peptide and non-peptide allosteric effectors of kinases. The invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library constiwction, and peptide libraries.
The use of cDNAs encoding kinases in drug discovery programs is well-known;
assays capable of testing thousands of unknown compounds per day in high-throughput screens (HTSs) are thoroughly documented. The literature is replete with examples of the use of radiolabelled ligands in HTS binding assays for drug discovery (see Williams, Medicinal Research Reviews, 1991, 11, 147-184.; Sweetnam, et al., J. Natural Products, 1993, 56, 441-455 for review).
Recombinant receptors are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson, BiolTechnology, 1992, 10, 973-980; each of which is incorporated herein by reference in its entirety).
A variety of heterologous systems is available for functional expression of recombinant receptors that are well known to those skilled in the art. Such systems include bacteria (Strosberg, et al., Trends in Pharmacological Sciences, 1992, 13, 95-98), yeast (Pausch, Trends in Biotechnology, 1997, 1 S, 487-494), several kinds of insect cells (Vanden Broeck, Int. Rev.
Cytology, 1996, 164, 189-268), amphibian cells (Jayawickreme et al., Current Opinion in Biotechnology, 1997, 8, 629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.;
see Gerhardt, et al., Eur. J. Pharmacology, 1997, 334, 1-23). These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).
An expressed kinase can be used for HTS binding assays in conjunction with its defined ligand, in this case the corresponding peptide that activates it. The identified peptide is labeled with a suitable radioisotope, including, but not limited to, lzsh 3H, 3sS or 3zP, by methods that are well known to those skilled in the art. Alternatively, the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur, et al., Drug Dev.
Res., 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160). Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med. Res.
Rev., 1991, 1l, 147-184.; Sweetnam, et al., J. Natural Products, 1993, 56, 441-455). Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama, Cur. Opinion Drug Disc. Dev., 1998, 1, 85-91 Bosse, et al., J. Biomolecular Screening, 1998, 3, 285-292.). Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur. Opinion Drug Disc. Dev., 1998, 1, 92-97).
The kinases and natural binding partners required for functional expression of heterologous kinase polypeptides can be native constituents of the host cell or can be introduced through well-known recombinant technology. The kinase polypeptides can be intact or chimeric.
The kinase activation results in the stimulation or inhibition of other native proteins, events that can be linked to a measurable response.
Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch, Trends in Biotechnology, 1997, 15, 487-494); changes in intracellular Caz+
concentration as measured by fluorescent dyes (Murphy, et al., Cur. Opinion Drug Disc. Dev., 1998, l, 192-199). Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS
has been described for these purposes (Schroeder, et al., J. Biomolecular Screening, 1996, l, 75-80).

Assays are also available for the measurement of common second but these are not generally preferred for HTS.
The invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to kinase polypeptides. In one example, the kinase polypeptide is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound. In another example, interaction between the kinase polypeptide and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound. In either assay, an inhibitor is identified as a compound that decreases binding between the kinase polypeptide and its natural binding partner.
Another contemplated assay involves a variation of the di-hybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published August 3, 1995 and is included by reference herein including any figures, tables, or drawings.
Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors. There are a number of different libraries used for the identification of small molecule modulators, including:
(1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as "hits" or "leads" in other drug discovery screens, while others are derived from natural products, and still others arise from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture.
These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol.
8:701-707 (1997).
Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit" (or "lead") to optimize the capacity of the "hit" to modulate activity.
Still other candidate inhibitors contemplated by the invention can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins. A "binding partner" as used herein broadly encompasses both natural binding partners as described above as well as chimeric polypeptides, peptide modulators other than natural ligands, antibodies, antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified kinase gene.
Other assays may be used to identify specific peptide ligands of a kinase polypeptide, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference. The two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs. The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA
binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast. The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein. The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene. The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene. For example, when the first protein is a kinase gene product, or fragment thereof, that is known to interact with another protein or nucleic acid, this assay can be used to detect agents that interfere with the binding interaction. Expression of the reporter gene is monitored as different test agents are added to the system.
The presence of an inhibitory agent results in lack of a reporter signal.
When the function of the kinase polypeptide gene product is unknown and no ligands are known to bind the gene product, the yeast two-hybrid assay can also be used to identify proteins that bind to the gene product. In an assay to identify proteins that bind to a kinase polypeptide, or fragment thereof, a fusion polynucleotide encoding both a kinase polypeptide (or fragment) and a UAS binding domain (i.e., a first protein) may be used. In addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay. Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein coding region being fused to the activation domain. This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein. The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
Other assays may be used to search for agents that bind to the target protein.
One such screening method to identify direct binding of test ligands to a target protein is described in U.S.
Patent No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states. When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state. Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand.
Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known. in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
Another method for identifying ligands of a target protein is described in Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporated herein by reference. This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein. Agents that bind to the target protein are separated from other library components by simple membrane washing. The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy. This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.
In preferred embodiments of the invention, methods of screening for compounds which modulate kinase activity comprise contacting test compounds with kinase polypeptides and assaying for the presence of a complex between the compound and the kinase polypeptide. In such assays, the ligand is typically labelled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to the kinase polypeptide.
In another embodiment of the invention, high throughput screening for compounds having suitable binding affinity to kinase polypeptides is employed. Briefly, large numbers of different small peptide test compounds are synthesised on a solid substrate.
The peptide test compounds are contacted with the kinase polypeptide and washed. Bound kinase polypeptide is then detected by methods well known in the art. Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with a kinase polypeptide. Radiolabeled competitive binding studies are described in A.H. Lin et al.
Antimicrobial Agents and Chemotherapy, 1997, vol. 41, no. 10. pp. 2127-2131, the disclosure of which is incorporated herein by reference in its entirety.
In another aspect, the invention provides methods for treating a disease by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide selected from the group consisting of those set forth in SEQ ID
N0:58, SEQ ID
N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ
ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID
N0:70, SEQ ID N0:71, SEQ 117 N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID
N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ
ID N0:82, SEQ ID N0:83; SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID
N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID
N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ
ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID
N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID NO:108, SEQ ID
N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID
N0:114, as well as the full-length polypeptide thereof, or a portion of any of these sequences that retains functional activity, as described herein. Preferably the disease is selected from the group consisting of cancers, immune-elated diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders. More specifically these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome;
neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-1, HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retinopathy, and macular degeneration; inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplant rejection.
In preferred embodiments, the invention provides methods for treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ >D N0:59, SEQ ID
N0:60, SEQ ID
N0:61, SEQ ID N0:62, SEQ 117 N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:
66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID
N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ
ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID
N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID
N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ
ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ m N0:98, SEQ ID N0:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ
ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ 117 NO:l 10, SEQ
ID
NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID N0:114, as well as the full-length polypeptide thereof, or a portion of any of these sequences that retains functional activity, as described herein. Preferably, the disease is selected from the group consisting of cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders. More specifically these diseases include cancer.of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia; manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome;
neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-1, HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retinopathy, and macular degeneration; inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplant rejection.
The invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ
ID
NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ
ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ 117 N0:77, SEQ ID N0:78, SEQ ID
N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ
ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID
N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:l 12, SEQ ID N0:113, and SEQ 117 NO:I 14, as well as the full-length polypeptide thereof, or a portion of any of these sequences that retains functional activity, as described herein.
Preferably the disease is selected from the group consisting of cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders. More specifically these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-1, HIV-2 or other viral- or prion-agents or fungal- or bacterial-organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others;
cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retinopathy, and macular degeneration; inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplant rej ection.
The invention also features methods of treating or preventing a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase polypeptide having an amino acid sequence selected from the group consisting those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ
ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID
NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ
ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ 117 N0:90, SEQ ID
N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ
ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:l 12, SEQ ID N0:113, and SEQ ID N0:114, as well as the full-length polypeptide thereof, or.
a portion of any of these sequences that retains functional activity, as described herein.
Preferably the disease is selected from the group consisting of immune-related diseases and disorders, cardiovascular disease, and cancer. More preferably these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome;
neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-l, HIV-2 or other viral- or prion-agents or fungal- or bacterial- organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others; cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retinopathy, and macular degeneration; inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplant rejection. Most preferably, the immune-related diseases and disorders are selected from the group consisting of rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplantation.
Substances useful for treatment of kinase-related disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (Examples of such assays are provided in the references in section VI, below; and in Example 7, herein). Examples of substances that can be screened for favorable activity are provided and referenced in section VI, below. The substances that modulate the activity of the kinases preferably include, but are not limited to, antisense oligonucleotides and inhibitors of protein kinases, as determined by methods and screens referenced in section VI and Example 7, below.
The term "preventing" refers to decreasing the probability that an organism contracts or develops an abnormal condition.
The term "treating" refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.

The term "therapeutic effect" refers to the inhibition or activation factors causing or contributing to the abnormal condition. A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following:
(a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells. Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
The term "abnormal condition" refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival.
Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
Abnormal differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
Abnormal cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated. A number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
The term "aberration", in conjunction with the function of a kinase in a signal transduction process, refers to a kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.
The term "administering" relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.
Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not S limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism. The effect of administering a compound on organism function can then be monitored. The organism is preferably a mouse, rat, rabbit, guinea pig, or goat, more preferably a monkey or ape, and most preferably a human.
In another aspect, the invention features methods for detection of a kinase polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID
N0:58, SEQ ID
N0:59, SEQ ID N0:60, SEQ )D N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ
ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID
N0:70, SEQ ID N0:71, SEQ 117 N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID
N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ
ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID
N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92; SEQ ID
N0:93, SEQ ID N0:94, SEQ II7 N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ
ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ )D N0:103, SEQ ID
N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID
N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ ID
N0:114, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probeaarget region hybrid as an indication of the disease.
In preferred embodiments of the invention, the disease or disorder is selected from the group consisting of rheumatoid arthritis, arteriosclerosis, autoimmune disorders, organ transplantation, myocardial infarction, cardiomyopathies, stroke, renal failure, oxidative stress-related neurodegerierative disorders, and cancer.
The kinase "target region" is the nucleotide base sequence selected from the group consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID
N0:4, SEQ
ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ
ID
NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ
ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID
N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID
N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ
ID N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID
N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ
ID N0:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID
N0:56, and SEQ ID N0:57, or the corresponding full-length sequences, a functional derivative thereof, or a fragment thereof, to which the nucleic acid probe will specifically hybridize. Specific hybridization indicates that in the presence of other nucleic acids the probe only hybridizes detectably with the kinase of the invention's target region. Putative target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database.
In preferred embodiments the nucleic acid probe hybridizes to a kinase target region encoding at least 6, 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of a sequence selected from the group consisting of those set forth in SEQ ID
N0:58, SEQ ID
N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ
ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:-68, SEQ ID N0:69, SEQ ID
N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID
N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ
ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID
N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID
N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ
ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID

N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID
N0:109, SEQ ID NO:l 10, SEQ ID NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ
ID
N0:114, or the corresponding full-length amino acid sequence, a portion of any of these sequences that retains functional activity, as described herein, or a functional derivative thereof.
Hybridization conditions should be such that hybridization occurs only with the kinase genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.
The diseases for which detection of kinase genes in a sample could be diagnostic include diseases in which kinase nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells. By "amplification" is meant increased numbers of kinase DNA or RNA in a cell compared with normal cells. In normal cells, kinases are typically found as single copy genes. In selected diseases, the chromosomal location of the kinase genes may be amplified, resulting in multiple copies of the gene, or amplification. Gene amplification can lead to amplification of kinase RNA, or kinase RNA can be amplified in the absence of kinase DNA
amplification.
"Amplification" as it refers to RNA can be the detectable presence of kinase RNA in cells, since in some normal cells there is no basal expression of kinase RNA.
In other normal cells, a basal level of expression of kinase exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, kinase RNA, compared to the basal level.
The diseases that could be diagnosed by detection of kinase nucleic acid in a sample preferably include cancers. The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.
The invention also features a method for detection of a kinase polypeptide in a sample as a diagnostic tool for a disease or disorder, wherein the method comprises: (a) comparing a nucleic acid target region encoding the kinase polypeptide in a sample, where the kinase polypeptide has an amino acid sequence selected from the group consisting those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID
N0:63, SEQ 117 NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO:
68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID
N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ
ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ 117 N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID
N0:91, SEQ II7 N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ
ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID N0:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
N0:112, SEQ ID N0:113, and SEQ ID N0:114, or one or more fragments thereof, with a control nucleic acid target region encoding the kinase polypeptide, or one or more fragments thereof; and (b) detecting differences in sequence or amount between the target region and the control target region, as an indication of the disease or disorder. Preferably the disease is selected from the group consisting of cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders. More specifically these diseases include cancer of tissues, blood, or hematopoietic origin, particularly those involving breast, colon, lung, prostate, cervical, brain, ovarian, bladder, or kidney; central or peripheral nervous system diseases and conditions including migraine, pain, sexual dysfunction, mood disorders, attention disorders, cognition disorders, hypotension, and hypertension; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Tourette's Syndrome; neurodegenerative diseases including Alzheimer's, Parkinson's, Multiple sclerosis, and Amyotrophic lateral sclerosis; viral or non-viral infections caused by HIV-1, HIV-2 or other viral- or prion-agents or fungal- or bacterial-organisms; metabolic disorders including Diabetes and obesity and their related syndromes, among others;
cardiovascular disorders including reperfusion restenosis, coronary thrombosis, clotting disorders, unregulated cell growth disorders, atherosclerosis; ocular disease including glaucoma, retinopathy, and macular degeneration; inflammatory disorders including rheumatoid arthritis, chronic inflammatory bowel disease, chronic inflammatory pelvic disease, multiple sclerosis, asthma, osteoarthritis, psoriasis, atherosclerosis, rhinitis, autoimmunity, and organ transplant red ection.
The term "comparing" as used herein refers to identifying discrepancies between the nucleic acid target region isolated from a sample, and the control nucleic acid target region. The discrepancies can be in the nucleotide sequences, e.g. insertions, deletions, or point mutations, or in the amount of a given nucleotide sequence. Methods to determine these discrepancies in sequences are well-known to one of ordinary skill in the art. The "control"
nucleic acid target region refers to the sequence or amount of the sequence found in normal cells, e.g. cells that are not diseased as discussed previously.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES
Figures lA-lEE shows the nucleotide sequences for human protein kinases oriented in a 5' to 3' direction (SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID
NO:S, S SEQ >D N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID N0:12, SEQ 117 N0:13, SEQ ID N0:14, SEQ ID NO:15, SEQ ID N0:16, SEQ ID
N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ
ID N0:23, SEQ B7 N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID
N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:34, SEQ ID N0:35, SEQ B7 N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ
ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID
NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ 117 NO:55, SEQ ID N0:56, and SEQ ID N0:57).
Figures 2A-2L show the amino acid sequences for the human protein kinases encoded by SEQ
ID No. 1-57 in the direction of translation (SEQ ID N0:58, SEQ ID N0:59, SEQ
ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID
NO:
66, SEQ 117 NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID
N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ
ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID
N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID
N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ
ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ
ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID N0:112, SEQ 117 N0:113, and SEQ ID N0:114). Some of the sequences encode predicted stop codons within the coding region, indicated by an 'x.' DETAILED DESCRIPTION OF THE INVENTION
The invention provides, inter alia, protein kinase and kinase-like genes, as well as fragments thereof, which have been identified in genomic databases. In part, the invention provides nucleic acid molecules that are capable of encoding polypeptides having a kinase or kinase-like activity. By reference to Tables 1 though 8, below, genes of the invention can be better understood. The invention additionally provides a number of different embodiments, such as those described below.
Nucleic Acids Associations of chromosomal localizations for mapped genes with amplicons implicated in cancer are based on literature searches (PubMed http://www.ncbi.nlm.nih.gov/entrez/query.fcgi), OMIM searches (Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/Omim/searchomim.html) and the comprehensive database of cancer amplicons maintained by Knuutila, et al. (Knuutila, et al., DNA copy number amplifications in human neoplasms. Review of comparative genomic hybridization studies. Am J
Pathol 152:1107-1123, 1998. http://www.helsinki.fi/~l~l www/CMG.html). For many of the mapped genes, the cytogenetic region from Knuutila is listed followed by the number of cases with documented amplification and the total number of cases studied. Thus for SGK187, the entry "non-small cell lung cancer (12q24.1-24.3; 2/5O)" means that the chromosomal position has been associated with non-small cell lung cancer, at position 12q24.1-24.3, which encompasses the SGK087's position, and the amplification has been noted in 2 of the SO samples studied.
For single nucleotide polymorphisms, an accession number (for example, ss1581624 for SGK187) is given if the SNP is documented in dbSNP (the database of single nucleotide polymorphisms) maintained at NCBI
(http://www.ncbi.nlm.nih.~ov/SNP/index.htmll. The accession number for SNP can be used to retrieve the full SNP-containing sequence from this site. Candidate SNPs without a dbSNP accession number were identified by inspection of Blastn outputs of the patent sequences vs cDNA and genomic databases as indicated, for example, in Tables 9 and 10, provided in Example 1.
Nucleic Acid Probes Methods. and Kits for Detection of Kinases The invention additionally provides nucleic acid probes and uses therefor. A
nucleic acid probe of the present invention may be used to probe an appropriate chromosomal or cDNA
library by usual hybridization methods to obtain other nucleic acid molecules of the present invention. A chromosomal DNA or cDNA library may be prepared from appropriate cells according to recognized methods in the art (cf. "Molecular Cloning: A
Laboratory Manual", second edition, Cold Spring Harbor Laboratory, Sambrook, Fritsch, & Maniatis, eds., 1989).
In the alternative, chemical synthesis can be carned out in order to obtain nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions of the amino acid sequence of the polypeptide of interest. The synthesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, "A Guide to Methods and Applications", Academic Press, Michael, et al., eds., 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment of the present invention.
One skilled in the art can readily design such probes based on the sequence disclosed herein using methods of computer alignment and sequence analysis known in the art ("Molecular Cloning: A Laboratory Manual", 1989, supra). The hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes may be visualized using known methods.
The nucleic acid probes of the present invention include RNA, as well as DNA
probes, such probes being generated using techniques known in the art. The nucleic acid probe may be immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.

The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid S extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
One method of detecting the presence of nucleic acids of the invention in a sample comprises (a) contacting said sample with the above-described nucleic acid probe under conditions such that hybridization occurs, and (b) detecting the presence of said probe bound to said nucleic acid molecule. One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue.
A kit for detecting the presence of nucleic acids of the invention in a sample comprises at least one container means having disposed therein the above-described nucleic acid probe. The kit may further comprise other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe. Examples of detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin). Preferably, the kit further comprises instructions for use.
In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like. One skilled in the art will readily recognize that the nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.

CATEGORIZATION OF THE POLYPEPTIDES ACCORDING TO THE INVENTION
For a number of protein kinases of the invention, there is provided a classification of the protein class and family to which it belongs, a summary of non-catalytic protein motifs, as well as a chromosomal location. This information is useful in determing function, regulation and/or therapeutic utility for each of the proteins. Amplification of chromosomal region can be associated with various cancers. For amplicons discussed in this application, the source of information was Knuutila, et al (Knuutila S, Bjorkqvist A-M, Autio K, Tarkkanen M, Wolf M, Monni O, Szymanska J, Larramendy ML, Tapper J, Pere H, El-Rifai W, Hemmer S, Wasenius V-M, Vidgren V & Zhu Y: DNA copy number amplifications in human neoplasms.
Review of comparative genomic hybridization studies. Am J Pathol 152:1107-1123, 1998.
http://www.helsinki.fi/~lgl www/CMG.html).
The kinase classification and protein domains often reflect pathways, cellular roles, or mechanisms of up- or down-stream regulation. Also disease-relevant genes often occur in families of related genes. For example, if one member of a kinase family functions as an oncogene, a tumor suppressor, or has been found to be disrupted in an immune, neurologic, cardiovascular, or metabolic disorder, frequently other family members may play a related role.
The expression analysis organizes kinases into groups that are transcriptionally upregulated in tumors and those that are more restricted to specific tumor types such as melanoma or prostate. This analysis also identifies genes that are regulated in a cell cycle dependent manner, and are therefore likely to be involved in maintaining cell cycle checkpoints, entry, progression, or exit from mitosis, oversee DNA repair, or are involved in cell proliferation and genome stability. Expression data also can identify genes expressed in endothelial sources or other tissues that suggest a role in angiogenesis, thereby implicating them as targets for control of diseases that have an angiogenic component, such as cancer, endometriosis, retinopathy and macular degeneration, and various ischemic or vascular pathologies. A
proteins' role in cell survival can also be suggested based on restricted expression in cells subjected to external stress such as oxidative damage, hypoxia, drugs such as cisplatinum, or irradiation.
Metastases-associated genes can be implicated when expression is restricted to invading regions of a tumor, or is only seen in local or distant metastases compared to the primary tumor, or when a gene is upregulated during cell culture models of invasion, migration, or motility.
Chromosomal location can identify candidate targets for a tumor amplicon or a tumor-suppressor locus. Summaries of prevalent tumor amplicons are available in the literature, and S can identify tumor types to experimentally be confirmed to contain amplified copies of a kinase gene which localizes to an adjacent region.
As described herein, the polypeptides of the present invention can be classified, for example, among ten different groups. The salient features related to the biological and clinical implications of these different groups are described hereafter in more general terms.
A more specific characterization of the polypeptides of the invention, including potential biological and clinical implications, is provided, e.g., in EXAMPLES 2a and 2b.
CLASSIFICATION OF POLYPEPTIDES EXHIBITING KINASE ACTIVITY
The following information also is referenced, for example, at Tables 1 and 2.
AGC Group Family members are described that belong to the AGC group of protein kinases.
The AGC group of protein kinases includes as its major prototypes protein kinase C
(PKC), cAMP-dependent protein kinases (PKA), the G protein-coupled receptor kinases (ARK
and rhodopsin kinase (GRK1)) as well as p70S6K and AKT.
Potential biological and clinical implications of the novel AGC group protein kinases are described in Example 2e. Novel AGC group kinases include: SEQ ID N0:58, SEQ ID
N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, and SEQ ID N0:64.
CAMK Group Family members are described that belong to the CAMK group of protein kinases.
The CAMK group of protein kinases includes as its major prototypes the calinodulin-dependent protein kinases, elongation factor-2 kinases, phosphorylase kinase and the Snfl and cAMP-dependent family of protein kinases.

Potential biological and clinical implications of the novel CAMK group of protein kinases are described in Example 2e. Novel CAMK group of protein kinases include: SEQ ID
N0:65, SEQ ID N0:66, SEQ ID N0:67, SEQ ID N0:68, SEQ ID N0:69, SEQ ID N0:75, SEQ
ID N0:76, SEQ ID N0:77, and SEQ ID N0:78.
Casein kinase group Family members are described that belong to the casein kinase (CKI) group of protein kinases. The Casein kinase (CK) group of protein kinases includes as its major prototypes casein kinaseI (CKI) and casein kinaselI (CKII). Both CKI and CKII are ubiquitous, constitutively-active, second-messenger-independent kinases, highly conserved enzymes that exist in multiple isoforms. CKI functions in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis (Cell Signal 1998 Nov;lO(10):699-711). CK2 functions in cell cycle progression in non-neural cells. CK2 has also been implicated in multiple signaling pathways in normal and disease states of the mammalian nervous systems (Prog Neurobiol 2000 Feb;60(3):211-46 ).
Potential biological and clinical implications of the novel casein kinase group of protein kinases are described in Example 2e. Novel casein kinase protein kinases include: SEQ ID NO:
79, and SEQ ID N0:80.
CMGC group Family members are described that belong to the cyclin-dependent kinase (CDK) group of protein kinases. The CMGC group of protein kinases includes as its major prototypes the cyclin-dependent protein kinases, as well as the MAPK kinases family, the GSK
family and the CLK family of kinases.
Potential biological and clinical implications of the novel CMGC group of protein kinases are described in Example 2e. Novel CMGC.protein kinases include: SEQ
ID N0:81, SEQ ID N0:82, and SEQ ID NO: 83.
Microbial PK group Family members are described that belong to the microbial group of protein kinases. This group is defined, for example, by the protein kinases that include ABC1, RI01, YG1R262, all of which have been initially identified from microbial genome sequencing projects (Proc Natl Acad Sci U S A 1999 Nov 23;96(24):13603-10).
Potential biological and clinical implications of the novel microbial group of protein kinases are described in Example 2e. Novel microbial protein kinases include SEQ ID N0:84, SEQ ID N0:85, and SEQ ID N0:86.
"Other" group Family members are described that belong to the "Other" group of protein kinases.
Within this group of protein kinases are members that have recognizable catalytic motifs that are identifiable by a hidden Markov model analysis, but fail to cluster with other protein kinases on the basis of their amino acid sequence homology over the catalytic region.
Potential biological and clinical implications of the novel protein kinases belonging to the Other group are described in Example 2e. Novel "Other" protein kinases include: SEQ ID NO:
87, SEQ ID NO: 88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ
ID
N0:93 , SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ
ID N0:99, SEQ ID NO:100, and SEQ ID NO:101.
The STE Group Family members are described that belong to the STE group of protein kinases.
The STE
group of protein kinases includes, as its major prototypes, the NEK kinases, as well as the STE11 and STE20 family of sterile protein kinases.
Potential biological and clinical implications of the novel protein kinases belonging to the STE group are described in Example 2e. Novel STE protein kinases include: SEQ
ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ
ID N0:108, and SEQ ID N0:109.
The TK group Family members are described that belong to the tyrosine kinase (TK) group of protein kinases. The TK group of protein kinases includes as its major prototypes the cytoplasmic and receptor families of protein kinases.

One family within this group of kinases is the "Eph" family. The Eph family, which is the largest sub-family of receptor tyrosine kinases in the human genome, has a stereotyped structure consisting of an N-terminal globular domain involved in ligand binding, two Type III
fibronectin-like domains which contribute to receptor dimerization, a transmembrane domain, and an intracellular tyrosine kinase domain. The Eph family is composed of two subfamilies: the EphA receptors which generally bind to members of the GPI-linked Ephrin A
family of ligands, and the EphB receptors which generally bind to the transmembrane Ephrin B
family of ligands.
Based on sequence similarity comparisons, EphA9 - to which the polypeptide represented by SEQ ID NO:110 belongs, is a member of the EphA subfamily of receptors.
Investigation of the Eph family of receptors indicate their involvement in a wide variety of cellular processes. Activation of Eph receptors can lead to changes in intracellular signaling, cell adhesion, cytoskeleton effects, and synaptic remodeling. These Eph-dependent cellular effects in turn contribute to changes in tissue functions such as border formation, pattern formation, cell migration, neurogenesis, angiogenesis, and long term potentiation, among others.
As a member of the Eph family, we expect that EphA9 will be involved in many of these functions as well.
Expression data for EphA9 indicate that it is expressed most prominently in the human central nervous system, the digestive system (especially in the colon and rectum) and the testes.
Therefore, EphA9 may be involved in organization and function of the digestive tract, including the colon, and could contribute colorectal tumorigenesis and other disorders of the digestive tract.
EphA9 found in the nervous system could be involved in synaptogenesis, neuronal development and regeneration, axon outgrowth, and synaptic transmission.
Therefore EphA9 might be important in neuronal survival and regeneration after injury, in long-term potentiation and memory formation, and in disorders of synaptic transmission such as epilepsy, depression, Parkinson's disease, and Alzheimer's disease.
Several Eph family receptors previously have been shown to be critical to several aspects of angiogenesis, such as remodeling, branching, sprouting and pruning of new blood vessels.
EphA9, as an additional member of this family, also may be critical for aspects of angiogenesis.

Thus, EphA9 may be relevant for a number diseases, including solid tumors, rheumatoid arthritis, and cardiovascular diseases.
Potential biological and clinical implications of the novel protein kinases belonging to the TK group are described in Example 2e. Novel TK protein kinases include: SEQ ID
NO:100, and SEQ ID NO:111.
CLASSIFICATION OF POLYPEPTIDES EXHIBITING KINASE-LIKE ACTIVITY
Two new family members are described that belong to the protein kinase (PK)-like insert "super family" of protein kinases. The PK-like superfamily of protein kinases includes the diacyl glycerol kinases (DGK) and the guanylate cyclases (GCyc), as decribed in the EXAMPLES.
Guanylate Cyclases (GCyc) Group Guanylate cyclases are ubiquitous enzymes that convert GTP to cGMP and exist as membrane-bound and soluble isoforms. A diverse range of agonist that include peptide hormones, bacterial toxins as well as intracellular molecules such as calcium and cAMP regulate the enzymatic activity of guanylate cyclases. Stimulation of guanylate kinases modulates multiple downstream enzymes including cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. The modulation of cGMP levels by guanylate cyclases contributes to the regulation of vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction (Pharmacol Rev 2000 Sep;52(3):375-414). As potential novel members of the guanylate cyclase family, disruptions in the signaling pathways in which SGK007 and SGKO50 participate may alter cGMP
homeostasis with pathophysiological implications.
Diacyl Glycerol kinase (DGK) Group A diacyl glycerol kinase phosphorylates the second messenger molecule diacyl glycerol leading to the formation of phosphatidic acid. Nine mammalain DGK isozymes have been described. The catalytic domain of a DGK usually is flanked by protein-protein interaction domains such as zinc fingers, pleckstrin homology domains and ankyrin repeats, as well as calcium-binding EF-hand structures. DGK's can be associated with the plasma membrane, nucleus and cytoskeleton. Experimental evidence supports the proposition that DGK's are translocated to and from these cellular compartments in response to agonists.
At these intracellular locations, DGK's are able to modulate lipid metabolism and PKC
activation, thereby triggering effector functions related to cell cycle progresion and differentiation (Int. .I. Biochem.
Cell Biol. 1997, (10):1139-43, J. Biol. Chem. 1999, 274(17):11447-50.) SGK093 - The Wnk family of serine/threonine kinases Wnk3 is a member of a subfamily of serine/threonine kinases which includes a described prototype, Wnkl, isolated from rat. This family is characterized by an N-terminal catalytic domain with several unique sequence features, most notably a change of the invariant lysine in kinase subdomain II to a cysteine, coupled with a change of the third conserved glycine residue 1 S in subdomain I into a lysine. The resulting enzyme appears to maintain catalytic activity due to this concomitant switch. Wnk3 conserves both of these catalytic changes and therefore is predicted to maintain catalytic activity. The long C-terminal portion of the wnks includes many protein interaction domains such as SH3 binding sites and coiled coil regions.
The wnk family catalytic domain shows the highest similarity to two families of serine/threonine kinases: The MEKK-like kinases and the Ste20-like kinases.
Both of these families can regulate enzymes in various MAPK signaling cascades, which are critical for many cellular processes such as mitogenesis, differentiation, cell survival, and stress response. The Ste20 kinases are also involved in regulation of the ras/rac/rho/cdc42 pathways and subsequent downstream effects on cytoskeleton.
Wnk3 shows high expression in human kidney, in kidney carcinoma cell lines, in prostate, prostate cell lines, and prostate tumor bone metastases, in colorectal tissue and tumor cell lines, and in human leukemia cells. Therefore wnk3 may be involved in the normal homeostasis and functioning of the human kidney, prostate, and digestive system, and may be involved in tumorigenesis which arises from these three tissues. High expression in human leukemia cell lines indicates a possible role in the development of that disease as well.

THERAPEUTIC METHODS ACCORDING TO THE INVENTION.
Diagnostics:
S
The invention provides methods for detecting a polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a polypeptide selected from the group consisting of SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID
NO:
64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ
ID N0:70, SEQ ID N0:71, SEQ 117 N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID
N0:75, SEQ ID N0:76, SEQ ID N0:77, SEQ >D N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID
N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ
ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID
N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID
N0:98, SEQ ID N0:99, SEQ 117 NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID
N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ
ID N0:109, SEQ ID NO:110, SEQ >D NO:111, SEQ ID N0:112, SEQ ID N0:113, and SEQ
ID
N0:114, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probeaarget region hybrid as an indication of the disease.
In preferred embodiments of the invention, the disease or disorder is selected from the group consisting of rheumatoid arthritis, atherosclerosis, autoimmune disorders, organ transplantation, myocardial infarction, cardiomyopathies, stroke, renal failure, oxidative stress related neurodegenerative disorders, metabolic disorder including diabetes, reproductive disorders including infertility, and cancer.
Hybridization conditions should be such that hybridization occurs only with the genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.

The diseases for which detection of genes in a sample could be diagnostic include diseases in which nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.
By "amplification" is meant increased numbers of DNA or RNA in a cell compared with normal cells.
"Amplification" as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA. In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, compared to the basal level.
The diseases that could be diagnosed by detection of nucleic acid in a sample preferably include cancers. The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to 1 S obtain a sample that is compatible with the method utilized.
Antibodies, Hybridomas, Methods of Use and Kits for Detection of Kinases The present invention relates to an antibody having binding affinity to a kinase of the invention.
The polypeptide may have the amino acid sequence selected from the group consisting of those set forth in SEQ ID N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61, SEQ ID
N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ
ID
NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ
ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ 117 N0:77, SEQ ID N0:78, SEQ ID
N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ ID N0:83, SEQ ID N0:84, SEQ ID
N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ
ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID N0:94, SEQ ID N0:95, SEQ ID
N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID NO:105, SEQ ID N0:106, SEQ ID
N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
N0:112, SEQ H~ N0:113, and SEQ ID N0:114, or a functional derivative thereof, or at least 9 contiguous amino acids thereof (preferably, at least 20, 30, 35, or 40 contiguous amino acids thereo f).
The present invention also relates to an antibody having specific binding affinity to a kinase of the invention. Such an antibody may be isolated by comparing its binding affinity to a kinase of the invention with its binding affinity to other polypeptides. Those which bind selectively to a kinase of the invention would be chosen for use in methods requiring a distinction between a kinase of the invention and other polypeptides. Such methods could include, but should not be limited to, the analysis of altered kinase expression in tissue containing other polypeptides.
The kinases of the present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction.
The kinases of the present invention can be used to produce antibodies or hybridomas.
One skilled in the art will recognize that if an antibody is desired, such a peptide could be generated as described herein and used as an immunogen. The antibodies of the present , invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting.
The present invention also relates to a hybridoma which produces the above-described monoclonal antibody, or binding fragment thereof. A hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam, The Netherlands, 1984; St. Groth et al., J. Immunol. Methods 35:1-21, 1980). Any animal (mouse, rabbit, and the like) which is known to produce antibodies can be immunized with the selected polypeptide. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on the animal which is immunized, the antigenicity of the polypeptide and the site of injection.

The polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity. Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or (3-galactosidase) or through the inclusion of an adjuvant during immunization.
For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such. as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Res. 175:109-124, 1988).
Hybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell, "Monoclonal Antibody Technology:
Laboratory Techniques in Biochemistry and Molecular Biology", supra, 1984).
For polyclonal antibodies, antibody-containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures. The above-described antibodies may be detectably labeled.
Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well-known in the art, for example, see Stemberger et al., J. Histochem. Cytochem. 18:315, 1970; Bayer et al., Meth. Enzym. 62:308, 1979; Engval et al., Immunol. 109:129, 1972; Goding, J.
Immunol. Meth.
13:21 S, 1976. The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues which express a specific peptide.
The above-described antibodies may also be immobilized on a solid support.
Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins such as polyacrylamide and latex beads.
Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10, 1986; Jacoby et al., Meth. Enzym. 34, Academic Press, N.Y., 1974).
The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
Furthermore, one skilled in the art can readily adapt currently available procedures, as well as the techniques, methods and kits disclosed herein with regard to antibodies, to generate peptides capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides (Hurby et al., "Application of Synthetic Peptides: Antisense Peptides", In Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307, 1992;
Kaspczak et al., Biochemistry 28:9230-9238, 1989).
Anti-peptide peptides can be generated by replacing the basic amino acid residues found in the peptide sequences of the kinases of the invention with acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.
The present invention also encompasses a method of detecting a kinase polypeptide in a sample, comprising: (a) contacting the sample with an above-described antibody, under conditions such that immunocomplexes form, and (b) detecting the presence of said antibody bound to the polypeptide. In detail, the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample. Altered levels of a kinase of the invention in a sample as compared to normal levels may indicate disease.
Conditions for incubating an antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay. One skilled in the art will recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion-based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of the present invention. Examples of such assays can be found in Chard ("An Introduction to Radioimmunoassay and Related Techniques"
Elsevier Science Publishers, Amsterdam, The Netherlands, 1986), Bullock et al.
("Techniques in Immunocytochemistry," Academic Press, Orlando, FL Vol. l, 1982; Vol. 2, 1983;
Vol. 3, 1985), Tijssen ("Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in _77_ Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam, The Netherlands, 1985).
The immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine. The test samples used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can readily be adapted in order to obtain a sample which is testable with the system utilized.
A kit contains all the necessary reagents to carry out the previously described methods of detection. The kit may comprise: (i) a first container means containing an above-described antibody, and (ii) second container means containing a conjugate comprising a binding partner of the antibody and a label. In another preferred embodiment, the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies.
Examples of detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. The compartmentalized kit may be as described above for nucleic acid probe kits.
One skilled in the art will readily recognize that the antibodies described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
Isolation of Compounds Capable of Interacting with Kinases The present invention also relates to a method of detecting a compound capable of binding to a kinase of the invention comprising incubating the compound with a kinase of the invention and detecting the presence of the compound bound to the kinase. The compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts.
The present invention also relates to a method of detecting an agonist or antagonist of kinase activity or kinase binding partner activity comprising incubating cells that produce a kinase of the invention in the presence of a compound and detecting changes in the level of kinase activity or kinase binding partner activity. The compounds thus identified would produce _78_ a change in activity indicative of the presence of the compound. The compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts.
Once the compound is identified it can be isolated using techniques well known in the art.
Modulating polypeptide activity:
The invention additionally provides methods for treating a disease or abnormal condition by administering to a patient in need of such treatment a substance that modulates the activity of a polypeptide selected from the group consisting of SEQ ID N0:58, SEQ ID
N0:59, SEQ ID
N0:60, SEQ ID N0:61, SEQ ID N0:62, SEQ ID N0:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ
ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID N0:69, SEQ ID N0:70, SEQ ID
N0:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74, SEQ ID N0:75, SEQ ID N0:76, SEQ ID
N0:77, SEQ ID N0:78, SEQ ID N0:79, SEQ ID N0:80, SEQ ID N0:81, SEQ ID N0:82, SEQ
ID N0:83, SEQ ID N0:84, SEQ ID N0:85, SEQ ID N0:86, SEQ ID N0:87, SEQ ID
N0:88, SEQ ID N0:89, SEQ ID N0:90, SEQ ID N0:91, SEQ ID N0:92, SEQ ID N0:93, SEQ ID
N0:94, SEQ ID N0:95, SEQ ID N0:96, SEQ ID N0:97, SEQ ID N0:98, SEQ ID N0:99, SEQ
ID NO:100, SEQ ID NO:101, SEQ ID N0:102, SEQ ID N0:103, SEQ ID N0:104, SEQ ID
NO:105, SEQ ID N0:106, SEQ ID N0:107, SEQ ID N0:108, SEQ ID N0:109, SEQ ID
NO:110, SEQ ID NO:111, SEQ ID NO:l 12, SEQ ID N0:113, and SEQ ID N0:114.
Preferably, the disease is selected from the group consisting of rheumatoid arthritis, atherosclerosis, autoimmune disorders, organ transplantation, myocardial infarction, cardiomyopathies, stroke, renal failure, oxidative stress-related neurodegenerative disorders, metabolic and reproductive disorders, and cancer.
Substances useful for treatment of disorders or diseases preferably show positive results in one or more assays for an activity corresponding to treatment of the disease or disorder in question Substances that modulate the activity of the polypeptides preferably include, but are not limited to, antisense oligonucleotides and inhibitors of protein kinases.
The term "preventing" refers to decreasing the probability that an organism contracts or develops an abnormal condition.

The term "treating" refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
The term "therapeutic effect" refers to the inhibition or activation factors causing or contributing to the abnormal condition. A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following:
(a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (, slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells. Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
The term "abnormal condition" refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation or cell survival. An abnormal condition may also include irregularities in cell cycle progression, i.e., irregularities in normal cell cycle progression through mitosis and meiosis.
Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
Abnormal differentiation conditions include, but are not limited to, neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated. A number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
The term "aberration", in conjunction with the function of a kinase in a signal.
transduction process, refers to a kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.
The term "administering" relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.
Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques.
The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism. The effect of administering a compound on organism function can then be monitored. The organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most 1 S preferably a human.
The present invention also encompasses a method of agonizing (stimulating) or antagonizing kinase associated activity in a mammal comprising administering to said mammal an agonist or antagonist to a kinase of the invention in an amount sufficient to effect said agonism or antagonism. A method of treating diseases in a mammal with an agonist or antagonist of the activity of one of the kinases of the invention comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize kinase-associated functions is also encompassed in the present application.
In an effort to discover novel treatments for diseases, biomedical researchers and chemists have designed, synthesized, and tested molecules that inhibit the function of protein kinases. Some small organic molecules form a class of compounds that modulate the function of protein kinases. Examples of molecules that have been reported to inhibit the function of protein kinases include, but are not limited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642, published November 26, 1992 by Maguire et al.), vinylene-azaindole derivatives (PCT WO 94/14808, published July 7, 1994 by Ballinari et al.), 1-cyclopropyl-4-pyridyl-quinolones (U.5. Patent No. 5,330,992), styryl compounds (I1.5. Patent No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Patent No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 Al), seleoindoles and selenides (PCT
WO 94/03427, published February 17, 1994 by Denny et al.), tricyclic polyhydroxylic compounds (PCT WO
92/21660, published December 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT
WO 91/15495, published October 17, 1991 by Dow et al).
Compounds that can traverse cell membranes and are resistant to acid hydrolysis are potentially advantageous as therapeutics as they can become highly bioavailable after being administered orally to patients. However, many of these protein kinase inhibitors only weakly inhibit the function of protein kinases. In addition, many inhibit a variety of protein kinases and will therefore cause multiple side-effects as therapeutics for diseases.
Some indolinone compounds, however, form classes of acid resistant and membrane permeable organic molecules. WO 96/22976 (published August 1, 1996 by Ballinari et al.) describes hydrosoluble indolinone compounds that harbor tetralin, naphthalene, quinoline, and indole substituents fused to the oxindole ring. These bicyclic substituents are in turn substituted with polar moieties including hydroxylated alkyl, phosphate, and ether moieties. U.S. Patent Application Serial Nos. 08/702,232, filed August 23, 1996, entitled "Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease" by Tang et al. (Lyon & Lyon Docket No. 221/187) and 08/485,323, filed June 7, 1995, entitled "Benzylidene-Z-Indoline Compounds for the Treatment of Disease" by Tang et al. (Lyon & Lyon Docket No.
223/298) and International Patent Publications WO 96/40116, published December 19, 1996 by Tang, et al., and WO 96/22976, published August 1, 1996 by Ballinari et al., all of which are incorporated herein by reference in their entirety, including any drawings, figures, or tables, describe indolinone chemical libraries of indolinone compounds harboring other bicyclic moieties as well as monocyclic moieties fused to the oxindole ring.
Applications 08/702,232, filed August 23, 1996, entitled "Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease" by Tang et al. (Lyon & Lyon Docket No.
221/187), 08/485,323, filed June 7, 1995, entitled "Benzylidene-Z-Indoline Compounds for the Treatment of Disease" by Tang et al. (Lyon & Lyon Docket No. 223/298), and WO 96/22976, published August 1, 1996 by Ballinari et al. teach methods of indolinone synthesis, methods of testing the biological activity of indolinone compounds in cells, and inhibition patterns of indolinone derivatives.
Other examples of substances capable of modulating kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines. The quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well known compounds such as those described in the literature. For example, representative publications describing quinazolines include Barker et al., EPO Publication No. 0 520 722 A1; Jones et al., U.S. Patent No. 4,447,608; Kabbe et al., U.S. Patent No. 4,757,072; Kaul and Vougioukas, U.S. Patent No.
5,316,553; Kreighbaum and Comer, U.S. Patent No. 4,343,940; Pegg and Wardleworth, EPO
Publication No. 0 562 734 Al; Barker et al., (1991) Proc. ofAm. Assoc. for Cancer Research 32:327; Bertino, J.R., (1979) Cancer Research 3:293-304; Bertino, J.R., (1979) Cancer Research 9(2 part 1):293-304; Curtin et al., (1986) Br. J. Cancer 53:361-368; Fernandes et al., (1983) Cancer Research 43:1117-1123 ; Ferns et al. J. Org. Chem. 44(2):173-178; Fry et al., (1994) Science 265:1093-1095; Jackman et al., (1981) Cancer Research 51:5579-5586;
Jones et al. J.
Med. Chem. 29(6):1114-1118; Lee and Skibo, (1987) Biochemistry 26(23):7355-7362; Lemus et al., (1989) .J. Org. Chem. 54:3511-3518; Ley and Seng, (1975) Synthesis 1975:415-522;
Maxwell et al., (1991) Magnetic Resonance in Medicine 17:189-196 ; Mini et al., (1985) Cancer Research 45:325-330; Phillips and Castle, J. (1980) Heterocyclic Chem.
17(19):1489-1596;
Reece et al., (1977) Cancer. Research 47(11):2996-2999; Sculier et al., (1986) Cancer Immunol.
and Immunother. 23, A65; Sikora et al., (1984) Cancer Letters 23:289-295;
Sikora et al., (1988) Analytical Biochem. 172:344-355; all of which are incorporated herein by reference in their entirety, including any drawings.
Quinoxaline is described in Kaul and Vougioukas, U.S. Patent No. 5,316,553, incorporated herein by reference in its entirety, including any drawings.
Quinolines are described in Dolle et al., (1994) J. Med. Chem. 37:2627-2629;
MaGuire, J. (1994) Med. Chem. 37:2129-2131; Burke et al., (1993) J. Med. Chem. 36:425-432 ; and Burke et al. (1992) BioOrganic Med. Chem. Letters 2:1771-1774, all of which are incorporated by reference in their entirety, including any drawings.
Tyrphostins are described in Allen et al., (1993) Clin. Exp. Immunol. 91:141-156; Anafi et al., (1993) Blood 82:12, 3524-3529; Baker et al., (1992) J. Cell Sci.
102:543-555; Bilder et al., (1991) Amer. Physiol. Soc. pp. 6363-6143:C721-C730; Brunton et al., (1992) Proceedings of Amer. Assoc. Cancer Rsch. 33:558; Bryckaert et al., (1992) Exp. Cell Research 199:255-261;
Dong et al., (1993) J. Leukocyte Biology 53:53-60; Dong et al., (1993) J.
Immunol. 151(5):2717-2724; Gazit et al., (1989) .l. Med. Chem. 32, 2344-2352; Gazit et al., (1993) .I. Med. Chem.
S 36:3556-3564; Kaur et al., (1994) Anti-Cancer Drugs 5:213-222; King et al., (1991) Biochem. J.
275:413-418; Kuo et al., (1993) Cancer Letters 74:197-202; Levitzki, A., (1992) The FASEB J.

6:3275-3282; Lyall et al., (1989) J. Biol. Chem. 264:14503-14509; Peterson et al., (1993) The Prostate 22:335-345; Pillemer et al., (1992) Int. .J. Cancer 50:80-85; Posner et al., (1993) Molecular Pharmacology 45:673-683; Rendu et al., (1992) Biol. Pharmacology 44(5):881-888;
Sauro and Thomas, (1993) Life Sciences 53:371-376; Sauro and Thomas, (1993) J.
Pharm. and Experimental Therapeutics 267(3):119-1125; Wolbring et al., (1994) J. Biol.
Chem.
269(36):22470-22472; and Yoneda et al., (1991) Cancer Research 51:4430-4435;
all of which are incorporated herein by reference in their entirety, including any drawings.
Other compounds that could be used as modulators include oxindolinones such as those described in U.S. patent application Serial No. 08/702,232 filed August 23, 1996, incorporated herein by reference in its entirety, including any drawings.
RECOMBINANT DNA TECHNOLOGY.
DNA Constructs Comprising a Kinase Nucleic Acid Molecule and Cells Containing These Constructs:
The present invention also relates to a recombinant DNA molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules. In addition, the present invention relates to a recombinant DNA
molecule comprising a vector and an above-described nucleic acid molecule. The present invention also relates to a nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in said cell.
The above-described molecules may be isolated and/or purified DNA molecules.

The present invention also relates to a cell or organism that contains an above-described nucleic acid molecule and thereby is capable of expressing a polypeptide. The polypeptide may be purified from cells which have been altered to express the polypeptide. A
cell is said to be "altered to express a desired polypeptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels. One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA
sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA
box, capping sequence, CART sequence, and the like.
If desired, the non-coding region 3' to the sequence encoding a kinase of the invention may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3'-region naturally contiguous to the DNA sequence encoding a kinase of the invention, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
Two DNA sequences (such as a promoter region sequence and a sequence encoding a kinase of the invention) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a gene sequence encoding a kinase of the invention, or (3) interfere with the ability of the gene sequence of a kinase of the invention to be transcribed by the promoter region sequence.
Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. Thus, to express a gene encoding a kinase of the invention, transcriptional and translational signals recognized by an appropriate host are necessary.
The present invention encompasses the expression of a gene encoding a kinase of the invention (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system for kinases of the invention.
Prokaryotes most frequently are represented by various strains of E. coli.
However, other microbial strains may also be used, including other bacterial strains.
In prokaryotic systems, plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
Examples of suitable plasmid vectors may include pBR322, pUC118, pUC119 and the like; suitable phage or bacteriophage vectors may include ~,gtl0, ~,gtl l and the like; and suitable virus vectors may include pMAM-neo, pKRC and the like. Preferably, the selected vector of the present invention has the capacity to replicate in the selected host cell.
Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
To express a kinase of the invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the kinase of the invention to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage ~,, the bla promoter of the (3-lactamase gene sequence of pBR322, and the cat promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage ~, (PL and PR), the trp, ~,recA, acZ, ~,acl, and gal promoters of E. coli, the a-amylase (LJlmanen et al., J. Bacteriol. 162:176-182, 1985) and the S-28-specific promoters of B.

subtilis (Gilman et al., Gene Sequence 32:11-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan, in: The Molecular Biology of the Bacilli, Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478, 1986).
Prokaryotic promoters are reviewed by Glick (Ind. Microbiot. 1:277-282, 1987), Cenatiempo (Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
Proper expression in a prokaryotic cell also requires the presence of a ribosome-binding site upstream of the gene sequence-encoding sequence. Such ribosome-binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981). The selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene. As used herein, "cell", "cell line", and "cell culture" may be used interchangeably and all such designations include progeny. Thus, the words "transformants" or "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell.
Host cells which rnay be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the kinase polypeptide of interest. Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-Kl, or cells of lymphoid origin and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR
332, which may provide better capacities for correct post-translational processing.
In addition, plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 195, and nopaline synthase promoter and polyadenylation signal sequences. Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459, 1988).
Alternatively, baculovirus vectors can be engineered to express large amounts of kinases of the invention in _87_ insect cells (Jasny, Science 238:1653, 1987; Miller et al., in: Genetic Engineering, Vol. 8, Plenum, Setlow et al., eds., pp. 2,77-297, 1986).
Any of a series of yeast expression systems can be utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes that are produced in large quantities when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. ' Yeast provides substantial advantages in that it can also carry out post-translational modifications. A
number of recombinant DNA strategies exist utilizing strong promoter sequences and high copy number plasmids which can be utilized for production of the desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian genes and secretes peptides bearing leader sequences (i.e., pre-peptides). Several possible vector systems are available for the expression of kinases of the invention in a mammalian host.
A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and the like, may be employed.
Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
Expression of kinases of the invention in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol.
Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982);
the SV40 early promoter (Benoist et al., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).
_88_ Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes a kinase of the invention (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the kinase of the invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the kinase of the invention coding sequence).
A nucleic acid molecule encoding a kinase of the invention and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome.
A vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
The selectable marker gene sequence can either be directly linked to the DNA
gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama (Mol. Cell.
Biol. 3:280-289, 1983).
The introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
Preferred prokaryotic vectors include plasmids such as those capable of replication in E.
coli (such as, for example, pBR322, ColEl, pSC101, pACYC 184, ~VX; "Molecular Cloning: A
Laboratory Manual", 1989, supra). Bacillus plasmids include pC194, pC221, pT127, and the like (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, NY, pp. 307-329, 1982). Suitable Streptomyces plasmids include p1J101 (Kendall et al., J.
Bacteriol. 169:4177-4183, 1987), and streptomyces bacteriophages such as ~C31 (Chater et al., In:
Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary, pp.
45-54, 1986). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis.
8:693-704, 1986), and Izaki (Jpn. J Bacteriol. 33:729-742, 1978).
Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982; Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon et al., J.
Clin. Hematol.
Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980). ' Once the vector or nucleic acid molecule containing the constructs) has been prepared for expression, the DNA constructs) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned genes) results in the production of a kinase of the invention, or fragments thereof. This can take place in the transformed cells as such, or following the induction of these~cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like). A
variety of incubation conditions can be used to form the peptide of the present invention. The most preferred conditions are those which mimic physiological conditions.

Transgenic Animals:
A variety of methods are available for the production of transgenic animals associated with this invention. DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al., Proc. Nat. Acad.
Sci. USA 82:4438-4442, 1985). Embryos can be infected with viruses, especially retroviruses, modified to carry inorganic-ion receptor nucleotide sequences of the invention.
Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention. A
transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilinington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc.
The procedures for manipulation of the rodent embryo and for microinjection of DNA
into the pronucleus of the zygote are well known to those of ordinary skill in the art (Hogan et al., supra). Microinjection procedures for fish, amphibian eggs and birds are detailed in Houdebine and Chourrout (Experientia 47:897-905, 1991). Other procedures for introduction of DNA into tissues of animals are described in U.S. Patent No. 4,945,050 (Sanford et al., July 30, 1990).
By way of example only, to prepare a transgenic mouse, female mice are induced to superovulate. Females are placed with males, and the mated females are sacrificed by C02 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts.
Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males.
Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice (Hammer et al., Cell 63:1099-1112, 1990).
Methods for the culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct inj ection also are well known to those of ordinary skill in the art (Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E.J. Robertson, ed., IRL Press, 1987).
In cases involving random gene integration, a clone containing the sequences) of the invention is co-transfected with a gene encoding resistance. Alternatively, the gene encoding neomycin resistance is physically linked to the sequences) of the invention.
Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E.J. Robertson, supra).
DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombine-tion (Capecchi, Science 244:1288-1292, 1989).
Methods for positive selection of the recombination event (i.e., neo resistance) and dual positive-negative selection (i.e., neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et al. (Nature 338:153-156, 1989), the teachings of which are incorporated herein in their entirety including any drawings. The final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females.
The resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene. Procedures for the production of non-rodent mammals and other animals have been discussed by others (Houdebine and Chourrout, supra;
Pursel et al., Science 244:1281-1288, 1989; and Simms et al., BiolTechnology 6:179-183, 1988).
Thus, the invention provides transgenic, nonhuman mammals containing a transgene encoding a kinase of the invention or a gene affecting the expression of the kinase. Such transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introduction of a kinase, or regulating the expression of a kinase (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes).
A "transgenic animal" is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA.becomes part of the genome of the animal which develops from that cell. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. The transgenic DNA may encode human kinases.
Native expression in an animal may be reduced by providing an amount of antisense RNA
or DNA
effective to reduce expression of the receptor.
Gene Therapy:
Kinases or their genetic sequences will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460, 1992). Miller states that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results. The basic science of gene therapy is described in Mulligan (Science 260:926-931, 1993).
In one preferred embodiment, an expression vector containing a kinase coding sequence is inserted into cells, the cells are grown in vitro and then infused in large numbers into patients.
In another preferred embodi-ment, a DNA segment containing a promoter of choice (for example a strong promoter) is transferred into cells containing an endogenous gene encoding kinases of the invention in such a manner that the promoter segment enhances expression of the endogenous kinase gene (for example, the promoter segment is transferred to the cell such that it becomes directly linked to the endogenous kinase gene).
The gene therapy may involve the use of an adenovirus containing kinase cDNA
targeted to a tumor, systemic kinase increase by implantation of engineered cells, injection with kinase-encoding virus, or injection of naked kinase DNA into appropriate tissues.
Target cell populations may be modified by introducing altered forms of one or more components of the protein complexes in order to modulate the activity of such complexes. For example, by reducing or inhibiting a complex component activity within target cells, an abnormal signal transduction events) leading to a condition may be decreased, inhibited, or reversed. Deletion or missense mutants of a component, that retain the ability to interact with other components of the protein complexes but cannot function in signal transduction, may be used to inhibit an abnormal, deleterious signal transduction event.
Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associ-ated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences (e.g., cDNA) erlcod-ing recom-binant kinase of the invention protein into the targeted cell population (e.g., tumor cells).
Methods which are well .
known to those skilled in the art can be used to construct recombinant viral vectors contain-ing coding sequences (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989; Ausubel et al., Current Proto-cots in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., 1989). Alter-natively, recombinant nucleic acid mole-cules encoding protein sequences can be used as naked DNA or in a recon-stituted system e.g., lipo-somes or other lipid systems for delivery to target cells (e.g., Felgner et al., Nature 337:387-8, 1989). Several other methods for the direct transfer of plasmid DNA into cells exist for use in human gene therapy and involve targeting the DNA to receptors on cells by complexing the plasmid DNA to proteins (Miller, supra).
In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection (Capecchi, Cell 22:479-88, 1980). Once recombinant genes are introduced into a cell, they can be recognized by the cell's normal mechanisms for transcription and translation, and a gene product will be expressed.
Other methods have also been attempted for introducing DNA into larger numbers of cells.
These methods include: transfection, wherein DNA is precipitated with calcium phosphate and taken into cells by pinocytosis (Chen et al., Mol. Cell Biol. 7:2745-52, 1987); electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane (Chu et al., Nucleic Acids Res. 15:1311-26, 1987); lipofection/liposome fusion, wherein DNA is packaged into lipophilic vesicles which fuse with a target cell (Felgner et al., Proc. Natl. Acad.
Sci. USA. 84:7413-7417, 1987); and particle bombardment using DNA bound to small projectiles (Yang et al., Proc. Natl. Acad. Sci. 87:9568-9572, 1990). Another method for introducing DNA
into cells is to couple the DNA to chemically modified proteins.
It has also been shown that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. The admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene (Curiel et al., Am. J. Respir. Cell. Mol. Biol., 6:247-52, 1992).
As used herein "gene transfer" means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expres-sion of a particular product encoded by the gene. The product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cyto-plasm from the plasma membrane or endosome. Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription.
As used herein "gene therapy" is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient.
In another preferred embodiment, a vector having nucleic acid sequences encoding a kinase polypeptide is provided in which the nucleic acid sequence is expressed only in specific tissue. Methods of achieving tissue-specific gene expression are set forth in International Publication No. WO 93/09236, filed November 3, 1992 and published May 13, 1993.
In all of the preceding vectors set forth above, a further aspect of the invention is that the nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above.
In another preferred embodiment, a method of gene replacement is set forth.
"Gene replacement" as used herein means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION
The compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions where it is mixed with other active ingredients, as in combination therapy, or suitable carriers or excipient(s). Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

Routes Of Administration:
Suitable routes of administration may, for example, include oral, rectal, transmucosal, or S intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.
1 S Composition%Formulation:
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable Garners comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Far oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurnes, suspensions and the like, for oral ingestion by a patient to be treated. , Suitable carriers include excipients such as, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, andlor lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g.
gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in mufti-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD
is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:DSV~ consists of VPD diluted 1:1 with a 5% dextrose in water solution.
This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the tyrosine or serine/threonine kinase modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions.
Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

Suitable Dosage Regimens:
Pharmaceutical compositions suitable for use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
Methods of determining the dosages of compounds to be administered to a patient and modes of administering compounds to an organism are disclosed in U.S.
Application Serial No.
08/702,282, filed August 23, 1996 and International patent publication number WO 96/22976, published August 1 1996, both of which are incorporated herein by reference in their entirety, including any drawings, figures or tables. Those skilled in the art will appreciate that such 1 S descriptions are applicable to the present invention and can be easily adapted to it.
The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient.
Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the ICSO as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the ICso as determined in cell culture (i.e., the concentration of the test compound which achieves a half maximal inhibition of the tyrosine or serine/threonine kinase activity). Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LDSO and EDso.
Compounds which exhibit high therapeutic indices are preferred. The data obtained from these S cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDSO with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).
In another example, toxicity studies can be carned out by measuring the blood cell composition. For example, toxicity studies can be carried out in a suitable animal model as follows: 1) the compound is administered to mice (an untreated control mouse should also be used); 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymorphonuclear cells. A
comparison of results for each dosing regime with the controls indicates if toxicity is present.
At the termination of each toxicity study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Euthanasia:229-249, 1993). Representative animals 'from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness or toxicity.
Gross abnormalities in tissue are noted and tissues are examined histologically. Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs. In general, the greater the adverse effect the less preferred the compound.
For the treatment of cancers the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness.

Plasma levels should reflect the potency of the drug. Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.
Plasma half life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder. Such measurements can be carried out. For example, HPLC
analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI.
Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.
Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90%
inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
Packaging:
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the polynucleotide for human or veterinary administration.
Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
FUNCTIONAL DERIVATIVES
Also provided herein are functional derivatives of a polypeptide or nucleic acid of the invention. By "functional derivative" is meant a "chemical derivative,"
"fragment," or "variant,"
of the polypeptide or nucleic acid of the invention, which terms are defined below. A functional derivative retains at least a portion of the function of the protein, for example reactivity with an antibody specific for the protein, enzymatic activity or binding activity mediated through noncatalytic domains, which permits its utility in accordance with the present invention. It is well known in the art that due to the degeneracy of the genetic code numerous different nucleic acid sequences can code for the same amino acid sequence. Equally; it is also well known in the art that conservative changes in amino acid can be made to arrive at a protein or polypeptide that retains the functionality of the original. In both cases, all permutations are intended to be covered by this disclosure.
Included within the scope of this invention are the functional equivalents of the herein-described isolated nucleic acid molecules. The degeneracy of the genetic code permits substitution of certain codons by other codons that specify the same amino acid and hence would give rise to the same protein. The nucleic acid sequence can vary substantially since, with the exception of methiorline and tryptophan, the known amino acids can be coded for by more than one codon. Thus, portions or all of the genes of the invention could be synthesized to give a nucleic acid sequence significantly different from one selected from the group consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID
N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ 117 N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID
N0:17, SEQ ID N0:18, SEQ )D N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ
ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID
N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ
ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ ID
N0:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID N0:56, and SEQ ID N0:57. The encoded amino acid sequence thereof would, however, be preserved.
In addition, the nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5'-end and/or the 3'-end of the nucleic acid formula selected from the group consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ >D N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ
ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ
ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ II7 N0:17, SEQ ID N0:18, SEQ ID
N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID
N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ
ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID
N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ
ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID N0:51, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID N0:56, and SEQ ID N0:57, or a derivative thereof.
Any nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence of selected from the group consisting of those set forth in SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID
N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID

N0:17, SEQ )D N0:18, SEQ >D N0:19, SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ
ID N0:23, SEQ.ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ m N0:28, SEQ ID N0:29, SEQ 117 N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:34, SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ
ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID
N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ 117 N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID
NO:51, SEQ ID N0:52, SEQ ID N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, and SEQ ID N0:57 which is encoded by the nucleotide sequence. For example, the present invention is intended to include any nucleic acid sequence resulting from the addition of ATG as an initiation codon at the 5'-end of the inventive nucleic acid sequence or its derivative, or from the addition of TTA, TAG or TGA as a termination codon at the 3'-end of the inventive nucleotide sequence or its derivative. Moreover, the nucleic acid molecule of the present invention may, as necessary, have restriction endonuclease recognition sites added to its S'-end and/or 3'-end.
Such functional alterations of a given nucleic acid sequence afford an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto. All variations of the nucleotide sequence of the kinase genes of the invention and fragments thereof permitted by the genetic code are, therefore, included in this invention.
Further, it is possible to delete codons or to substitute one or more codons with codons other than degenerate codons to produce a structurally modified polypeptide, but one which has substantially the same utility or activity as the polypeptide produced by the unmodified nucleic acid molecule. As recognized in the art, the two polypeptides are functionally equivalent, as are the two nucleic acid molecules that give rise to their production, even though the differences between the nucleic acid molecules are not related to the degeneracy of the genetic code.
A "chemical derivative" of the complex contains additional chemical moieties not normally a part of the protein. Covalent modifications of the protein or peptides are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, as described below.

Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized~by reaction with bromotrifluoroacetone, chloroacetyl phosphate, N-alkylmaleimides, 3-vitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylprocarbonate at pH
5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect or reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing primary amine containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine alpha-amino group.
Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tetranitromethane.
Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-vitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimide (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Derivatization with bifunctional agents is useful, for example, for cross-linking the component peptides of the protein to each other or to other proteins in a complex to a water-insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of Beryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.
Such derivatized moieties may improve the stability, solubility, absorption, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein complex and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).
The term "fragment" is used to indicate a polypeptide derived from the amino acid sequence of the proteins, of the complexes having a length less than the full-length polypeptide from which it has been derived. Such a fragment may, for example, be produced by proteolytic cleavage of the full-length protein. Preferably, the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence.
Fragments of a protein are useful for screening for substances that act to modulate signal transduction, as described herein. It is understood that such fragments may retain one or more characterizing portions of the native complex. Examples of such retained characteristics include:
catalytic activity; substrate specificity; interaction with other molecules in the intact cell;
regulatory functions; or binding with an antibody specific for the native complex, or an epitope thereof.
Another functional derivative intended to be within the scope of the present invention is a "variant" polypeptide which either lacks one or more amino acids or contains additional or substituted amino acids relative to the native polypeptide. The variant may be derived from a naturally occurnng complex component by appropriately modifying the protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence. It is understood that such variants having added, substituted and/or additional amino acids retain one or more characterizing portions of the native protein, as described above.
A functional derivative of a protein with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art. For example, the modified components of the functional derivatives may be produced using site-directed mutagenesis techniques (as exemplified by Adelman et al., 1983, DNA 2:183) wherein nucleotides in the DNA coding the sequence are modified such that a modified coding sequence is modified, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above.
Alternatively, proteins with amino acid deletions, insertions and/or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art. The functional derivatives of the proteins typically exhibit the same qualitative biological activity as the native proteins.

TABLES
AND
DESCRIPTION THEREOF
Table 1 documents the name of each gene, the classification of each gene, the positions of the open reading frames within the sequence, and the length of the corresponding peptide.
From left to right the data presented is as follows: "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family", "NA length", "ORF Start", "ORF End", "ORF
Length", and "AA length". "Gene name" refers to name given the sequence encoding the kinase or kinase-like enzyme. Each gene is represented by "SGK" designation followed by a number. The SGK
name usually represents multiple overlapping sequences built into a single contiguous sequence (a "contig"). The ">D#na" and "ID#aa" refer to the identification numbers given each nucleic acid and amino acid sequence in this patent. "FL/Cat" refers to the length of the gene, with FL .
indicating full length, and "Cat' indicating that only the catalytic domain is presented. "Partial"
in this column indicates that the sequence encodes a partial protein kinase catalytic domain.
"Superfamily" identifies whether the gene is a protein kinase or protein-kinase-like. "Group"
and "Family" refer to the protein kinase classification defined by sequence homology and based on previously established phylogenetic analysis [Hardie, G. and Hanks S. The Protein Kinase Book, Academic Press (1995) and Hunter T. and Plowman, G. Trends in Biochemical Sciences (1977) 22:18-22 and Plowman G.D. et al. (1999) Proc. Natl. Acad. Sci. 96:13603-13610)].
"NA_length" refers to the length in nucleotides of the corresponding nucleic acid sequence.
"ORF start" refers to the beginning nucleotide of the open reading frame. "ORF
end" refers to the last nucleotide of the open reading frame, excluding the stop codon. "ORF
length" refers to the length in nucleotides of the open reading frame (excluding the stop codon). "AA length"
refers to the length in amino acids of the peptide encoded in the corresponding nuclei acid sequence.

r B ;g ~ ~ 5 a ~ ~ ~ ~~ ~ ~

Z ~ i$ -r ~~ ~

~ Ngl a m..~~m'.id:.:8 of '1 m ...
~,~m U ~Yo m m n w m ~ v p u.~ ~ ~
n m ~
N

m _ ~ m ~
~

Y In t~ o~v o m ~l' ~ E c Y ~
a '~
~

n ~n $ ~ s ~ ~ m ~ n Sr .
n~ ~ ~~ r1 ~

m U
o 4 ~

_ _ ~ ~ ~
_ N
~ ~
N

N m rN~'- ~ O~ ~m ~~5~ ~ a~ a~

Z U
m O V N
m N

t0 r N

a o N m E o n m 0 0, ~ m o C'N O t-0 O

r I

m N N N

L

m N
m C

N

f a~

O C
~t 3 a T y~
d _ I- a ~ ~m m m ~ l z io ' r m m '-a E ~ a u o M
~.

O

d a a a c~ a t r m E a a Ya a a N

A
j ~ ~ 4 U

A
N ~ t0 tD
_.

C
N N V

m Y

~ U U' U ~ o c o Y Y Y Y
N N N

m a z r 0 ~A

a ~ ~
~
~m~A

mR

y ~ ~ ~ o ' a ~ m a ~
~

~

~ a = on M 'u ~ o m Y
y y y c~
o J - M 7 m ~

A~ (n fri ~ t0~ IO ~ ~ ~ ~ a ~
d L Z '- ~

O
A ~O

a z p , N N
m N , V O
O ~ m N

O
t9 ~ o N Im N

d ~ 'w m t0 V. ~, m J W

~

d L

t0 C

N

r d' O

t0 I o t f,. n Z N _ d m U
a a m - a a ~
E
A

-N
w N
j ~ U

m a O

m m E
m z m c m o m O .~ o Y Y
~ O

N

m ~ '~ ~ $ n o z ' ~ ~
-' >

> Vl m$) ~ ~ F~Y
Y ~~~~ ~ ~ Y~~

'r~.~a ~~~~~ m~ c d m m ~ 8 E E ' p : ~ ~' 8 E

$H

_ Nmn ~ Y v of ~~,j o ~ of o U ~
0 ~ uW7 ai m o ~

0 O ~ N t ~ Ih ~
m O Q~1 N ~ D ~
Yo~ Y7 O' m~~ O ~ I~ I goo~om Oo I 0 ' ' y I,~ N
cn ' E

m U U' a$ ~ O a omvor- .- m m ~ U 07 (n C fn Ch O U (A !L ~I ~I
1 O ~ h m C Cu~~ ~ ~ ~ O N
~~ ~ u~
(n lL I p=
m fn ._. ~
S
' ' n n ~NZO~USe3oW Zr~.- n v7n~ oi.-mov m~~c n Nvo 0 ~ o r~

O ~ ,R

O t0~! N (yrf h O N ~ OOD N N
N ~ ~ N

E ~ '~ n ~ c~
m N O O O
C

m O O O ~ O
n n ~ r r m M n m M M
~ ~ M ~

!r IA t 7 ~) O

o O h o ~G 1y m ~ m o0 0 0 ~ o o ' r H

o a r ~ N cN7 m ~

Z O O O
C
m m Y Y Y Y
a n.

Ii j Q Q U U

Y Y Y
E

U' a U U ~ U

m E
a a a a a N
~

m U ~ ~ ~ tJi. IJt m m r~ ao m o m a Y y Y m m U a U
c U

f'J~ 'gin N ug m Y Y Y

C9 (7 C9 U' U' N N ~ N N

N

a I

N E S

n Y w ~
m ~ N

.. m ~ a m ~ 9 .~3 a g Q Q ~ g 2 d ~ Q ~ o ~ m ~ o G~ '~ m do m ~ o o o h NI at V =
t ~

v - ~ ~ .~ ~ Y ~ N t $ I
l Y I p r N v Y o Y o U a o v ~ m c r y1 Y ~ o .L
Y I m ~I Y $ ~ ~ Y Y ~ ,~ I ~ I
N n -n d $ $ m ' O O ~

C7 a N .- _ ~ U W7 a C9 C9CO a p Y m E ~ U ~ o o o $ o U p U

9 o m o cn a: co c E ~ n ~ I ~ ~ ~ ~
' ~ .
~

A N tpl NI Z d .~~-A .~- A~ A N U
~ U C ~ O r1 ~ r N N Q t00 A~
- N ~ Z M A ~ N
A A Z
O

_O
O N O !'r1 Or0 m VNI !r'1 OrD m V
~ O

U U ~ ~ o U U
~ ~

o ;i c, co r ~ ~ ~,.~v h r r ~o r ~n c~ ~ v o'5 O ~ N N O~1 ~ ONO
N
p N m ~ 1~0Vr1 00 O O O ~ O O
N r O N O O

t m ~,~, '~ o~ ~ r r N
y V ~ r ~

W
r m O O ~ r N ~ ~ 07 C 4 7 N N N N N r N ~

K
O

r d 9 l j r m ~ N a0' r N Of C K V t0~7 N N N N m ~

~ O

r O

.a t !

$ _ N r N m ~ O7 Z N ~ N N N N
y Z' Y Y Y Y Y Y

W w W W W ~ W

N

U U U U U U U U

W

~
E d a a a a a a a in .
:

U ~ ~ ~ Z c ~ ~ V
m m m A
n ~ ~ ~ ~ n N (~ ~ ~ tO r m Q1 N

N
Y

2 ~ Y
U

m d ~ r ~ ~c a' c~f v N ~ ~ O m O O

N ~ N N ~ N N

~
m ~

m g ~

s~

tp ~ ._ ~ ~ ~ m C o ~
NVI C.N ~J~N r 0 J um1 0 U00 ~ m'W
S 'y C m O m C 0m!
U

9 . ~ !~ ~ N, _ O
~ Y of N ~ ~ r_ ~ H O U
n cn~ m ~ LL y ' ~ C ~ ~ _d ~ m m t'j J O ~ N V1 ~p 0 E m O
~ UaO
~~ N C ~ O O C ~ a ~~~ N
N ~
~ m m ~~

~t ~ W/7 t C y 0~ .
~< j ~a E
-DD UI ~ O m O d O ~iN Y OI ~ E
Om ~ O T ~ N t0 O !O
Q ~
N . O r- IA ? u1 ~
~

A O~ t0 /1 ~ N OI ~ .- ~ N 1~ t~ 7 A
J '- N U W d m A L ~ 01 t0 t0 J ~ m C1 d t0 41 01 f0 ~ L ~ ~ C
= N d U

O
O
O ~ O
L
Q O N

a r O V N N N
m f~0 r m N ~
M V m N
C

m O O O O
r n N 1~ O
' d r t0 M ~

V7 t0 V O
m N

C N O
N N

W ~ O

O N

_. _...

N

r K
O

.a .~ m f0 0 0 Z O M O N
C N O
m N

_T Y
Y

-~ U U
t~ w a Y Y
O U U

d ~

aE 0. a a Ya H

A
m m O N N M
N N N

m U

m ~ a Z Z

m <j ao m o M ~

Y
N N

N

w B _. :

2 K ~ g T
A$~ ~~m~_~N~~~~BA

..i~C ~.~~ ...LS~ m ~m 2 UQ t3 ~~ m m m b g p ~~'~~~~ ~~a~

m ~ .5 ~ z v " ~ ' ~ 'N o o (~ (~ ~c cmv .

Y Mg~ncaio~YEE~n U'a U'a U' U'D_~,~YU'~Nm r~' ZuW~N ~, m~ a~eZUrn~~$ n~~Nn~5i.~25UCtn n~=.co S c~

m m O N
O ~ ~ M
m N

O

" D O m (n ~ O ~ Z

f0'1 N V (D ? N

M ~ a~0 ~ ~ pN7 O~7 OI N N O N t-0 p Y~7 t~~7 M m ~ ~N
O O O N O ~ N
O O O
O n ~ ~ ~ ~ O
O

p O m m ~ m GI C N ~ In A N

( t N 1~
m p J .~ ~ ~ u~7 Wi C a A O m N m N
N

O W f~~. m n N r N
1~ O m C ~ N m N
~ o a r, O
~

m a ~a r O ~ N amD um) Z N N
N

Y Y
Y

U Q Q
N

U

Y Y
a a U U U a a o C7 U' U' a a '~'r U U U U ~ ~ . O
n ~

T

Y Y Y Y Y Y Y
a a a n. a a a N
y-a j a aNp m ~ ~aNO ~ m m N N N N N N M

W
N Q' Q

Z Y ~ j m Z !L U7 C

N !'7 1m(7N N A

U' U' U' C7 C7 C~ C7 fO N f0 tn In tn m b _ ~ ~ ~

N ~ ~ ~ ~ ~ ~ N
~
~

Q

g g d ~~~ 8~ ~ v'~ ~ ~w~~~'~~d~'~~~~c~ m~ ~ ~'o~~o ~'Ly a o N Y~ ~ v ~o~ v j o yU' ~ ILL YO Y~ ~ Y-~~oJ
' LdY; o~ ~ Q Q ' o o u.
U Q o t c ~ ' ~ Y O Z m tn U' c x E c a v x . O O U C7 O
E t~ o N m u! "'j y o o c%7 u) a of ~
o o o ~
m v o o d ~ o m o eo o '~ o a m m ~ x O O c9 c9 > tn ~ v y ~

a C7 U O O a o n n n n W n ao ~"
n ~ N ~ C9 a.; U m ~c v a ~ N O v Z om v m H . N

a O ~ 7 0 L ~ ~ 007f~0 n N r O
N
~

r0 m ~
O

r O O

2 ~ 2 U Z
Z

OH o~'1 N M V N

O7 t0 C ~ 1 O m O O

U r r O

O O
p O m ('~I
~

O O O O
O O

N y ~ o, G> ~ 'o N o v c m r d !Q

N O O N m V N

x ~o ~

d ~ W ~ rn u7 m r c~
<O

O N N Om1 ~ C

r d O

H a , ~ ~ N ~ m m Z Q o N N m m m m U Y Y
Q

D D D

U

'u 'aS m d v f9 O O O O O

nE
a a a a a m j ~ V

A
O aoo m oo~ 07 M ~ m m A

Z c tn m ~ Y
c C7 ~ ~ n m m Y Y Y ~ Y
N N N N

B ~ ~n bn y~~y~a~

i ~ o_ g ~ ~ ~ g . ~ L
~ ~

V C tr!1~ N .. O ~ 01 m ~ m M ~f7 g g ~
~ ~

A .-a Y Y ~ Y ~ Z
.Q o ~ I $
Z , ~
O O ~
~
m ~

~ C U' V, p ~,~p d 'J ~i o ~
c N ~
O o OM ' ~
n ' ~ ~
~
~

n z f/7fAl~ U7 fn N 47 fn( n ~ n MI~NN n n n n L
~ V v , E N o n m E n ~ m 5 m o .-~ o ~ n m m C ~ N
O O ~ m m Y ~ , 3 m o m ~ o~ ~ ~ i ~ m ~n O
m E
~

n ~O N ( ~ O N N
O o < ~ '~
~

O o f ~ ou _ m n ~ N
~

~ a m ~

2N Z~tNO Z 2 H

O OD m m 01 m m M m t~ ~o m u~ o m ~

o N c~ n ~ ~ N
O O ~ ~ N_ V N m ? OI~7 ~

O O m N HMOO N
E m ~' ,m ~ um, O

C Q O N ~ N O ~ O
O M
CJ O S O A O ~ r n m H ~ "' ~ m 41 m ~o m a~ m ~ ~ ~ v v V.
r c _ M O M

J ~ O a v O o V N
III ~ d N D N 7 C N

_ p e-G1 a I

W ~O .~ 10 m O m N
IL ~ N ~ mV OM7~ ~ N

V N N

p a G~ p ~a ~ ~ ~ ~ ~ ~n Z ~ N m V
C

O ? N

N
7' m Y u. a N d'> j a c a ~ ~

z ~ >

~ L L L t ~ ~ d O _ _ _ ,t p p 0 0 p 0 p p p p v.
a d Y Y Y Y Y Y Y Y Y
' a a a a a a a a a ~

N
u.

A
j ~ ~ ~ ~ 4 ~ 4 m . ~ ~ ~ m $i 0 0 m C m M ~ M v v v e o E ~ d.

z a ~ a N ~ N O N ~ m Om7 r ~ ~ ~ N ~ ~ N N ~
~

t 4 t m .s d n ~ ~
~ w J a~ c a. o'~ ~ a m , = c o U 0 y O r ~ ~ LL N ~ D p m t C~ 0 O
~ p (b v o1 ~ ~ ~ O t~
~ ~ O
E ~
~~ ~ ~ N
~ d =

m E u_ ~ y - ~ c y U
y ~ ~ v E o ?:~
U
' 'gi o ~ .
~ ~
"
o ~ ~
~
d ~

n d ~ t d n n n t m Q ~1 n U , o C n $, ~
a N
e5 d , o t n N y n N ~ .- N 'a ... o~ ai o m ~ m O ~ m ~. .- ,n ~ Z
U a ~ of 'o v O
o n m m r a ~ v ,~I N
p~7 O p n a a a z z O N N
H

O ~ ~ o ~ m V N

C

d O O 00 CJ O O O
n n n d _ _ m I W n m ~

C ~ 7 M
fp 4 O

Ir N m ~ N

f p a~

O
~r r-a O

.Q

H
z d a, w w w z z z w w w c~

;, r ~ a a a E
w N

A
V J J J

A
O O O

A
C < V V

m E =

z d c c~ ~ m m Y Y Y
N

N

_ _ '~ 8 ~ m~
a ~ A~o o ~ 'Py $~ s ~ m c, ~
' ~ z ~

o~~ a 'ae L7 ~ ~- ~ ~
m 1 ~
ii g ~~

~a~ ~$ n ~ m n9... ~09 ~U~

i$

~

m m Wr m m O ~I m !L u1 ~ ( CW O p p ~ r p ~ (~O =~
=
m N ~
~
~

_ O tJ ~ l (] ~ r 01 YQa YQo d Y YmN Y INS
r Y~~
~
O C ~
u1 ~om~
~~ off Iom' o ~
'Q~~I
Y
~'~~-noa ~

a N N N m t~r~ N w . c%f o d a ~ ml' U o o v ~ a ~ E o m 5 c ~c ~ C~ ~
~ ~ m m u! ml ~I o m d ~ U

n uW nm~U' a.,.Q.~a m c~!9S n n.-Nn v n n Sr n moo m m hr.xx v o~~ 3 ~N ono .

O m N
Z' N

O o ~ m ~ o ?
~ N

t~ 10 r a ~ ~ z ~

O a a O

N

N ~ V fD Or1 N 1~ N O) ~ O' N

y O r N t0 m ~ m O
r ~ ~ ~ m ~i ni C O O O O O O

O
m O O O O O O
(' O
r r r N I m m m m o 0 0~
d ~ M N N ~ O 001 ~C

m C r ~ V m r r ~ ~' ~ g ~

C m , r N
fll .~~

O

t0 W
c~ ~ ~ ~ m o r ~ ~ ~ O ~ N

M

Q

O N
~r ~ r r ~

d O

.0 !0 I o~ ~ m ~ <n ~ m u~ O

m yn N

a e- ~ ~ O O ~ m A ~C

",m m m m m a ;, r Y a a ~Ed a a a a N

j ~ U ~ m U

A N m r m m O
O O O O O

m 01 O N l'1 V V n0 ~ N 47 ~!1 a i m W

m oN u~ o m ~ o v O

Y Y Y Y Y Y Y
N ~ N N O N N

t / I
/J

a z~m -g~~

m m m K

d rn r N

~ ~

A~ A~A~
~ ~
N N

O!
O
O m ~
t O N

N

O H a o ,Ny n m b f'7 N aD
D N l~N
N

E N
O N
C

O O
m O
O O
n C ~ m V

J

C ~ ~ c'v _N

X O
T

N N
r. ~ M ~ V

~

r K

_d r ~ N

C
n jy U U

O

a c 3 a O o O

d m d y Y Y Y
~ Y Y Y
~
,Ep N d O.O.
w.

m m U ~ m ~ U m a A
N c~0 A
h ~ N

N
E
m z m c d o ~.o Y Y Y
V N jj I r Table 2 lists the following features of the genes described in this application:
chromosomal localization, single nucleotide polymorphisms (SNPs), representation in dbEST, and repeat regions. From left to right the data presented is as follows: "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family", "Chromosome", "SNPs", "dbEST hits", & "Repeats". The contents of the first 7 columns (i.e.,. "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family") are as described above for Table 1.
"Chromosome" refers to the cytogenetic localization of the gene. Information in the "SNPs"
column describes the nucleic acid position and degenerate nature of candidate single nucleotide polymorphisms (SNPs). For example, for SGK386, the "SNPs" column contains "835=M", indicating that there are instances of both a C and an A (M = C or A) at position 835.
"dbESThits" lists accession numbers of entries in the public database of ESTs (dbEST, http://www.ncbi.nhn.nih.gov/dbEST/index.html) that contain at least 100 by of 100% identity to the corresponding gene. These ESTs were identified by blastn of dbEST.
"Repeats" contains information about the location of short sequences, approximately 20 by in length, that are of low complexity and that are present in several distinct genes. These repeats were identified by blastn of the DNA sequence against the non-redundant nucleic acid database at NCBL(nrna). To be included in this repeat column, the sequence typically could have 100%
identity over its length and typically is present in at least 5 different genes.

' a -~~R ~~ ~~ ~m s"~

m N m ' ~t !p m~Z/7Y~ZaYm~~~ ~ "15 d ~2~
~
~

m ~ ~
t~ 9~ m ~ Sm ~-~~N ~.r~--.K
~

N

N m ~ ~ D n m Fee <

d ~ _ N ~ N
N
m O

N I O! 01 I M O
O
~

~ Y Y Y
s z ~ I l c~a as ~
~~

U C7 ~ r7 n n ~n n M
~
N lit ~p ~ m o v c ~
n v! I I a,r 'o m Y

N M ~ O
Q ~ N

O Np O 1 t m ~ O O 0 D
N ~ ~ O

U V m U
m ~

.. ~ ~ ~ Z ~ U
m o O v N ~ O
N

O

I
O m V N ~ f~ 00 00f0 d N O O

~/ O ~ O

IEm N m N

g !, N

a ~~

z L m~~ m ~ g ~

a mm~

v m Z

V -g - ~ ..

a ~~r~~m ~~ o N ~
N 8m~~~ F

_C1 N~m~ ~ ~
d _ E
H N ~ N

O N ~ _ O N N WO N

t U

-E~ ~ a v 23 lio U ~ o U U U U
a a a c~ a t ~, _ m a a a a ~
E

~w j ~ ~ ~ U

m N

c N l''1 V

m Y Y
U

M
O

O
Y Y Y ~ Y

(7 C9 C9 - (9 tn fn 4! Y (n 3 ~ ' Na ~

N
8' ~o K
n $ ~ ~~ ~ ~ r ~ w~
p N ~ ~
a m~~~~>~~ ,n db 8 ~~ az N

a N W ~ r a n a m n ..in N
'~

w m ~ g Y g g ~

~ ~ ~

A

M N ~ ' U! (h r ~ m 10 - V
O m O M t'm7 I O ~ ' p ~I ~ N
ml lL d C a O LI O C Y N in N
n O M ~ O
r 9 I O ~ p U N I ~ OI N ~
~ ~ m ' ~ N = i N Q OmD ~ O ~ IA
p p E N m ~ ~ ~ x ~
O 'm M

x M Oo Y YO Y~ o m t ~ I~ ~UU
V o N N 7 M
S . g Y I I N~ mM ~' Y I ImQ~r~in~
o m ~

x c~amo E cv E ~ c~ c~o~o~c~<o IU c~a .
~ci IN fn f~ m m b~aNc~~-= c~o~ ~a tO W m o o v ~ m fn o'f N W n o o o o -~ ~ Y d m r o ~ ' . E m tn ul ~
m S S M of a~ . a M o m ntn to ,r (7 a~ n n.-m~rn ~NZ~~e~cnN to a~mvomn U I lo>ouo~.-oaNaz~ N

_p~ ~ ~ o O O ('m~ M N V

Wn m u~ o r r O ~ N f'7 N !~
N

m O ~ O
I N m N O m M
!'7 O M O

V N m m M
m aI ~ O O O O O
r O O O O
r r m W y ' m Hl e, a ~ a ~

vi a ~
o L ~sa ~
z ~ I d v ~

<n g g 3 ~ iu w m mm m v maa Z

U ~ >

N~c>m ~e>e o m ~~m ~ N
~ ~ V

(n r N Y ~ ,' ~ R
N
N

N

M
O M ~ M

O N M N
m O N r O x 01 L O
U N

a. ~ ~ d d Y
U

u~,a j j Q Q U

U U U Y Y Y
' ' O G U U U U U
a a a W

y ~Ea a a a a a ~w m j ~ '~ ~ uJ.

N

W n m r m w o Y

m E Y

o m o ci v m m ~ ~ N
C U
m a ~ ii t t n U

~ ~ t5 ~
m Ig o m N
~ S ~ ~

a .. ~ w' ~, g ~a o a ~ p nm~ >~a~ M
m Y' ~ I~ ~y > N II Z t ~I
~ Z ~ ' ' ~~
~

'~' ~a eg ~ ~ ~~ ar R ~
a m m m m a, m d m d g ~ ~ g g g x 1 ~I E Z ~ . a N N oy.~ I I
In o m v ~ ~ N o o < w r I r lo~r2 mc~ ' M to ao~~
I y O r I ~ Z O ('J O O O U M O O U' Il i O r1 N N W O p 1l ~
V ~ 0 0 I I I
~y I
a y N~a y to coo yo yo y~ yln y y~ o. ~
x y Im U' o ool C7olrnNoaroN NU~ U' -N.Q
C7aW ~~~o~o~ ~ ao ~ U' C~QoY~ ~z~r ao ' E

fn W N ~ N r fn u) _ N N M U a II fn A th ~ N r ~ ~ (A ~ A N (h o U A
~ l1 aD N o o t~ A A ~ N (/1 o A U I A A O (O ~r N U
A (/I I M ~ M ~ r y Z x ~
I U ~ ~ (p M ' r Q ~ N A ~
N
Q
V

N r r r 47 m O ? O M r r r m N
O m OD V7 OD m M
O O

~ M
~ ~

N (m0 07 t107 ~ ~ O p t10 t~

Z

o to ~ m to r ~ ~ M 'd' v N

o ~ r r w r JN.1 O ry M m t0"7M t~D ~7 ~

VIN OOD N N ~ n W N
Q. E .M- O N ~ a~DaM0 ~ N I! ~
~

~/ d O N O O O O O O O
O N O O O O ~ O O O

M

A d d d v d E E g E

i ~

yp ~ r ~ d C ~ t'a fn ~ $ ~ ~ ~ g N
M ~

, _ ,~ ui ~ w 3 1u<a 3 3 a a ~a~
a a aa a m a v m m II

o ~ $ rH ~ m I _ II
N

!d H d E M
M

O N N N N N N N 'p M
O X ~ M M M N O

t In r U

Y Y Y Y Y Y Y Y

U U W IL tt~lLPL u~1 ~ ~

IL , u 111 Y , Y Y Y Y Y Y Y Y Y
a a a a a a a a a a f~U U U U U U U U U U

m~

E a a a a a a a a a a Nw A _ j ~ i A
r l~ n ~ r~ n 'r ~

M wn to r m m o N

at N Y Y
E ~ ~n m m ~

m ma N ~ ~ a'o ~In N o d ~ Y ~ Y Y Y Y Y
~ ' M

(7 C7 m U C7 C~ U' C~ U' U' C~
m (n U a (n O
U

!n (n N fn (n (n fn N
Z

f a N ~o m tS

.N.
d g ~~

m a m t ~0 0 0 ~ a N ~ m roe ~' ~ m a ~ c m c L ri d o o ~ a C O
(D t0 ~ N O 'N
~ O N ~
~ (p (D f0 UI ~ Vo H ~ N ~I
l ~

u~ O J N p _ O 47 U
~ .. T ~
I~JJ00 OD Y
JJ~On p t0 C
OC~IL -d CO ~ Qp N ~ ~ C ~O
O N C C O N U
~~ ~
~
~

a,N y ~ Y
U rf Q Q _ m ~ Q u a y o 0 o v ~
Q .- ao o ~ o . a N -o o m o v a ~C9Nm Eu~ioic~vUro~'-Euyo d ~ o ~mc~Ya E m t~ v mo U
~iuiN m'o o a N~~ta m ~
a r ntn~ o~dr~r~~2vU'o>v~n~n ~N rnaw Nr~~nU wm E 8 m n ~ N oi.-a aua (~UZC~

o o m o L O ~O

a O N N N
N

O

u1 ~ N

O N
C O O O

O O O
O O O

d O N

N

Z
L N d X17 I O y M N W g g a a ~
a U

N

N
z _d1 R

F- d E ~ ~ ~n o ~,~ r~
L

U

Y
Y Y

U U

a Y

Y Y
V U U

v ~

Y Y
a a N

A
a U

m c N N

O
E

N

d C Y Y
~

N ~ N

w a V Y

H ~ ~ ~ ~ ~
~ ~ ~
(7 n d ~ ~' H ~ ~c' N S ~ Z U' ~ wa ~ ~ ~ ~ ~ o ~
~ ~ N

E ~ ~

~

~ a~ :a = w ~
Q aro aal d ' ~ m V d 01 E LS ~ ~~ g E
N '~
~~ ~
~
~

nI o m a ~ M M N G ~ ~ ~ N N N N r M
UI ~ ~ (NOD u a~g ~ ~ o d ~ Y Y Y Y N Y ~
x ~o ~ ~ Q a ~ 7 .~ C7 a C7Y ~
a U C7 CO C7 n Y E 'a ~c v a < ~ i ~
o m y ~ E o o ~n 2 N N.=-. NYZ ~A A~~NA~ A~=C~ A~A~"O
E U~ ~N C~

O
N N
O N
N7 0-D t'~~
O N M O

(h ~ O U Z
U O

Q N M 'NC(00 ~ N
H

O 1~ f~ N N
I M 1~ a~D a M ON7N
W r- in v d E i.' ~ o M ,gy m d o o o o ~ o o 0 0 0 0 ' 0 0 H

N
liJ ' w d N ~ d d N

m o ~ g a ~~

~ .
t NN ' m ~~~~~ ro I ~ ~ ~ O ~ ~

N ~ ~ u7 N g N
M H m ~ w ~ < ~ m N
~ m ~

aaa ~ ma mQammm m~~
a 'a Z

r ..
r~ ',~. ~ m $
rW r ~

d R
E- m ~

E ' N

_ G M

O a 2 c ~ m n U

t U

Y Y a U U o N

U U ~ Q Q x cUo N

U

U U U
C7 U' U' pY pY pY

c7v U U " '~ " U
a a ~ a a a a a a a E

N

A
U J J J J J J J
l1 LL lL l1 IL LLIL

N
O m m ~ m m m C
N N N N N N M

D
m Y D
E

z w V ~ N M N N N r C7 Y C7 U' U' U' U'U' (n Z v7 fn tn tn fnfn S

H
.

$
8$
'3 ~9 n.'.ao~~ n~~..'>

~ ' 8 ~

8 E ~ ~ ~

_ -a l ' ~ U
Z

y M > c c ~ ~ n ih a~ N m v ~ yn P c o c ~ ~ ~ a U' ~ d l .o T'S n ~ ~
J~

aa ~
~

o( oC7oUr vp ~ ~ n v m I $L ~, M o ~vo ~ I Q
t ~ o o ~moQC a 7~UQU' ~

d m~YCa7aoCr9 3 0~ d c '~ y U' C7aU' poo O
tp t a tn tn mo E~' f 0 f~~ ~G
E ~m Eoa~o o c Y~~
c~W

0 ~ = o v0 U~o o o A /~ A ~o A Z A~
A ~- N w U d ~ 07 O U a N CJ ~ N A f/) I ~
O IL IL fn r C Z N N !0 Z N O .~ O N
C~ O Z O N Q

m O N 0 ~ m M
N N

p N V N O f ~ ~ c0 ~

n o n o ~ o ~
" o 0 ~ 0 l o a a a a a a ~ a, o a Z Z Z U Z Z
m M
~c O f'M'1 N M V N M (p H

M t0 M p N
(p m ~ O O

G1 ~ o ~ " o 0 0 a E ~ ~ M ml ~ W m d O O O O N O

O O O ' ~ O

M

r y m m ~ N y !C a~~ g o a r a ~~

o O N N ~ ~ M u~ ~ N
~
~

H O ~ ~ ~
N

r W W W
mad m mamaQ~

Z

Z ~ N

d N ~ o: 'N'3 ~ a n M

(~

E "
N N
M

E N N N N N C
O n N ~ O

t p U

U
E

r r >
,~N o 0 o z Z d U

a O L_ L_ L_ U t_ t_ O
(7U O O O O 0 v a ~
E a a a a a a a ~w m m U J ~p J J J J
~ U ~ ti ~ ~ m m m m m o~ m m c M PN7IM'10~7 m M
E c tn A ~ Y
Z

m ~ i m ' N a 0 0 0 ~ M

N (n fn tn 47 fn In ~

H
N

N ~
u c, ~ ~
~

f N
~
N

a b~
o ~~r~~n n c~
N

$N

N $d N C N N N
' n ~ <gag ~ g g a m m_ o o m . o d Nm ~ ~ V m O Nm C ~ ~
~ IN

A I
~
o1 = ~oomn~j~ U' U' U'V' C7Cl~Y
ov oN u1 ~' ' v) r m tp t~ !/J(O (p a m r m N v n o n n in n n ~ v S ci N v . m ~ ~ r n n 5 r ~ c V~
m I

m NI OI I ~ O
N 7 m N O N
N

O ~ M O ~
r O C N O O N O th 7 O r m j~ N N N m a a a a O z UU ~
a m z z F u~
~lE -r m m w m m ~ m O r n o u~ a N

M r ~ N N
W O_ yn ~ N Oro I O t0 ~ m !'7 V N OD ~ m N ~ r d N O 0 N O

O O ~ O 0 ~ O
O O O r r r H

N

g g g a c r N
I

~O
a r -o o I d v d d ~~ v E 8~

a Z a N m ~ m W

N o8 ~u'~'m'~

fn N N N lD
N
N
~

._.

N

O N (h N f~
O N N N C C N

O N N N O m L
U

_!~ a N N V
E ~ a '~ Y > > r>
~ ~

~i ~' ~ > j a d d d 'ma~ v v O L_ L_ L_ L_L_ L_ L_ ~
T

N
- a a a a a a a ~
E

Nw m_ j uJ. ~ a a A
m ~ ~ ~ m 0 0 A
M ~ v v c v a d E cYn Y

A ~ a z " -r of Q r m m m N ~ N ~ 07 m r ~
O
D

O N
~

O C7 U' V' C7C7 C7 C7 Z

N fn fn U)fn In U7 Q fn N g a ~

,~~ m~z,a, i'v mz ~ nN

5o oa m .".8N 8~
~

tSm_ "~ c m ~ c "
~m ~~m ~

d p'a~ m a m ~

rn_ ~ y rn_ o ~ a~
d O A N O TO~ E C ~ N~p 'O O P W
TO NS
M

d ~~ O
Y oNr 3 c m Eoa~o~ r MV N E y~M a a ~ m Eo ' c ~ ' yYC~'>
am "~~ EZ ~
~

~

I c y C7 E n~o o E ~ n~ n~
a W W

na oC

~
~
Z
n~
~
~
m m Q

~ ~ . ~ v - ~
t c ~ ~

~ n in U a rn v ~ ou c a ~

o ~' w t'~'1O
O O O
, a a as z z C N N o p N !'7 H

.NV D tN0 r O

t0 _ m rn a' y d Q' 0 0 0 0 c o 0 0 d o 0 0 c9n ~ o H

N p W y ~ a d H

'~O ~ ~ g 1! ~

a ~~

Q lV
LI a N
m H

M ;
m~

c Z

U

' N 3 ~ ~MN~

_d ...

la F- m O ~ N

E
O ~ M N
N

L
U

7.
Y Y Y
m Z Z Z

a W w w c7 L
a.

d Y Y Y
E a a a ~w N

m a J J J

N
N M

A
c d E
A

z a d Y Y Y

N ~ N

v ~ ~- x H U ~ m ~ ~ m ~
oc~

Q > ~ ~ ~ B ~ S
~
H

Q ~ (7 H Y a LS ~ '~ r To O ~ ~

~ ~ 8 ~~ya m~~~~~r~-~ <

~1 y ~ ~'o~fZ/m~K~' ~~~~(7VU~U~(~7 i~ ~AE

9'" m.. C7 V. QaCJUr U ~~.-.~ m ~
N NS'NO'm.~~.'~.N

O ~

N M N ~~ N N ~
~~

p m W N N
A na g ~ g < N
' <
m~

, ~n ~
~

ai ~I E m m y ~ ~ - o ~ O N O n d f0 f0 n D I 07 O
n O U ~ U f0 L U! ~ O
N ~ C n OD V
C V ~ C M n ~
h ~ VI
' O C O _ O O ~ O U O
n C N U U V Y ~ 7 O
O 07 J '3 ' m I O . N UI C i M ~ O U
O . ~ - O ~ UI Y Y ~
~ ~
~
~

2 C7 C:J ~ u. c v E o m ~ m ( Q N Y N Y p Y
~ ~ '~ Y U' U I Y Y
c o v O 'v~ c a N Q U' C7 C7 N U' ~
~ 3 c N v o ~ 5. C7 a p ~ o c O o U' ?~ N C7 <
o _ A~ ~A ~ A~ A~ A~ A~ A~
7 ~ d E ~ OI C (p !0 a ~ 2 r ~ ~ C fn r ~A
U 01 N ~ W .t.. E N N OI ~ p1 N r U ~I ~I U d ~ Q U ~ N

O aIOO N
' ~

O n V N n p !h ~ O
~0t7 O n0 n ,~ N M n ~ M O N N
~ N V O

(p OD ~ M I (h ~ N
a U ~ O (D
~ z Q U d a a O H
H a O N M N N .~ N 0) ,n N
N

(D n IA 07 n th N
~N.I D tnD N W N p m N OD
I N M

U ~ nN N (p Q ~ ~ N ~
! n n ~ mn N O ~ O

~ O N N
O

O O O O O O O O
O

N ' m n m ' m m m a P

- a ' g ~ ~ O' 0 0 c~
' ~

O z o , a a o~

I m m m m m m ~ d o ~ m ~ ~
N

A~ .'-M~
M m E ~ g ~ ~
o ' a g ma<
maa v V
>"
~

N _ In'c~ a ~ y ~ y ' ~
i N Z ~ ~ a g ~ a a m y / au Y

N , ~ M n , Y

N

N

E <?

N N ('7u1 ~ n, V
O M N ~ = M ~ ~ N
M

O n N ~U U N M M O
t m U

T ~ r ~ N N ~ j y U
C U

lL~ N N N N ~ ~ U' c 7 11J lL 111lL lL Y Y Y U
~ ~ U

(/7 r~ f~ tn ~ Q U' U' _T Gl N
N

Y Y Y Y Y Y Y Y
N d d d d d d a Y

Y Y
a d a U
~ U m m_ U ~ ~ ~ m m ~ U

m O O ~ O O O N th V

N
C C V ~ N N N
l7 fi 1f d E

N
Z

C O ~ O M n Op O 001O
N O~ t~

~

U U U U U U U U U
U ~ U N N N U N U
W N
N

Table 3 lists the extent and the boundaries of the kinase catalytic domains.
The column headings are: "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Profile start", "Profile end", "Kinase_start", "Kinase_end", and "profile". The contents of the first 7 columns (i.e.,. "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family") are as described above for Table 1. "Profile Start", "Profile End", "Kinase Start" and "Kinase End"
refer to data obtained using a Hidden-Markov Model to define catalytic range boundaries. The profile has a length of 261 amino acids, corresponding to the complete protein kinase catalytic domain.
Proteins in which the profile recognizes a full length catalytic domain have a "Profile Start" of 1 and a "Profile End" of 261. Genes which have a partial catalytic domain will have a "Profile Start" of greater than 1 (indicating that the beginning of the kinase domain is missing, and/or a "Profile End" of less than 261 (indicating that the C-terminal end of the kinase domain is missing). The boundaries of the catalytic domain within the overall protein are noted in the "Kinase Start" and "Kinase End" columns. "Profile" indicates whether the complete or "Smith Waterman" (partial). Starting from a multiple sequence alignment of kinase catalytic domains, two hidden Markov models were built. One of them allows for partial matches to the catalytic domain; this is a "local" HMM, similar to Smith-Waterman alignments in sequence matching.
The other "complete" model allows matches only to the complete catalytic domain; this is a "global" HMM similar toNeedleman-Wunsch alignments in sequence matching. The Smith Waterman local model is more specific, allowing for fragmentary matches to the kinase catalytic domain whereas the global "complete" model is more sensitive, allowing for remote homologue identification.

Mv co mcmnon rocoa~ca N o N~ u7N MNM ~u7~ ~M

t' O

d U

~o M coovo~ o nnco D ~N~ ~ (DOa0O M M
OO

c0~ Nm tI7NaO 1(7 (OMM(D
~O~ o0NO ~N~ aD

U O a_D OM OmO) N c01010op N M ~ ~ V

V~ O ~ V~ ~~ O VM
O O O O

_ _ _ C OOn O OO OOO ~OO OOOO ~' N N

O~ O nO OOO ~ON OOOO
O O O O~O O ~OOO

O O

> (~ J ~ J ~ Q
O + a~~~Z~U, x J ~ Q-w~?~~a> >-' z r ~~+ Y

~~~~Y~=ao~Y>x a "

~=+Q~~ ~ ~
a zao'lYaz~c~n~~Y

v c ~
C~

~
+ ~ >
Z
a ~ u ~

a ~ C7 H
~
i o m ~ ~
Q x w Y Z Y ~ > Cl ~ ti J l1J + -Z + N. U' > ~ fn >

ccc m mmm ~~ ~ ~~ ~~~ ~~~ ~E~

a~a~a~~ a~a~a~a~a~da~~ a~~a~a~~

___ _ __ ___ ___ _~~~ _ EEE E EE EEE EEE E E

UUU U UU UUU UUU U~~w U
~

EEE

N

c ~i VM M I~ OWtnf~ON 10 d t0InV CO LON NI~ 1~f~N~OV ~O
MVi~N ~V MNM ~NN MnMO M

C

Y

A

r N

I N~O07~ M ~~ OMM N1nV et~ O
O N (DNf~ ~ I~ ~ Ln N ~~ N~ ~

Y

Y

M

M ~' d NNN N NN NNN NNN N~~N N
O

a H

r N
dl~ ~OOM ~
m ~
O
' a ()JJJ
~ ~~ '~~ ~

> ti~~ U U m u ~mm ~

m aDWO NM V1n(OI~aDQ)O NM ~
~l!7(DO OO OOO OOO nnrr n R

NM V tn(Oho007O~N MV~c0h D

N
M

rO f0~N if) Y O

d Y ~ YY~_ V OO)OYV'~~ r~UO M
Y

W C~ N (O UN Q(4 N~~a0Z
d ~~Y Y YY Y~Y VV~ YYYY

M
V ~~ ~ N U N

' 'Y t V~ YY ~~~ Y
/JJ

U UCY
', ~~Y

C~

- M

O

d U

n O

_ I r a E ,~o c o d o > Y = ~ Y Z W Q fn fn U' ~ U a Y ~ J ~ '~ w a Z

.7 dH~QZY>~>W( D
~

D z ~ j ~ u.1 a C7 Y ~ Y > ~ ~ d J a J
a w J
a a w d ~ a Y J ~ (~ ~ a g ~ a +
E J 2 J U t~
~ a O Q U
W J a LL J ~
~

= Z g lL
o Y J > - ~ d c~~u- ~~aYZ~ =~Y ~ualau~l~~~a~~~
> a~z~

C7 U U' ~ Y Y S H D U' d Y !n Y Z Y U Y D C7 1- ~ +
+

a~
d _a~
w O

a U

N

c N

C

Y

_ v1 r N

Y

Y

M

M

N

_ ;~
O
a' r m ~I
~

o a m () J

A
r m D

Y
E ' .o z d o d Y

C~ C~

V M
VN

O

d V

~

D v ~N

_ u~ ago urnv O N O
d O OO

O OO

J ~ + 2 + ~ W
J

U U + Y

C~ LLJ + + lL Z d 4 ti + ~

n + a ~
cn ~ J ~ ~ +
Y ~ Y ~ ~

. + + J
w +
- Y + Z
' ~ + 2 l0 +
Q
+

V~ (~ w Y Z W d + ~ ~
Z + ~ U + ~ + +
J ~

Y + C7 Y
Y f7 ~
t ~ C7 +
!n C7 Q
Y
+ ~ ~ Q

a~ a>a~

a~ a>_a7 .

E EE

vv v c ~

i N NIn C

Y

C

N

~

N M

Y

Y

M

M ~

O

N NN

O

a H

r m i d w a ..
m j U ~~

m D

O N

N

YY
' ~

U
J

t0' O fn Y o~p C~

N Y

CJ Y

~
~

N

N ~~OM NO mM Ohop m ~ r M
N~M~ C't0 NMN Vu7M OM

O
N

d U

O)Wf~ h if7N 0Ic0M~O~N
N Na0Q~07 m ~m mop ~0OOM ~,.~N O ~DOWOO~M
O

p 0M~(7CO m N (O OOO ~
v o~u~ rmnOv ~car~nnoo>~"~o O

NOmN u7 N (OOV OOO o0 1~ M1~u)O M~00M~M cDM~op~N

tn a0tnMM OD_IncDaOc0M~_ h c0 ON NNOO N N NNN O aOO OO
O O O

O OOOO O O OOO OOOO O
dO OOOO OOO OOO OO~O OO
~ O

O OOOO O O OOO OONO O
h ~ ~ 1~I~MI~~f~

~Wr-N

Ow ~ Y ~ Y I a ~ O 2 D ~ ~ Y U a J
~

Y O Y W a O ~ U ~ ~ Y J ~ W 2 ~ ~
Y
W ~

aI
L
~ Q O I- Y 7 ~
U ~ C7 j ~ ~ ~ ~ Z
Y

Y
~ ~ ~

z j ~
~
a +U' I
CJZ~U' C7~~~a -a ~~
~
~a YU' CCC C
tDf0N N

a~ ma~a~a~8~E Ea~a~a~a~a~d a~a~

'a~ a~a~a~a~~a~a~a~~a~a~a~da~a~a~

a ao.no.mmm maa anaa a-8 0000 '~~~ ~00 0000 00 a UUUU w~~ wUU UUUU UU

EE~E~E

c ~

i dO (OVM NIn00MON Mo0Vf~ N
O 0aOON M IA NV OM oD~N
N NNMM InMM 1n~M I~NMN Om Y

,.~NOf~CN (OM
NVV'.-NN~ VOO ~NNN ~D

1 f M

M d' dc0 CDf0t0tDf0~t0Mc0COc0Oc0(O~(D

_ ;w,N NNNN N N NN NNNN N

a' r N N

O
' a UJ jpJJJ JJJ JJ
~~~~ ~

u_ Uu.~ti~u.~ ~~U ~

a m 07 OONM VLO(pI~c0O~OONM ~tn M MW MOM WMM m Om mW

W
N MV1O(Dt~00O~OMN M~47t01~op N NNNN NNN M M M MM MM

l1J

d ~ Y O ~ N O
Ao ~a~W N N~~ ~Yo~r Y ~ fn g c 'Z'p ~

C7 U U C7C7 U'pC~C7YC~N U'U' N Uoc~Y ~t~y UY~ !~ U UN

O O Y

M cp V

U

c0 O~a0cDMc0NNO
~ Vu7V ~NM

O
H

d V

I~ V W N c0N(D
~ ~'~N~~ 0~O

4 c a O tI~Q) _7 ~~ 0~ 0 N I~ In VO O M 11)f001 N u7O~N NaG

a0N f~O(O00 O M

d ~O ~~O OOO

a C7 d ~ ~ a f~ > Y ~ ~ Z a U' >
J J > I- Y o a ~ C7 Q

~
' a '> J
U
~ ~ U' C7 ~n ~ ~ a' w W U' J w CJ Q: ~ U' C! ~ ~ '~

-~
~
~

Vg a ~ > Q J ~
Q ~ ~ a W Y
~ > ~
~
D
~
~
~
~

a ZQZZ~z~c +YY
nzz>a YZ
Q>>~Q

m a~ a~E a~a~a~a~a~a~

a~ ~~ a~a~a~a~a~a~

oa am aaa na-E E~ EEE EEE

U ~ UUU UUU

E

c ~

I _ (O N O~M OhO1 N NM OV NNN

M

C

Y

N N- ~a00~ CNM

A

C Y

~
Y

M

r M

d ;"N NN NN NNN

_ O
' a r m _N~ ~M ~
N

w O

a m J J~ JJJ JJJ

R
m m~ OOO OOO
O O

R
O OV NM~ t0c0I~
M V VV~ VVV

Y

M
D H

Z
EY N~ cflcod no0 ZQ yO OMOjoOO

d HY YYr YYY

N ~~Y ~~~

N ~, %j ( Y

N

O

N

d V

cDr D~ ~ ~

I

O rNN

ON O N
CO O O

d O O O
~ n J + ~ W ~ C~ J ~ ~ ~ (n ~ ~ l1J
k g p Ya aoo pc~=Z>Zaa Y~

'~a a ~ ~ ~ p ~ ~ Y z ~ = d j a ~
o ~ g ~ ~ a Y . Q Y

E~dwdrJ~wJCU7a~~i~V~Qcnwa~~~I=-~aC7 Y

U' I J IL ~ J W 7 ~ Y
~ Z ~ J a J U~ ~ z ~
-~ H C~ (n d lL

_ d > O Y Y
Q Y ~
a > w ~ ~
~ +
' > a ~ Wa z ~

c m > ~

m a>

O

U U r E

c ~

i y 11110M N N

C

Y

R
N

~

I "I_ r C
Y

Y

M

d i dN N

O

a H

r NI ~ N

w O
' a m m VJ m r ~ U ~o m D

A

c Ov W
n m o u~-~o Zo o cU' C7C7 dN ~ U

~- N

O

N
V

47 ~
~ N

l v O O N

C

d O O

~ > U' Y J C~ Q Y ~ J ~ ~' U ~ a '~
a N p ~ j Y ~ w ~ > d Y Ct WiJ > Q p Y d w d .~~ ~~ ~~n~~>a~~w~
a+c+
aw ~~+a~

n z > ~ a ~ a m z o ~
~ ~ ~ g a ~ ~ ~ n >
> u V u ~
d u~ a Y ~ ~ Y C~ > ? ? a c9 w ~
~ _ ~ c9 Y a ~ ~ z a Y Q Q Y ~ + c7 o a ~ z > ~ c9 ~ m .~ > ~ u. U H ~ + mu a~ a~
d ~

a~

a o 0 U U

c ~

I
d M N

R

Y

m N

r N

r Y

Y

M

M

N N

O
' a C
m N~

N

O

a m m U

m U

a m D

m c D

d M ~

Z o 0 Y Y

c U' U' N ~

~ V NNm V ~ ~

t' O

N

d V

f~MNe0 (O m N 47 ' QO~fM1f7 M N J r N

V c~( ~ o Dao0 n r r ~ ~n~n y r7 u mnc~

C O N NO
O O OOO

d 0 0 000 (' r ~ ~~n cu>1 ~ j ~ ~ + ~ C7 d ~ U tn + + tn + ~' z~ ~g~Z ~'~~~' ~

~ z w +
+ ~

'md c7 w J ~ ~ ~ ~ ~ ~ > U >
> J U (j ~ C7 In E~ Y + ~ > a w ~ ~ ~ ~ V + J
a ~ Y ~ > ~ U
~ a a +
' oU + c~ Y + J Z + ~
~ Z + a IL w C
U O
= ~

VJ~a +UH~j +U
+~~~u ~Y+ aC7Ua Wn U J ui a +
a o a + V
+ ~ rl U U
O C~ +
Y rl +
d D ~
~ + +

CCC
m NNN
~ ~~E

_ _ a~a~a~
~ ~

mmm E E ~~~

U U tt_r_ EEE

c ~

i OM

aD t0Mh Y

N

r I IA ~ P7M

c~0 M M(D

Y

M

r -~

M d' d. ~ ~ M~~

d ;,_ N N '-N

_ O

a H

r m N

~I
.

O
' a A m U J J J

> ~ ~ ~Um m O ~-Nfh R

M ~ ~Oh O
L

E n o or-o Z ~ o o coo Y nYY
Y

~~

Y f~f~

Table 4 describes the results of Smith Waterman similarity searches (Matrix:
Pam100;
gap open/extension penalties 12/2) of the amino acid sequences against the NCBI database of non-redundant protein sequences (http://www.ncbi.nlm.nih.aov/Entrez/protein.html). The column headings are: "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Superfamily", "Group", "Family", "Pscore", "aa length", "aa ID match", "%Identity", "%Similar", "ACC# nraa match", and "Description". The contents of the first 8 columns (i.e.,. "Gene Name", "ID#na", "ID#aa", "FL/Cat", "Serial #", "Superfamily", "Group", "Family") are as described above for Table 1. "Pscore" refers to the Smith Waterman probability score. This number approximates the chance that the alignment occurred by chance. Thus, a very low number, such as 2.10E-64, indicates that there is a very significant match between the query and the database target. "aa length" refers to the length of the protein in amino acids.
"aa ID match" indicates the number of amino acids that were identical in the alignment. "%
Identity" lists the percent of nucleotides that were identical over the aligned region. "%
1 S Similarity" lists the percent of amino acids that were similar over the alignment.
"ACC#nraa match" lists the accession number of the most similar protein in the NCBI database of non-redundant proteins. "Description" contains the name of the most similar protein in the NCBI database of non-redundant proteins.

a ~Y ~~'iN.~~,u C70 p ~ O~NZYQ~~~
+NY + W d IL Y~ 2 Y<+ Ii ILUU' 1u NYIt Dz a J~ IL2 ~ U' w Y +~+ ~ J~u1 J7 ~J~>~V, 2 ~w~u~lD U' a uu~lNa O>~)~ t ~ ~Y UYdNY<-UgV, I
O ~ <p N O ~a ~ p O ~ Y ~ U' ~ g ~ > ~ Y ~ J U ~ ~aa ~ Y d Y H C7 ~ Y D W K K
Z ~ ~ ~J Y
H ~~Y~~ j~ + ~+ g ~~Oa.NCJ~ J j0& ~~Up ~ J O W Y~~dpt~p ~Z~~2 a IL YS p(7, dZ' ~ O p Z Z g ~ Z u1 ~ W ~ Ky a U' ya D Y ~ Y Oa N W ~ ~~'y ~ 1-1N1! IL IL Y Ur U' ' E Z~N~~~da~Q ~Y ~JdOa~~WN.~NZ~_a~ZaNW ~Y~ ~3C+7~NQaY~u.~~
V ~ ~ N d J ~ ~ 3 a c o ~ ~ da' ~ ~ V a ~ (7 O ~ U7 ~ Y U f.~'.1 ~ ~ H g a d ~
a ~ Y ~ Y
Qod;;~~au_~Na~==~p dw~~'a~w~~o~~~rc~~a~YZ~Soz~~>*~wao~~~~>'~a f Y w a D V ~' ~~ EK~u2~~ i~~2+ONODKYYJC7~p~NY~V IYL i ~N1~-ZO~_U~' N(7Z~I~uYNa ~ uJ Y U' Y J J J 4 ~ Y % O O t d N ~ E ~ W Y 1J ~ ~ J J Y N Y- 7 Y N ~ N W Ii O W (7 Z ~ Q Y Z ~ K J C7 Y
O
O
N_ O p N
O (NO
O
C ~ a ~ 7 ..~.
n ~ N Y " Y O ~ M m C C ~ c p d' ~ ~ p ._ a ~u ;~ ~ ~ U' m ~ m d L°cE o 0 0 ° a y tn ~o C7 ' c L
, N L N O tND
CI "' N ~ O
C
R N A
.E m o~
H
c o ~'f ~ v m m o M a T
,L
D_ _u ° to N, ~ sf N
A
r ~ 01 ~ ~ N
A y N ~ N
m w a a ,L o a o UU' UU' CU7 a a a °' E a a a N ~
A
U J J J

N
N tpD
N
C
N M
M
O
O M
Y
A

d y ~ ~ o Y Y Y
N N

Y W ~ 4 Y ~

~zo r_w~3 ' j ~ (7 =
a ~
>

p ni Y ~
a ~ ~

>

N ZJOY W~~~
1u a . O
Z ~ C7 C7 ~ ~ N
f E wo~E~w Z~~w U ;
~

z ~ ~ ~
~ ~ N
o d ~

r OO~ ~_i~>
~ ~

fYC70 aU' N2+ E

M
l N
m u E N
C O

N O
(' O

N
C C
N

~
C
U

d =
~c .~
C

C
~

d~

I
N
L~ O
L in c a I~

U m E

Q U

N

ft _C1~

d M

IL
O M

~
I N
N

~
~
E

L
NI N
~

W M
d m y M
V W
a o a E v o ,L

j U
O

G
a.
d _ _ ~E a ~o N
u -A

A
c 0 d E
a z m C N
O
Y

C~

cj w a "' a_xc7+w~ c~,ioJZa~~Z~Y~vp S~aaa~'g~Z~~
>J

w ~ a ~ ~~s> Yya c~Naouaa- a~yoat7~v~
> a ~wz~c~~~~'Uytt ~ ' ' a a + a u1 J Q a d x N a z Y O O ~ u1 ~ N c~ > a O N Y U Y U ~ ~ - g ~ 4 ~ Y ~ a a ~
~ ~ J J ~ a + U ~ g 111 g ~ ~ =
U 3 ~ d ~ Y W ~ J Y p + ~ N N
~

O d ~ Y y J O p Y Y C7 Y N W ~ + t IL IL III f p N ~
~ + +' + > ~ IL ~ U' p ~ ~ ~ ~ ~
~ ~ > p d + a p N > ~ U ~,' ~ Z N p yy ~- _ * Q a _ ~+ a 2 ~
~ a N ~
~
~N
~

>O ~ O H>Za II (7 a +>N d .~QI'J ~c7~~,n N + a U ~p DNy ~ ~ + J p a N p N a 7 W N
~ ~ 2 N a d ~ <
N

~ [J p C7 p p + J p Z + ~ a W p.
p D J w J + + ~+ dY+++WHaW II ' ~U J J ' a C7 U' ONY a IIIp+a(73a ~ I
J Yda >aQ> E d N ~~
~~

a !7 (7+Y Yd' ~ U ~
p ~+ ++aU' O >p t e + i+ C7p C7 U' /l u> 3~ JU Z O N .7a IL
(7!'J NppUKZ Y ~t/a! U~t70~~~J
a ~NYw ~
JdZN Z
~

J U l _ ~ f J d ~ d ~ Y N ~ ~ 111 ~ ~ H ~ ~
N U' ~ ~ x ~ a~gC7 = H ~ J
Q K + 3 Ia. w w a ~ > =
+ a Y > J 111 Z ~ a ~ ~
~ ~ I ~ 7 O Q
~ J

~ O U' W J i > p + W Y ~ Z
N O J p i i ~ K U a > C7 U' ~
't + H a ~
Z > O a E a ~ + p p (7 Q
W J

tN0 O ~ OMf y N aND N O
c~ 0 01 m o , r ~n m E
m ~ M v c o o 0 o~ o o 0 o o r r N C
C ~ O C N V (0 Y ~~ O C O
~ j ~

U ~ O

d G O C N Y Z N d = C x C
y = N L ~ a j N d N
p ~ N C p Y ~ > m N
~ ~ '~ ~

d N a Y G
N Z

(n d l0 O. R ' ~
X U

i M N r M N
C~ N ~ m In v o M n o a '~
~

C a z z U

~C N ~ ~ ~ ~ o _G1 a ~

O d ~ ~ ~ o ~ M

N N ~ r W
I

~ 1~ M M
W
A

L
10If~ ~ n N 100 m N It) M C7 (7 tp d N N N N
V O O O W lL
a ~ co 0 N

T ~ j U a a n Q Q

Ii ' > j O U' U' UU' Y Y

a a a ~ v t a _ " a a a a a N

A _ j m a A
m ~ m A
c N r W m n d Y

Y
Z o 0 U
d 1 of v d N N N

w_ a x> aaa 3waaYp x~_~~vww_~>Y~ i ~ _+ "' +
J J J ~ 'd. ~ a ~ j ~ ~ ~ ° J Y O N J ~ a ~ U Y t Y a + J + a d' a ~ a a z ~ Q K E o ~ C7 a J ~ W W ~ K ~ Q O C7 J J ~_ Z ~ W Y i Y a +
~ >°aN~ y y~ all°la g3~5,Y"~oaN~ 'S~~>,~~F~~~ao y m x a+ ~ 'S
O J> Q ~ QY ulx3Y N = aaQx ~g~U~Y x° ~~ > p0~ yU + 1jY+
N u1 Z d. d IL Y d p = ~ (7 > ~ >U, d ~ Y (~ 1u u. N U Y a W + Y W U N Ii + +
~ x u. O C7 A ~ N W ~ Q W ~ ~ p U ~ ~ ~ N ~ N ~ ~ J N ° ~ ~ ~ ~ ~ ~ > Q = d U Y J +
x + W ~ N a - ~ ~ ~ f E ~I° a a ~ x > Y N a a ~ ~ O W a W W p V a W Q ~~ Y Y N ~ V ~ C7 p ~ ~
° = + _ ~ ~ ~ J ~ d 2 a U a d 3 c'~ ~~~~a a~ NU'QY~ ~a~»>~H~°~ ~UUxp~xN~>IIIOU,NY pZ,~>W+Y>a iLW~ Y yQ+
> QQ Q C7 ° 3 ~ ~ y 2 C7 Y ~ C7 ° O 4 a C7 ~ W ~ U K ~ ~ u1 ~ N
C7 ~ W K Z a ~ = U' N ~ + a J ) + f t +
d~~'~+a~~iaa~y W a ~'Jaa~U~~ ~~a~Jwgaaa,~,~_+;9~CN+Q~~~(73~0 '~ a ~'o +a~+
N > U Y a' d C7 ~ ~ N Q 41 ~ ~ Z IL N N a > 111 = J p p d J C7 Y J 111 W a 2 U' U + + C7 O ° U
u1 x ~ d d ~ p Q O ~ C7 ~ > U' a J C7 Ya a a Z c9 a J U ~ Ia_ + + H ~ Ii = 1~
p y ~ W U d' Y +
~>a~c~xulc~m~apo ~Ild~zc~c~zayaN ~"d'~ac~ozo~ac~p+mJo+>+=o~>++ ><E~ az z~+Y
o "'' ~ " I~co' o o Imn u1 N ~ ~ 1'~n 1'~n m N N
N ~ ~ OD
d ~ O N O O O O
O N O O O O
C ~ N f0 C ~7 ~ N ~7 p E C N C a ~ ° ~ O
c ~ '° m Ip m c _a ° ~ _a ° c ~a°y m c 8 Y Z U v N m 7 Y o m ~ a1 n1 N ~ n1 ~ ~ ~ c o d + a o + ~ m N ~~ ~ o ~ ~ a1 N o N N o '~ ~ " ~ d c m ~ Y E V I U M U in U N U
U o I
A
L° r ;° o ~ N ano ano I~ n c O N n v E
a C
_ ° p ~ ~ ~ n a m m ° 1~°n v 1~1 ~ M
Ir _ O _u o rn n V ~ V ~ t'~1 N !'m) A
Z
AIC N t~ ~ ~nM ago m y N ~ p ~ n 07 Q ~ N
o ° ° ° °v, °e ~o ~i o.
Y Y Y Y
Ii U U U ~ w ~ w Y Y Y Y Y Y Y
° a a a a a a a v T
~ E
a a a a a a a ~ A
'° m m m c f z j a i ~ ~ m m m a A
° r~ ~ r~-A
O N M ~ N ID
a Y Y
U c~ U
a ~ m ~- N ~ ~ on ~ N p U' C7 V' d C9 C~ V' U' fn (n tn a fn N 47 tn IJ ~ J O ~ ~ j ~ ~ r ~ ~ y -~ z a Y a a ~ ~ f. y o ~ U a N w w C1 J Y d' N r O a o ~ ~ ~ W J iu - j ~ a 0 ~ I
y ~ 2 J L (~ N
U r 111 2~
H ~ J N

J~U~ WQ(>'Jy~ ~- y ~~ ~ ~~~ ~,,7 W (n Q
Z O U
ul~~~ aQ('JHa6-i ~Y Y
N
u~u K I iY- aa~ ~ A wOONON.a~a x~N"aJY /l - HY
> j ~~y~~o Y a ~<oY~a S
Nx~a -" j C ~
O , J j Y m + , NN
N Z Y N D ~ K x U Z m c7 u a a J U, > c7 ~ ~ O ~ + IL _ I ~ Z d Z W t + Y y > ~ ~
Q ~ U' ~ 1u Z ~ ~ (7 ~
f U U W ~ ~ ~ x > N J
~ f IY > ~ ~ U. (7 ~ J d Y O ~
d U w U x Y Y ~ F ~ N ~ g ~
+ N ~ Z > J ~ Y U' J ~ a ~ Y U U (~9 (7 M + U N 1u J a ~

p Ip D Z N a (7 ~ j 4 Z m J Y > ~ u1 a a a a 3 J ~ ~ ~ ~ ~ C7 1u J Y J y C7 U 3_ > m a N iu (7 ~ y = ~ v~ ~ ~ ~ N
U ~ d ~ ~ 3 3 t = N O 4! a (7 ~
O Y Y N W (7 C7 a V

E O Q Y H Y Q O U Q J ~ O N
~ 41 Z N = ~ ~ W y > U d N
~ ~ W ~ ~ r ~ ~ > J Y U ~
~ ~ x ~ ~ Q ' ~ ~
~

+ N ~ a Q N
d Z + Q U
D H Q Z W > ~ + ~ ~ U ~ ~ N
W ~ a N ~ Q ~ f d W D
I> W ~ a R ~ ~ W ~ d > ~

C7 U ~ ~ O ~ + d f7 ~ -t a a ~ ~ ~ 2 Y f ~ + O 9 C7 ~aJ ~ J O O
' ~ > r $ga~~awgo :' a w a+~ ~~a Z ~

am is~~~og~3 E ~ ~
a N + ~ a a x~~wo~ ~ ra< m 9 N
iu > + ~ z O Ja_~ Y
~ z Y ~= 9 : O a Y Y
~ ~ N
a ~

Y a c7 z x N c7 o c ~ a . J a (+7 c _ > ~ _ ~ ~ O ~ C
J u1 ~ Y f K 2 Y C7 K r ~ K H ~ C7 7 > IL Q 41 N U C7 J ~ Y Y (7 H
a a + Y 3 9 Z U' ~ ~ o o v C9 H >
a J C7 a n y ~n O_ ~ a m N

c O N m ni O N O

d ~ O O O O
('N O O O O

N
c ? N E

0 ~ ~ o -o.z c o d C = G C ~ Y V = C ~' ~ 2 C
N ~ ~ ~ d ~ E ~

c Y Y v m o ~ E ~ o m' ~

p a ~ Z N x v ~o ~ Z ~ > r ~ N
~ o r-O N O ~
V n! O
O t0 O O
E

Q Z ~ v U
~

A

cn o 0 0o n m o m m ~ r a d n ~ c m m t a M
H

~, , N ~ W

,ev n n u~
A

L
~0~~ ~ (N0 (gyp N
~

N n n N n (p d O
O O N O O
y N
a m T
Y Y Y Y U

Ii~ W W w Y Y Y Y Y

O
t7U U U U U

Y Y Y Y Y
a a a a a j ~ ~ U

m n wn ~ n m n n n n m n w o~ o N
N

N
mM ~ ~ U

O
a d M o o m m U. o 0 U Y

U

+ ~ Z ~ J H w U Y 4 U ~ ~ O ~, U, y + N= O ~ a U Y
U Y > U
Y
J Q

d ~ ~ Z N Y Z > =~ K y ~ 2 Y
J d'~ '~>1_l(~~YI-Q~.Y K 1- + Y
N = 3oaJ >~+u~. ~ O
O 111 > w + ~C7 ~OZW~~a ~ Z U3J
~ 7 rc C C7 Zlj ~ OJ~ p ~U Jk > ~~WZ >( JJZ Y+
O (9 ~ ~ Y + 4 1a_ C7 u1> O Z O +
U. + + N ~N~UOYY J O
~ T d J >a ~- y ~ _ N N ~
~
~

Z Q , D UJZ NZQ~y~ 5 Y ~ZZN
, ~' N=Z , ~ N
NY~ Ofn(7J~~O~Y C7~111 W t WIR1~U ~~
~ a + ON~ ZIII~~ +
U3 ~uDY ~Wd ~ ~
2 Jau ~ u YWu Y 0 _D +
uJ ~O _ 2 ~ Z
u ~ u p Y~==(1=
Y ('7 Y
N U ' Z ~
~
~

E . ~+ a~aT ~~ >3~Q~U~ZZyJN
uljYY ~OZ~y(7~W QNJDS I Y ~ IL T
f_d U ~ U y +
O U ~yUW a ~ U + Z
J ~ O Z a (7 O U
N a ~ ~ ' g O J g I U IU a N -U U t > f ' ~ Y
+ t C7 C7 d. E
t1 O ~ a ~ 2 a ~ Y ~ Y U Q ~ ~ ~ O
E Y y ~ ~ d a N
~ O Y a a Q
~ Y +

a ~ ~ Y 111 + ~ ~ Y d _ d N U' IL Q + K J ~ ~ K W
. ~ ~ ~ ~ (7 ~ U Y Z ~+ p U K U
- N C7 Q ~ y a Z O ~ + ~ >
~ O 2 K Y J
~ U ~ > =
a d ~ U

+ ~ ~ C7 O (7 U =
U t u U +
U (7 d = 2 > . ~~Y
Z < U d ZY
Y d 1 ZQ ~~
~~~ dQ Z2~~
O ~
K~
Y

t Z+ WO ~ ~U' i ~=WOJ> Ua+
1i(7 f Y>Y O (7 d' U' f ~
F +=d' ~ a -U' D ~ ~ OND m M
-I f0 !''> 1~ N
V n N ONI N N
E ~ N

N a N ( M
O 0 O ~7 O

N o w N
a m E a o m E Q
,Np = N a N 2 u~ m o v1 ~ w Z

r y=d of'm wv ~'=v Z
zE

V C~ 01 O O a" N N~ g o ~ o ~ ~ D N
N ~ W

G o. Q c ~ ~
' U Y U ~ Z
a~

I N
L N
N
- ? f v ~ a ~
Q

z z z A

E m ~ n N
r a d C

d m 1"'n ~ m h ~M a IL
o c to v a ~

I O t0 VI N t0 A c~

~ c N
E
m r I r- 0 0 o, ~ 0 'n M
A
y O
O ~ M ~
a v m m o.
'E Sc o p p a U U U U

U U U

U U U U U

Y Y Y Y Y
a a a a a A
j ~ V

A
m m w m A
N N N N N

m E Y

i .w w o ~ ~ ~ ~ cmn Y Y Y a Y Y

N N N Z ~ U
N

I
d ~ ~ Z ~ U u1 ~ a ~ U > D f > ~ Y C ~ N ~ (7 W ~ 111 N U + J Q ~ 9 y ~ Z ~ N J ~ U x ~ ~ ~ d a ~ ~ N ~ ~ N ~ a ° f/1 O ti ~ ~ ~ Z K N C7 ~ N
O* C7 + >
11 a r ~ Z C7 N d + N ~ N a > a d O ~ ~ u1 _ (7 ~ a > ~ a ~ C7 N a w > C7 d N u1 ~ O + a N x + ~ U H
'wo~~ Jaul~,'Slld° °~ulo~N» i~ao$1~°ua~jrcY a'Na~alig+ *w ~ N ~~>
V7 Y J N r Z ° ~ a ~ d -~ Z ~ > d ~ ~ 4 ~ ~ ° d ~ Z N d ~ ~ ~ ~
d N ~ a ~ N ~ ~ N ~ ~ H Y
~ONU~u J°~ (7 U' U' 1uJ r >O ° ~' ~'r~ N 1~. x111 (7 d N d~u.Y~~aN 11Y.N a d~pl~jN9 agaSYOY U' W~ ~W N JJJFN- ~W~ ~JZN
E IZ ~ ~ Q> j W C7 J Z Z ~ ~ O ~ ~ K ~ a Y a ~ ~ ~ ~ a * 1i ~ U ~ Z N a, O QCll~u~d~~~r > U Ya ~ OW a°(7ND K~4+d° ONa(7NU13da H~vY
U I° J > m E ~J o ~ ~ ~ ~ ~ ~ J ~ a ~ = 9 > ~ ~ ~ ~ ~ a ~ ° ~i ~
~ m a a ~ ~ o a z ~ ,~ a y > w d w 3 ° a ~ z > a u~ ~ ~ 73I- a a a + ~ ~ x ia~~mzolxl.~~°axzN
J~~~~Cx7aJa~°arc°°~adawa~o:OUao'n aar; m $>xs N1~~Y> Ya +~c~o°N u.x~ JU doZ°rrc°d'S~J a a>
1S+mw~~+'S J+
IJ ° ° ~ ° x K N 111 C7 m a' > N N Z = I~ U' ~ ° 3 J W r Z H Y a d N E d O ° t a N (7 N a + U' + Y ~ y~ 7 Y a °
QKa'NIIJQUO~°O=JNa'J>YU1 'Zau1(7u1111°U' ZZxC7~d°+O:OaN~a+W°HO:W +UEaC7 n VI N
D ~ (~ ON7 N f~~1 I
M ~ aND M
m 10 m m O
O ~ O O O
O O O O
C O O E
O 1p O V1 N
o ~ o N E o N v ~ ~ E ~ ~ N y z o ~ ? c nE'oltN ~~ aQm C .- C .- > ~ -d T ~p N tU ~ N ~ O N _t0 E ~ E ~ C N
G = 7 .O Q > O S C N
d a N = Y o ~i LPL NON7 N~ W
C O ~ ~ m I N m M O O
U E o D o m t> a ~ n.
z z N _'m E °o °o °o "'1 n n H
r a °o °o °o w M
I
OI ~ m o1 N
cm.! .°-' a v m m L
I m o1 m _ N ~ N N O
m d n m 1n n m N r M N
tit tit o tit tit r°~ °0 1°n °
(~ m m N
N N
O U
a a N N
U U
m m m .
'o ~ a o a ~ a r_ r t' ~ ~ ~ U O
d~
E a a a a a ~w m j m m m m m m n n m m o N N N fh C7 d D Y
N

z = 3 y 1m0 N 1AN
O O
N N ~ N

+ t~Y a~'~°~a~x~Y ~~ NZ~oo~aN3 °~~a~~~2~Q~3 t~~°Jn~~i~N°g~
Yw g >a ~~>'~~W~a>Y~ ~~ ~w3p ~~~Z?>axN ° o oJ0(71-a a wJ J a c~~c>ui=4z~>~~ ~x ~~~ oN ~NC72z"'w c~Y~ ~cs~c~'~ mz~a~
C t ~ ~ > d 3 + N ~ W INt ~ Z J a Z ~ N ~ U d U N ° ~ a ~ U N U ~ Y ~ 1-~ O W ° Z Z 2' U ~r ~ ~ C7 Y
O I~ 2J~+ J ONYyY_ulOY4~~~ ~QNUW u~~~ (7~gfUJC7UuW' a aYYY>4 JxC7pa 'NC ~~ Z W Y ~P N ~ J ~ Y a a a ~ Y ~ U x a a 2 J n. Q j ~ Y U = ~ N(~ N ~ J ~
O = >U' ° ~ Q Y ° ~ O
U+' ~_ ; + JN-~U Wd~~~9 = YOIIIN ~ 2= UUHY U~W~Y ZZ(7 ~ W Y~~ZNU~=
E a Y ~ a Q ~ ~ ~ a O d Y ~ ~ ~ Y O ~ N > C7 U d ~ N N ~ Z O N ~ N U ~ ~ ~ Y Q
U > J Zp ~ W > ~ Y Y a j H d j + Z ~ Z 4 > - ~ W CJ Y > > x ~ Z ~ ~ C7 u1 u. r Q d O N d U' ~ ~ H °
Z > Q Z - p U Q > U 41 U. Y
U ++~~: ~o>UY°_~ J~ ~~~a~°~~~~wc~~ a~~JU~~~~,~ ~a=~~Nd~3~~
~p~°a~'~a~~
~ ' i ~c ~ N O a T a a a ~ Y ° w ~ x ~ N a ~ > N o ~ o a w N ~ a ~ x Y
z ~ ~ a a ~ ~ a ~ ~ N = ~
> N + ~ + ° > N 3 + ° U U N Z Y ~ > J N O a > U, > K 2 O = Nli (7 j ~ U > ° W ~ ~ U a U ° ~ a f ~ ~ ° d ~ ~ ~ ~ J W Y ~ Y U J V U d C7 ~ a 7 ~ d C7 U U' Z ~ Q ~ ~ ~ ~ ~ ~ ~ ~ N J ~ ~
Y a > W Z ~ la- ~ a N ~ + J + d > + + u. u1 - IL ~ ° U' Y Y a N 2 ~ 3 U' d ~ H ° x Y
IU Z J ~ ~ (7 > Ii O J a a J Z J a ~ H > x ~ U' 2 Y C7 a ° J
(O m ~ O O
u1 ~ n r~ o o~
Wn ,°n ~I ~ o m N O ~ p d O O O N O
(' O O O O
N

C N O C C_ V O O
O ~ J O N .L~ .E .N ~ _O N = N
'G (n U .Q ~ C ~ O L N Q_ N
d T E ~ H ~N .L.. j T ~ ~In ~ ~N
o g o ~ '; : x a °.

~, of of L~ L O~1 ~ m ~ N
C Y N (h m OD
I~ ~ O
V E O ~ o m °
a ~ ~ z v C
E ~ °o °o °o °o ~ r c ° o a, 0 0 ,f, o m o 0 r .~ d. ,L
N -°, N ~ N ~ M O
A
L
~I ~ N ~ 07 '_n n0 ~ M h ~ t00 O
N O m ~ ~
N N
a w w w tii o a °o ~°y ° o0 N t0 IA
T
Y U
,y ~O O O Z Z
a p L_ L_ L_ G_ L_ (9 0 0 O O O
~E a a a a a N w A
J J J J

N

a0 07 n7 M M ~ M M
m Y
c ~ ~ m o d N
C7 U' ~ Y Y
U N N

y ~+m a= ",ca >=a YY o c~ (~ z m d + L7 fn a ~ Z U N ~ Y ~ =Y 2 ~ C7 K N N Y > a ~ C7 N ~ ~ D 9 =
+~'; > a ~~~gxJ~~'~p~ C~'!°--3-~ UpYw -a,a-~x~~S~~~~w ~+', >
c a Y= ~ a ~>>o YzaJOa ZY ~ ~ Nr a =~>o~Jx> rc acsao +~ O~~~aO~-x N +>~ J ~+ ~ ~ Jaa os'~~oo~"~ ~a~ Y ox ~oc~~aa~~~~~ ~+f x U Y D Y 3 J a > ~ O 2 d' N > = x N ~ ~ + ~ IL J ~ 0 ~ d O Y ~ K C7 N W Z y ~
1u +
U + + t a ~ (7 J ~ a Z fn y Y u1 > IL ~ a d' N + ~ a a + ~ U I ~ ~ H Z ~ N >
c.,~~= a 'J'a+ + + a~n'Y'w~ax~oa ~ ~N ~~_ a J~mw~~>Z=~~ S~,'u E c~ + + ~ ~~+ 3ua~~~a~-~~ ~ a~ x aNa c~~ ~ d a Q' + y J U K > x ~ J M O ~ Z M U' 2 ~ aY H O H x ~ N 111 + .J
V C7 J~ U+w W d~+ fU, (Z7~Y~~OJUa~Y ;>Y1=~YJO~~UJ'Uid UZ~~F~~~ J~NZJ2 aNJ(+7 N Z + O U O a 1L + U ~ ~ a x Y > ~ ~ ~ ~ J ~ x YO ~ ~ ~ O N a a a (1 l~ u1 Z N
Z + x C7~>Da~ ~+a~ a~~ ~NUJ~~Q ~ O ~aU Jd' ~~~ +O~O(/!~~>F1 ~Ja +++i m0li+~°z >+~ ~c7 ua3al~~.~N-UO a+oo~z~~>~Y +lyu~ ~~~111~ °'> +
1~1u Y+c~ln>u1++alnfl-3J1u3aoc~Yxa~aam>u1a+~ac~~~ x~Jl~n~»J~~°zao ~=+1°n o ~ m 1mn ~ ~ m v N f~
o m p o m °1n° ~ 1°°n C ~ ~ ~ N O
O
d O O O ~ O O
O O ~ O O
O N N - C
o '~ m v ~ 1n ~ o ~ ~ ", ~ a E ~o E ~ u' 'u c ~= a~' v=c nQld.o~ymvzc a2c coc V d N V C t~ d N p y V C 01 N N O Gl ~ Y ~ t0 V Y N 1% -~ N C ~ Y ' (p UI ~° L G a - 41 U ' a c a '~
~ E o E ~ ~E ~ ~ N ~ 'p E o ~m v u~°, ~ a ~1 c Qo ual~ EE~ ca ~o.
3 v ~m a I
_.
L° L ~ 'o° o ~ cNO N
m ~I io ~ ~ 1n ~ o Q E ~ a a a a a~ am z z z z A
° 1n ° o°! ~ °o °
T a .0 R ~ .'~~ coo H ,t~, M a>' r ~~ ~t I L
O W o ~ ~ o v m ~°.~ pn m L
(~D N r N d O ~ ~ M ~ N V
p ~ ~ O!
N N ~ N Q
o tit o w 1n w 1u a °' 1°u °v N ° o0 O 00 f0 O N M
'' Y ~ a ~ ~ > >
Ii a ~ ~ ~ N .c .c <n >
a O =_ L_ t_ L_ L_ L_ L_ ~y O 0 O O 0 O 0 r T
y ~E a a a a a a a ~w m m J m ~ ~ ~ ~ a A
m ~ m o m c r~ w w o r1 c~ M v ~ v a Q1 ~ N
E
A
z m _ d v N o cs1 c m rn Y Y y Y ~ o Y Y Y Y
N N N !~/7 H N N N

~~a~ ~!~9 p~ ~ x ~Jy w~2Ni~w~cy,d',!~~
(Z7 Y' ~ -.~~
f ~

~ p (71- YpO~Q YW ~ ~ ~D~ NWpZU?~7JwwC
W> ~ J
W
x(7~(U7K
J~ ~

C U' ~ ~ ~ ~ ~ - ~ a N 2 U' ~ > u)1 J Z s Y Z x ~ ~
tJ'J ~ ~ ~ N Z O a ~ ~ U N

p ~ N
N U J Y Z ~ O x U ~ ~ C7 Q p K C7 ~ + U' K U ~ Y ~
J + W = Z ~ I w J N ~ O d' O 1y ~ N
Z Y Y Y ~ 1u g ~
(7 J

a d d O U U C7 ~ Y O _ > y w J + f s ~ N - > K ~ 4 N ~ J ~ N t ~ Q O + U. N W ~ p a > ~ U' d ~ J W u1 ~ ~ Y J ) u1 ~ Z N IIY! Y a Y IL
~ U -' 3 W
~ ~ ~
~

E a U, JQ: ~111 ~
N)U W> Y N. 0 N ~N=~
O11l~ N 1-~~ ~ N U ~
7 ~ N U - J ~ J ~
w d' N IL Z Tp J a K a ~ ( 3 111 E ~
~

Q + y N
~J~NU Y~~~ >Q.V Q U~4DN~111 7 u1 C7 +U+' 0 ~NZ~d ~pOUW>Y W
~ ZIL ~
U l x J ", ~ z a + Z Z > ~ s H N w 5 : Z E H
p E U p ~ a 1u ~ ~ ~ v ~ + N + ~ ~ ~ I
+ u1 * u~ >- ~
~ 1u ~x~>ua >oa~a Ya J= omiN ~>aiw~OYN~eoz~aO~adgoa~aai ' a x ~
~
~

~ x~luc~ osa>~"xY rczma NUN
+ ~~~~a w aJ ~
px cs~~a ~' ~
~

Eap) ~>x~Y~aa~+f~lu~>+a ~xzaal E~i lax ux~l c u~vl= ~rc v laa D_ n ~ ~
I ~ ~ m V N N N

C
d O O O O
f~ n o ~ o N v 0 C N N
. 7 O l0 C

O N N > c c O C

- Y C p ~- V ~ N ~ OI'Y N
' v Y~ E~m N_do ' ~ c ~ N ~ ~ ' o o d Z ?n Z'E~2 g via' a AI
N (~j O
a m O
O

Q ~ m C
z m m r ~

o>

M a Ir o o to r1 v ~

f a r v v m t I V O N (O
~ V ~ ~ 0 N
d N ~ N N
M

2 In o r ci m U Y Y
Y

Z Z Z

a O L H H f~-' V, p tn f~ N

v _7.

m . a a a a <n m U J J J J

N
O O O

A
C V V
t d Z

A Y
z a d d n M 1 m Y

N N N

''tats j°~~a c_src J °_z~ ~~N ~ O ++
O ~ N ~ ~ _ ~ ~ ~ ~ > Y a J p j J ° (~ 4 N
w a m > U m SJzo g~~~ ~~°~ ~ oa aN p°n~ ~ ~+>cs~ J
Q !- Y ~ N (7 C7 Q Z ~ y ~ W ~ Y 3 ~ Z ~ + Y ~ ~ ~ ~ C7 Y C1 (7 y a + ~ w J W
N Z O C7 a' w ~ OJ f/1 41 ~ = a 2 Y J ° K N J ~ 7 m O 2 u1 K c~ Y z w ° c~ a A J -i = ~_ C7 N ~ ~ ~ Z ~ ° (O ° U~ ~ Y H C7 J 7 ° _ ~ ~ ~ W Y ~ ~ ~ ° u1 °< ~oZ~~a>~OJ N>~i ~m° + ma~dZZ vo"p ~ lu+~~~'Za ~ °J
E d'a +~4JK NU' F- C7gJ Y YOY~Y_Z ~Z1- ~ ~°C7+JZ- U' ° ~ u! Q + ~ Z 1- ~ U u! a C d ~ Z S a_ ~ a 7 -~ Z (r9 d a ~ ° ~ a g ~ ~ ~ ~ ~ ~ ~ r U t ~ Z ~
a ° J a ~ U' Y U W ~ d O J p 4 ~ J IIN1 ~ ~ Z d N ~ r + ~ ~ ~~ N a O (7 9 J u1 d Y ~ >
a ~ Z
Y Q u1 f Y 2 Y ~ (7 C' ° IL O Y ° u1 > ~ d ~~ C7 ~ (7 u1 Z U Q Y J ~ ~ ~ Z < Z H (7 J O Q U ° ~ J E J (7 ~
° Y Y ~ > + U ~ O H
~NQ~IIJI(Z72~NY~Y~mIL~O~' ~~2U J a~~YQJJNa~(~/1a3 a+H~_~~ ~N~+m~
H IJ Y m Q J O IJ J J IL f 3 ~ U Y ~ ~ d III Q ~ ° d Q Y + > IL III C7 J U' ~ F- + + 111 + (7 Q = > K ~ ~ J
D_ tn0 a 001 N
o n ~ ~ m '? n m N
E

o °o o °0 0 n n m o o E ~o E a a~ E o Otl1 a=NNN ON =V!
V 2 d N U N D C N N Q N
d w ° mg~~~
c ~ j a ~ E a °.
m~
r L ~ ~ a~D ~ N
C~ r 10 ~ ~ O7 m M M_ N
M O
Q ~ m ~ O z E
m rn ~ m °m N
~' C
m m Inn m cno M
L
m n m m n ~I N M m M N
,~ E
m~ ~ ~ ~ m m o N ~ t~ N N
m m N N m N ~ N
m d ° o° °o I°n~ o ci of ~ n v T O O
~ N N
IL fn fn fn fn (n a N
c7 ar_ E a a a a a ~w N N
N W U
N
O O O O O
m m O N
0 ~ In In V7 N
E
A
z C O N O tN~7 m N N

o y a a + w rc > x m a > = J
+ 3 ~
~
~

+ z v~ a Y ~ d r + ~ v > + N > a " a o + a ~
m Yo5"' ++ ~ ; a ~
"~~o~'a~~iia ow c~~rc~ >+r~+ ~o~e '~~ ~ >3 wo v 'ors = au ~_~
ac~wg>++ ~ ocs ~wr~' +

Z W ~ ~ ~ (7 ~ O ~ ~ U' +a o > Y ~ H N 3 ~
Z ~ Y = N N
~ V > ~ O

c N J + a ~n N c7 Y ~
U + + ~ ~ ~ ~ ~ w ~
(7 ~ N a J N ~ N
u a w d a c~ w + Y ~ 2 < ~

o ~ ++a 'noYZ p+
$ ~' o=o ~~~ ~ =
~> ~~

c~ J O~ w U ~a i~ , ~nio'ua3o ~j "
a "'~~~?~= ~m~Qau ~~'?wg~~>+w ~ 'na; a~oa~wo~YN

~ , a 3 3 japo Z ' N ' ~
H ~~WnYa a ~ ~ ++3 NK rcwg J a+ Z c?, ii ~ Wua ~ n~ Kw ~ cs++~++++rcQ v w ~
U ~ c70F'u~wwav~uzw wa3oc7Y'~Lara ~'Sa+xc~+~n+U

I~ C7 N m C m m ~ M
-I07 N N 10 1~
O n m E ~ N

N I N M
O f1 O O O
O O O O O
O

Q j f/1 N N ~ 9 ~
C

O ~~ ~t/1 ~~0Ø V" Qm~01 - g 7 i0 U U UI V ~ d p ~ N VO
I

a _ ~ O h ~U . ~ O ~
~' V G O N N ~ d ~ O
N
o a ~ E Q ~ m x ~ ~
' c d ~ m" ~
x w v m o _ m U' ~ Z
o.
a I N
N r V m 00 m C o U

~ V f~0 N a p a o ~

a z N A

_ d7 _ M ~ N

M

IL

Y m ~ m cp I N 1~ O ~ N
~0 "
E

I o o coo ~ m m o m m v A
a d 01 N (~O O
O O N
d N N o07.
V

O C
E Y > ' U U
~ cU7 a c jy V U
c~ c~

Y Y
d - Y Y ._ ._ ._ d d y Y Y

um a a a m m j uJ. ~ na. U m m npO N t'7 A
C N N N

Q
t Z IL
N

C m ~ ~ O N
N
Y Y Y Y Y

U' C7 C7 C7 C7 (n N (n N

Table 5 describes domains in the proteins outside of the kinase catalytic domain. The column headings are: "Gene Name", "ID#na", "ID#aa", "Extracatalytic Domains (AA
boundaries)". Extracatalytic domains were identified by performing hidden Markov searches of the amino acid sequences using Pfam, a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains. Version 5.1 of Pfam (Sept 2000) contains alignments and models for 2015 protein families (http://pfam.wustl.edu/faq.shtml). The PFAM alignments were downloaded from http://pfam.wustl.edu/hmmsearch.shtml and the HMMr searches were run locally on a Timelogic computer (TimeLogic Corporation, Incline Village, NV). The PFAM accession number, the length in amino acids and the number of proteins used to build the profile are listed below.
The CNH domain (Pfam PF00780) is approximately 300 amino acids long. It is built from 23 members and found in NIK1-like kinase, mouse citron and yeast ROM1 and ROM2.
The PKC terminal domain (PF00433) is approximately 66 amino acids long. It is built from 235 members and found in protein kinase C from multiple species. The phorbol esters/diacylglycerol-binding domain (C1 domain) (PF00130) is approximately 50 amino acids long. It is built from 269 members and in found in protein kinase C from multiple species. The RGS regulator of G protein signaling domain (PF00615) is approximately 125 amino acids long.
It is built from 103 members and found in RGS (Regulator of G protein Signaling) family members that include the GTPase-activating proteins for heterotrimeric G-protein alpha-subunits The PDZ domain (PF00595) is approximately 83 amino acids long. It is built from 721 members and found in membrane-associated proteins that include homologues of the MAGUK
family of guanylate kinases, several protein phosphatases and protein kinases. PDZ
domains are also found in neuronal nitric oxide synthase as well as in the subfamily of dystrophin-associated proteins, collectively known as syntrophins. The Octicosapeptide domain (PF00564) is approximately 30 amino acids long. It is built from 47 members that include NADPH oxidase subunits, sorting nexins and PtdIns 3-kinases. This motif may be involved in Ca++ binding. The cyclin domain (PF00134) is approximately 267 amino acids long. It is built from 233 members that include cyclins, TFIIB and RB/p 107. The RNA recognition domain (also known as RRM, RBD, or RNP) (PF00076) is approximately 71 amino acids long. It is built from 1335 members that include a variety of RNA-binding proteins such hnRNP proteins, proteins implicated in regulation of alternative splicing, and protein components. The motif is also found in a few single-stranded DNA-binding proteins. The myosin head domain (PF00063) is approximately 409 amino acids long. It is built from 310 members that include the motor proteins such as myosin. The ankyrin domain (PF00023) is approximately 33 amino acids long. It is built from 2220 members that include the ankyrin family of structural proteins, CDK
inhibitors such as pl9INK4d, and other signaling proteins such as the nuclear factor NF-kappa-b p50 subunit and Bcl3 (b-cell lymphoma 3-encoded protein) among others. The ankyrin repeats generally consist of a beta, alpha, alpha, beta order of secondary structures. The repeats associate to form a higher order structure. The ephrin Receptor ligand binding domain (PF01404) is approximately 171 amino acids long. It is built from 52 members that include the Eph family of receptor tyrosine kinases. The fibronectin type III domain (PF00041) is approximately 85 amino acids long. It is built from 2468 members that include a variety of transmembrane and membrane-associated proteins that include fibronectin, cytokine receptors, receptor tyrosine kinases, receptor tyrosine 1 S phosphatases, etc. The SAM domain (Sterile alpha motif) (PF00536) is approximately 110 amino acids long. It is built from 64 members. The SAM domain is an evolutionarily conserved protein-binding region that is involved in the regulation of numerous developmental processes in diverse eukaryotes. The SAM domain can potentially function as a protein interaction module through its ability to homo- and hetero oligomerise with other SAM domains.
The DAG
Diacylglycerol (DAG) domain (PF00609 ) is approximately 166 amino acids long.
It is built from 27 members from the diacylglycerol kinase subfamily of protein kinases.
This domain is assumed to be an accessory domain in diacylglycerol binding. The ROI1 domain (PFOl 163) is approximately 570 amino acids in length and is known generally in the art (see, e.g.
www.pfam.wustl.edu). It is built from 14 members and is believed to encode an atypical A-Kinase.

n N ~ c0 O O O

O O
O ~ ~D

V O
a0 N N

N O O
O O O

O O O
O

_ _ ~I ml n1 M M

I I I
D_ O_ D_ I I I
a a a w w w ~ ~I ~
I

I _ v M mn M m m o~ <o r v N u wn M v M M

N

d U

Y

N

~

J

E

N v C +Y

(O M O M n I m I N m O
-' " f Q ~I' o ' o con o~ o co I I

~EN ~ ~ ~ V M ~ N ao o a~ m .- n o u~
N ~ ~ N O O N

d N O ~' O O O O O O

~ O ~ n O O O O O
n n n r o~ ..

N

r o~ ~ p ~
o ' v ~
0 n M M
1 C C m ~M N p MO
E E

c ~
D
O

., ~ ~
A

U U

O ~ c a 'ca v u c o ~
U ~

a = ~ v ~ E 'o o 'm .o c .

v o o ~ o o v v yn ~ E o n N ~ - M
V
O

O d ~ ~ 'C ~
o .. ~

_ ~ a ~ N E d m ~
c m m m ~

C ~ M C ~ Oi N
~

E o ~ 'm ~ % c 'o c ~
c v o N ~ ~ _ c ~ o a ~? ~ .
O a 'Q

D ~ ' d T O

N O N O r ~
O O (O

C N N U d O N
~'N ~ ~ ~
~ N
' ( c0 ~ C
10N 4 >. Q M N
U O ' E C
C -T U

V ~ . ~ v Ol (0 U

C~ CUB Q' m QN
~N ' ~

cp N N

-K Z o L N o o ~
o W
a d ~ o ~ c c o N ~ T
O ~ ~ Y
cO

-a a a a A
o ~ ~ ~ ~ ~ ~ ~ m m A

N M 1f7 M M M M M

M

Y n M
' O a Y

E ~ 2 O c Y N
U 7 ~

m C ~ Z

Y

O O pj ~ O N ~ O
d Y Y N ~ o y o ~ Y

(9 N

N
r O
N

d U

cw ~ + ~ ~ + ~ C~ d N +
+ U

+ ~ wg>~
~

.a a +
~
~
~

~+~ ~ ~aw ad~w~
Y~
+

' ~+x ~ OZ+aU' Y~~+~~utl+
O~ +~U+

VU ~

C ~ w a a ~+
o ~
f~
O
U' ~+
Y

Y
d D
~
~
+
+~
d +~
+

O

_ n N

O O

O O

i~

V

L M ._ ._ ._ m N C

W(7 fO ~ N v O
'm ~M vE

C C O~~! ~ C ~ C

o E M ~ 'm x E a 0 o o c E v "

(0 f0 U

t C C ~

N . 0 0 ~ N

O v N ~
C C ~ N oLf (0 0 m.E v) c N m O C Y
O
.

a h Y
f0 O v O
U

N O ~ C U
d N ~

C ~' ~ p~ ~ >.

d N
~

V _d c p C~ p O

a p m ID
O

O L

E a o z u' m Y

m Y
C~

Table 6 shows the results of a gene expression analysis of the kinases presented in this application using a microarray of cDNAs derived from 469 tissues and cell lines. The cDNAs were spotted on nylon and probed with labeled kinase genes, as described in Materials and S Methods below. The kinase probes were PCR cloned from genomic exons. Data presentation from left to right is as follows: "Tissue": tissue type of the cDNA; "Tumor sym", indicates that the tissue is derived from a tumor, "sym" refers to the fact that the 5' and 3' primers used to make the sample are the same; "Normal Sym", indicates normal tissue was used to make the sample, with symmetric primers as described above; "Tumor 10", indicates that primary tumor tissue was used to make the cDNA; "Tumor cells", indicates that these cDNA
samples were made from cultured tumor cells; "Normal", indicates that these samples are derived from normal tissue or cell lines; "Endos", indicates that these samples are derived from endothelium-related tissue sources; "p53" refers to the status, mutant or wild-type, of the p53 gene in the source samples. Normalized expression values are presented for each gene referred to by its SEQ-ID#
on the subsequent columns. Genes represented in Table 6 are: SGK187 (SEQ ID
NO: 1);
SGK124 (SEQ ID NO: 9); SGK386 (SEQ ID NO: 21); SGK003 (SEQ ID NO: 22); SGK093 (SEQ ID NO: 31); SGK074 (SEQ ID NO: 32); and SGK396 (SEQ ID NO: 43).

r M u7(O~~O OD~N ~r(Dr r~~O O rO O MO OfmoN V0 O WOOM ON~ OON
f O a001f0r M N MM N N rc0 aO N 01 a0 Y ~~t0t0 r r r a 0~
(D r r NWMM OW OG7C7 f~~f0st~~( O rW f7 V r~~tO 0W e0 (O a0a0NtDO(J
O NNN t0 O a !

I N ~ ~M~ fV-Q
Y

~

fn D

p O a0NN MoO rtDtDaD n001~M r rN OaDr a0~DN ~D~r ~Na0t0 N 10~~ ~ ~NO ~OM ~OInN ~~~0O c0~r M f0M WOra0!r M O07mi0rrm O~Or N o N1~ t0a0o M ~~ N~~O a0O NOf~N M(DO ~OVa0 ~ N W
V fD
~

MI ~ OO7rN Nra0c0OO1~ U7O0M ~O~r ~ a0W ~ a~N Ot010N 1~a0a0r'~~ N ~ a0OM
ap MNr ct1~r f0~rV a0W~ NVM O NV M ~M 0Dr~N Q!M O NN~r ~O ~OMM
a0N ~ N M a0 M

Q N a0rc0aD N N(DOr Mor ~

Y
Z

it H
D

O O~N~ OOO OO O c0O~O OMOO tDNO N NM 01~OKIM(ON ~OOON ONO Oa0 r u7 O m N~aD

rn0Q~ O N ~ f7(O r ~ N ~M f0W O ~Oa0p 10 O ~ tOc0rr M- N O m ~ r M ~M M
~

C9 r M f0 M

Y
Z

it N
D ~

(OOMM O n17O~ ~ONOd0O NOMM ~ a0~OV ra0OOOO OOf~OO47OO rN OMf0O
O r oO O O~ M 07a0 ~ O 07aDa0 N
M O W
(O

N M ~ NVcMD NO ~OW V ~ f0~ 4707f0r M~ ~ O f0 N U7M(OM N~
N n ~

NI N N~ (V M '-fV

O OOOO OOO OoOO OOOO OOOO O OO O OO OOOO OO O OOOO OO~ OOOO
O O O

t0 N

Y
I

C~
m N
D

0 0000 000 00~no ~oeo~ o ~oo M a~<or 0o roov oo N o0oM a>N~noo~o 0 r r(O O r o M O o O ~ON ~NN
n0 10 c ~10 rN ~ ~a0 10 O1 ~ N ~
M

N M (D
Q

Y
~

~
D

N MOOV fDOO OOMO Ma0OO f0OOO (DOO N OO ONOO OO OOOO OV'M ~f)ap~m O M a0O f7 O~ 0 t h O ~!7~ N~ NM M N a00 y ~ ~ ~( ~ r 0 I

m Y

~
'w9 ,~
~ ~ D

N

M a a c w m z ..
O
N

V

H

O

r H

O
N

Z

a.
O (Ot0ro7r a~01O ONMW Ot0ra007O~N M ~(7(Or oO NNMV 1nf0a0~OMN M~~l7(Dra00 EN N d.~~p ooO OOO OOOO O~ ~ a~~N NNN Nr N l M MMM MMMM
NN

H

O

~r_ N
C1 ~#

7 N#

N
N

NOM
'M

NU~ g U
~~ U ~ ~

p o >or7 O ~ 00 NMt0 N O N Q N cI11y f7~ MN
N N ~ ~ 7C ' 'N
V

rd~ ~ j j ~ c~ a0 N O1 Q VaD ~ N N 2 G) '7 V YmaO >~~ UOYUZ UZUN U~iotiO >U > >O ~Zav ~m ~ CSUU~ UY~ OO~U
~ ~ ~

H str2 Ot1!~ ZNZcnZ!nZO Z!n~~ 2 OU O O~ Icn~Q v)U ~ n.QZr ZI-tn~U~2 In Y D

a~ON0747M 01 h~Oh hO~O ONaDsf~h N ~OO~M NNON hONaDMaCMe0MMO stO
N~N MMIWO O 1nONNO ~eDa0 01mO~ h MM O~~O~a0(Da0~~ MhO~O haD N cD

O f0 a0 ~ NO h ~t0 N ~Nt0 V~~a0t0Ot0a0O OO N Oa0Nha0N hNNV O~h '- N
~

p ~ N ~<OM VN 07hepN ~ N~ ~ 47(ONN C~ f~
I

Q
M

Y

~

(n O

M a0OWM (pOM h ~-a0h ~Nc0a0a0MVN Nh~O1hO~MN O e0O~cDNN~ ha0aOO~ha0 M (D1At1700~ a0O tDfDN~ h Om N NWO ~ If!~ M W(7 ~V t h 0~a0O~(D (Da0 N ~ ~(D~JN ~VNO Nu'7NtDM~NN ch~OM Oa0c0(O~NVh OO~h ~aGO~ M~~07OIAV ON

MO N (ONVt0hIA r aONo0(DhOOC'7f~t0~N NaDo7(Va0Mhh ~OM(V(DOi O1(7: cDV'O
I N NN~ OM~ 07Nh~ MIA N Nc0OO t0ON~ O1~N07W'COr Mw(1N hMV0Dh~O N'-N
N cf Q ~ ~

OOO ~OOO ~OhO ONMO I(7 ~O a0VNO a0O O NOIn O OO OOOO OOO NO
a0 O a0OM MNo0 hOt0 u7 N t ON M47 s M M ~ O V MN~ ~Na0 N~a0M O (') 0 NN oD~ t0 i ~ V

O ~ ~ a0 p N
Q

M ONON MWhh NhOt0 OMN hOf0fOOOa0N O NW hIAO(Da0 M ~ MV Na0f0MOO

N N t0 t0ha0tDa~h h ~ ON O7 ON NN N~Oa0MOt0M MMf0WhOO O~h ~ O
M ~ a0 M ~a0 a0O1 W7 ~ ~In~ N~ sfa0ON fDa0(DM apW~1~stO~of1y-OV1~U7~ ap h N

O ~ e'N ON r~ O~~t0~ (V~ MNN~ N~ VN N N N N
I

G

O OOOO OOOO OOOO OOOO OOOO OOOO OOOO OOOO OOOO OOOO OOO OOO
r i0 N

~
I

MQ

N

01hOO ~ NO OOf0O MM07e0MOOO hM~O OO01O t0ONcOt0~ OOOM O O NON
p NO N hMa0 N a0ONtnO~ OO a~hO 01fD f0 NN IfJVa007M ~ iph O~
N

I N~ ~M r OOh M~ NO N fD ~N ~~~ N M 1n~
r a Y
z ~

N
D

O Of0u7O OhO(OOOO O01~O OOOO OMmO OOc0O OIDhO OOOa0ONNM OO(DOOO
N M N (O W M Otn tD Mh hh h ~~ f0 N h ~N O M M h fhV Mh M
~I

fn ~ Q ~(f ~N

j~ Y
z d r N
D

N

M a d M

c W

~o z L
O
N

V

H

L

r ~N MVM(Dha0O~O ~NMV N(OhaDWO N MVu7h e007O NM
~NNN NNNN NNM MMM

O
N

Z

L
O O ~NM~ OhON MNhQ1 M
~ V ~V~ ~~V ~N~N DO

O

H

d N

L
N L

L . L L
L

~ CL 3 LCL ~ N L

~ M ~ L L S 'O
N tn N N ~ L Lt ~ ~ L

N a CO O7p m~' N jOIC C p~OE y L L L ~ L L
N ~ ~ ~ ~ ~

ILNW W f~a<L~2Z f0C E LN N~~' D~>~'C~,0 C L LU~u!U N
U D E aC Y~ D

~M~ LLLLhaa a CN N C(~$a~ .._(6 ~~ df0~U)~l9N7 tJJ tLNUN
UUv ~n d E. m~ ~ ._> m cc d mE-E Q H L 2 w Y ~YOY QQ_U U~nOD D~j~E omm~ ~~~ ~o~- :yyE ~'adQ coin>.
~~ '~ Z ~

(nYIn~~ ~7~~ ~2~~ ~I-m2~aEaa cawa ~dwH wV)LN Y41~(n~N~~ ~ ...2 E

Q
z -r ~~ ::. f y~ ;,. ,,,. ,w~;~~: -;~ A'-': °: t~,=h:~:K-'-z~.~ -_.,»;, : ".<.x.r:; ,.~ Y":""«~'~.:~' ~,"~';' ~ .=ys"'~"".,' .»$' r.,',~~'~s " ..>~"'~o<,' - ~ ;, ",~",~~'~,~';'r.;::,'',;'a~~; ;z;."»., "",... ,.:~~ ~W_~_.T;,~~»,'.a;"
_... ' o ~.',ru-%H'"' ~-.~z~"' ~ ", .>W..., ." ,~, , '~", V'° _ ~rrs'~ n.=n:~_ ., ~ , »,»s ~ym~ > ~a "':;; >-~ ~; ~-p ~ _ ; ~ ;;
I~~a~~ ~: '"i"% s ~_~"n."»" . m ~.~_~~,--.~.
'~ >~,.. _-' . ":~:' ~.;,, ", ,;&;
_»,.~-. >:
~:"~r.
,.",.j H . ",'~i , F:,'.
"~ u~ ~" : rN-~".>.
'~-,'~".",.~" "y,T":,~,~" i ",.
,_~:" r7,~
H ~ ~.~~,', , '~,:;e;,rrr" ; H~'~~" z , ~,x ra~, ,r >',,''d,':~ .-""
x r"
." M~ ~ ~ " S.
',' " ~'~ yy,, .... 4 , w .»
a M,.,~, (Gam"
;ywy~..,'fe,':=, k,E"~9,":.~~~~.'rSx,R~.,',~~~.~'~r~ .....,ri?~~~i",." >P;i' ~,~1 Y,,, '~ ' u,',°i -,..,...;n~:~~,:~a'« '~ »y ~~ P ..
-c, a'~~~,°,~. y ; ,,.. nfi ", r;:.'r~" _: .<~ :z ' ~._:z,:.-x;w;,,~,.x:," ,'",%' .'~'~,2';'".,s;;,.,~,.;ri d ~,s. :~,'.'. , ;;'~~~~' V ", N a ra',~.",~~~~ ", ~f~ r,5~ "~ ";s: ~ ~, E>,.;,.» ,.'.~, n s. ~;'-,~~ >,'= , ~",.~ °.
~.~~,;.~: .-- ~,a :==n°'~~w-e,:,aE~;W. n::~'~" ~V ;,s, _; MT,, "s,~,~
°<~;~° ~ ~~<''-'r-;-~~,~~~::' "
d "__ ,~ <, »~~;,.~
~,; ;l ~, ,'_ s,..»~
~» ~ . ; "n ~,~.,.
a"Y'- ; r~
<'~~ gym: '-;~"; ..
GI F'?, r;~ " , ~; , M, Q
t;-'i:'"
,n x! "
';,,~ , " ~~, . ..
°''~;,azi; ". ;rc~ <a~~, ~y .:.:;~ s ~,. '_s ,~~' ~.,. , ~.~'~~''.":~..;,~' .a' O ",z, , ~-Wi,T~.f~,~;Y,.., °.,c:;?;"' a..~~'°'Y.',='u,:a,., 'rn _ L ~',~ ~. Z '" yA~F'.~ ' '"'. "'~5 ':,rv,~ ~t a rF'"'S;,i ~ ,s;,M 01 3 z~
"."s". .sue :~",~,i,v ; 'j, _ .~,~...... "~, ~i-r,-~";
, ...; t«,-,., _, iiN<s =.'-" , ~"ty'.'~":',~~i?r .;~"s~ra~,.y;
~Y 2, ,j;;,, >, -'z ~"r'~ ' ~ x .. ;,~...y H :_~., ,.» ' ,. ~f":' n::'"i<:z,~,'r. T~,F~,,,,a',~ ~!', ~ ». x.»
O »;:-,.>."."".~"'...".» " ~ ii" ,:a;;s.,s ~, w > ~ ~ :, a F urr, f ' ,~4 ~ ?ran yz"..~~~~z ~~
M;.~r,z'F;;;r ,:,.~n~ ~ ~ w.~ " "c:.: ~.':
~~3 d ~ r »~..x ,, ,~~~, .,; '~. , k?' ~"
~'."r,,'-'''f', , a' ~t'r _a'a'. ' 'ii n girl S.ly" ~~"~~ ,','r',, a ~,fs' .. ~~ ~ , ",~, ~
"aF.~m;;f.
:..».:~~: ;,~,~~,:ir:" .> a ,~' :x"""",.,..., t.»»... ,~"~" ,,~.',~. s ~''a"»,ra:~r.»~,.. ~,.a, »
M"::.>s~
P.,;-x;~.t.~. ~., » ..";,~;;a,r"~'' ,s'~r>, ~~'~~'.,,z:''~;-;;s ",~.,.,.~>"..~~~~:~. vif '~»;-;~r;M;,,;>a".,""." "";~iM~r"
;.. ~,- ,I. r" r ,~":" F»,F.~~x~~ " !;:,,x,, ~ ~s "~ w :, 3.~~"- "
L O ~'~",~ ft ~~ ' 3 < ~.'F' ' '~i P 3', 5 '....
Q ..>".:~' -f~'"~,»:»~<°x~ .,v,";'~:"tf.,~:..K.u.~.-°<~k, ~
~~-;zs,.,o~
>.s »
»->"~ar". ~ ",~.
:,' ~a , ,~~I",", t,~~.~
»:;~..»»,», a;;:<:,.i~:;:-,_, r x~:Y.
"; =~ ",.
,."~>,..."r, " ,.~: ~.. ", : ' ~;."....,s. ,." ,ssa~>"."?" s ~.".~' M r _. .a~, ,a ~;" ". ; v "mss ,.,," s,F~
c;,"r~ ' ' ,.a, r "~.r»,..~xt'~,','=" ~'7~ a "' :: k~ ,,,~, / '~T:_'~<~"
' »;';,. _ "~ ~,».,~, ~, avv?;" °.:
-~.
t0 . .,a_.
(/7 »,-" r ~».x_ gt~~e . ' . .
--:r u~: "".=
:~':f 1.-.x'i Q "-. ". ~'~~'.2',,r,., J,,, ~.., 'i M
O a~~~
x ~', ~~ , '',',,:.,t ., ';"I~.~r".'u.,x.,,,~.,~, ., ,c,.~>~~~a>.;,": »ry, , .a',' '. , ; ~, ct-- ,~ ' :.':-'-'-~'.."~.'~:, m;Mw..»» .r: ~,' .,_ "...,,-.
,.m:,r":..o:,.~;'.~.. ,sc~;',',,e,;, "".,._ v ~,~~ _ ~" »x"
_. ~ ., --_.~. " ». _ ,m_ _ .". ~.~» . ~ . . ,.. __ m" ~.... _ ° R ,.
a' »,°,- ° .,~.~~:-~.',;~'°'_°'".< .."~'y -.;,,..~",° »~~a, ,»: ' ~?~,.: ;_-s;;:W.: ~-~_~~-,.:~,'"-,-'.;:ya:,<<..:"", -.'~". :.,.,'_"""~,,.,'r ,,~
~~» s ~ ~ F ;',t ~' s. ~
fn ~;;'~'m ~ ~,,, ~'.-, ,',,,r":;~,~'~"~' ; »';'~, ,; °~ "' ~.,.,~'~"' :, ~,~, ;r~;
~"a= ~" _ ~ N 'a rt U ;.. > ; ;;, '"».»."r .,:, r ~;i ";rarx »
".~ ~ " »~ >1r :°" .",'",a";, '~ ~" ~ n:xe,t', <~,'-~-~-~"''.y,s .,»,n ~~ _ ~_ ",~~.;~ » ~::: ":, 'r??,~r , hr ",, y , x s,.~4~
'if' ,dn,""~~,...",~&m, ~;";;>w ~,~
~ ""~ ~. ~ .w;. gx", '.. x" ,~ .;3s'~,. , ",'~'~, ,~'>"'""»&,r~
, ~;x ~»»s»»»~r1 ~";1 s,,~sra<m;;;~t' ,m, ,~"~ ;~, ( d:, ,F;;.; "'.'. xa.':
V ,',;~'' '.,r v~ r ~, i.~~m ~; -~. , ,_~z..;";. =n >.. ,"i i"
> , , '.. , "'" ", ~........ : ~ ',»» » ,;"',,' , ,..,.» s" '.r a a"
.";,;s.~, m:=:., s _ .~.,'~~;:,~, . -.m:;,.,~., ,.,-.,a=:r.:z..
°~.y~,."o., ~ rz-r~'~%.".;., .;'%''~~'r'":~s';, "..~;:~.
a,~~v.= "'_ -.--M.;r",., '.N,_y' ~» _ ~-.:,'-~ a~.~
",<.yw,"vi~
»M fix=
i.~.~. ~3'~:~;', :r r F . ,."
'%,:> °~~,ar",ss,.,>,.~."'.n. .;~-~~%t'ss~
x°~i5~ a ".
~;!""., .,wly 9 '' s Yes' ~?i».~.," »e..,~~y.
~~~,,,i$~r°-e~n:';,,~»r~,,"~r~,;'n":,~h~~. ~st~"s', .~,e~'n.,~,.°;;','g-~; ;;$:;a, ~~ o°~o~; t'',';~'", ~»~~p~' ~ y ,Vr x~1'ev rl ~~N N N,_fV~N N N~N,.ri,r~N ~l.,eNmN ~~.'~rN~~"~t~1 C~~N~~::,~,~~,:e"1,":f!M N'~en ~, o , , ", p,~:,:,~
_G1 ,' . ~' : >"~",y.~:~,x:Yr' p°: ' ', H ,, x-,~ .'y>:~ f~ ~ ,.,~,,.~~d~
A ~~~~;,s ,~u~,::3fs;~;~
V "~'~i"~~~"
,","".%~v",.;,ii:, ; %,ri~~> ~~r~'"' ;. ",~, , ~~",'~"~~'"~~~.
Z ~'~>c,' ~ ~' H, "' . ~ ~ ~ ~ . ~ 1 . . a . I ' : ~ "A
d N
A
d d OQ!O N V'V7OOf0M MW O01W nN(DM OOnO N00OM fD(DOO ON N O~Oa0NOVM In N a0 W O~On NO10a0NW~ NW(D~ O a0~ON nO ~Of0N ~~n Ot0nN N

M ~c0MstN ~N ~NN N~ MN~ ~NnN '- NM~f~~~M ~ 1 a0 ~

N N M M N ~ N

a Nn~ ~YVO WQ W 0W~ NNN(D MNt0OOO~~ ~10~M N~O W1nN~ aDsfO MO7O~ n N c0MIna0(ONf0nfD~M NcDc0O OO OOMO~MO~ m O~OW1~OfM Of0O~ n~a0M OM M
0 ~

~Net01Ma0O ~a0n~ NInN NoInInN N~ (Dn47fDO ('7O n~~W NC7(D~ n1~~(O t(1N
M ' I rp7(p~N a0ON1nM h0D(VM~OcMnO(Dr NO Mt0f0cDI~(D O O(DOO N(ONO a0 a0I~~
N~ f0M~W nMVa0(O~M Na0aD~ ~(DN~ M1t~~M ONO~ ~N~M ~a0M~ OV ~fno7f0n ~

Y
Z

~%
D

f 000 000~ No00 00~ 000~ ~~oo no~o 0oMo ino00 00~o No00 0~00 0 M M ~ O N M ~ N M
M

as I

Y
Q

t9 ~

(n D

oNo ~ocoNinmooco0oM aoino0 00oaooao00 0~o~o o No 00oM o~omo nnoo ~
ao ou~v m o> o ~cv n o~ m ao> ~oto n N m v o M ~ N~O~ ~ M m N N N ON tnM ~ M(D fDN
N ~

N f N

N

OOO OOOO OOOO OOO OOOO OOOO OOOO OOOO OOOO OOOO (OOOO OOOO O
O

r tD
N

~
I

M
Q

OnaDOO~(OMOO OOG~NOMO 07OV'O (DOOO NOO(DOOOO Oc0OO (OO~~f7O OMOO a-a0Q~ W(O~ N O ~ ep V M ap OfD W ~ ~ 10n N N NN ~ c7 Y
Z

~

N
D

Of0NOOO nOOfO VMO OOO10OO07O OOOO nOOO a0OOO O~O O MOOO OO~ON o7 L O> N at O WW N ~ c0 W n t0 M

~ r N nN M
L h ~ M N t1~ nV- '-~ n M (O~ t17 M
I

Nl ~ ' N
Q

M a d 9 n C N N

W

R

E O ~,.~~nO OfMInn O MN 010~N MVInN ~(Da0t0nOfON ~t0ne0N
nmOOf01OOO O N M(O(On nnmO OOMO O

O ~NNN NNNN NNNN NNNN NNNN NNNN M

Z

O
N

E
d V

H

O

E

R
Mf~07MMM~ V~VV

Z

L
OE

N

N
N
(0 Z

(J
t C

N
dl 01 d N d N L 'O L L

m V LLL L L, t Ut f0(0(0 a O L

LN 7 L ~'. >,LL N H. ~7 L L t E~~ C3NC
n L

NC E'LL C O ~'m L ~ nL~O NEC C ' L OO"' mtEN

Uy f~jnN ~~L j L L 41MZ'Ly_ EU~ ~V= CCCU (0_C
> > ~N ~ ' 'N NNN E~~ d 111~L O'~7 ,~ !UN Y' C' L CO7~l9_!L~Od~ UamN _mC'Cna0O~ CO,- f0~'N 'O
Q d E >._ G) d M > ~ ~ N(UC ~
~ ~

U(6E Yr~41T 7dfEN ~~~ ~;~CE TWf0Nf0Y7 ~~U(6r~ONN F-F-Hd GN Q~N (p ~ vOYE ~S

.
=IL~'fnw=r ~lL!n~ ww.v-LY>?' r2fn~dU~ =w=O.~N=11=__= N.DNN NU~d D

O ~Oa0O NO O ~O~N ~O~O O~OO-~MO1M N-NN N M~ Oa0OO aDOt NOOa0Of O
1~ ~ ~ NN (D O V~hM ~O aDNOaD~ Mt0fD I~ON ~O O NN

O N O a0~ 1~ I~c0~~ m1OM~ O~NM ~~~ ~cDa0~a0 01.-N
N N

t~ '-M ~ M N

t0hN4Dc0c0 aD01NInNa0 ~ MNNN Oa0t0VN(D (O(D n(1a0O1f047c0O t0MOa~t0 oD
N f~ Wn V'47~a0N oO ~u7N ~a0NO)O70 oI~f~~ NVI~I~O V~cD0101N Oa0Of~~~o c0~01NM ~?~flN OM07OoOI~~- Nf0O~ N~a0 07Nm07a0~a0a0N47a~I~Ma0a0t(1N

aDVOa0a0f~OM~ t0~N(OM~t0 ~ OM ~OMt0OO~h OmN WI7a0Oi~ a~~a0N Nma0 f~N~~ NNI~f0Wf7M(ON 07N MaDN~-~f~~M ~~ t0N~NV ~Oa0 47a01~O~VI~c01~f0m N In(D t0I~ 0707OOM~ c0 ~MM ~(Ot01~~N~

1~~O~O O~OO OOOO O~Ot OOOO ~OOO On~O OOOO OO~O OOO N~OOO OO
O

r ~ N N _ ~ N
CS n 0! N
I

O
Q

I~Of~W OcD o OONM OOMO OOt(7O f0Ma0f~N07IDc0~Oa0N Ot0NM oIn~ c0oOt0Nt0~
O~ 07O ~~ ~M N ~ O01MM MhOf0 hNO V'tDM NNO~M a0t0 N h ap N~ (D I~~ N tn~~a0f0NON cD~f0M O~1~M (Oa0f0C~a0(O~ N~N
~ ~

1NOf~ OOOiO a0a~M07Ot0~-M MM~ aD~ apNI01 ~N~~ at~MM ~~ NN M ~ ~Nc0 O

O OOOO OOOO OOOO OOOO OOOO ONf~O ONMO OOOO VOOO OOsfcDOOm MON
fD O of c0 O N ~ I~ W

<O toN
N

~
I

Y
Q

~

N
D

o ccovo o~noao00~0 0ornv o~nu~o ~aoor-000o aoovo 00~M o00 0000 000 M N M a0O a0~ NN ~ f0 ~ c007 (D V ~N N t~ (DN

Y
#

N
D

ofOO1I7OO01~f100O O117(OO OOf~O ~0N m OMO Of0 O f0OOO OOf007OOO ~O
N W f (DN N~ Oa0 ~ a0f01nO~I~ M h~ OV' lDaD NI~

L ~ N I 7 ~O N Nf~ O~ ~MO(D~ N ~ M M~ a0 cO
L h f0 V
I

V

!C

d r N
O

M, L1JLilLUL11LL111u1LJJcccc cccc cCc ccCc ccc ~ J77> 7J77 ~~roy0mc~a~a~oc~g~,o~,a~0c~g m ~,a~0c~g M ~ aaaa aaaa 5 3 > 5 a d xxxx xxxx EEEE EEEE EEE EEEE EEE
~

H

'00 L

I

i0 M 1nI~Of~ ~t0a0O f01~apON~N Mif!

L O OOO N NNNN MMM t0f0(D
O M MMMM MMMM MMMM MMM~ ~MMM

Z

O
N

V

H

O

O

O
N

Z

a.
O

N

H

N

N

H a o~

N t f..a L L f0 N
y O

a L ' E Nt L LfEl0(0 ~NM~ ~(Of~ap m Lm ' c ~ o ~ U E~~

, d ... ..
~ ' f0LLa CLCC ~ 7CCC ~
N ~' - p ~(n~n fn~ n 07 f~ ~NM~ ~t0hap ~NM ~WOh a0~N
Z' 2' 7 'n a ~Ut ~~~~ ~~~~

E d ~p v~,~ o~ ao r ~~ap~... .... ' ~ ~ XX

E C~~n> ~Vad EM~ ~d!-C ~ NN JJJJ JJJJ ~~~~ ~~~~ MMa Mf~a7MM ~
' (p.Ogyp~'(p~W I-~'HN ~Ed~ ~I-f-NNNN N41NGIDD D O D MM MMMM M
E aaaN ~..~x x~x~ r?~~a c~x2x xxxx xxxx QQ~Q ~Q~Q UU~ UUUU Ut11L1J

OOMW MNO7~OI~O1N OWM a0ofN(ONMm MMOM NMO O7a0O mI~~O Nu7I~M t0MMf~
~ ~ ' N (O M M M MM ~

~a0OO Of~IAO~MM~ ~_ OO~N ON07M IAMOa0W!7aDO1In~(7 f0NN OO~N ro7IM
OI~a0~O O O ~ N a0u7OO (O u7N NNr N O rO7~

O Nr rNNr rM N N rM rM
O
I

Omt(7f0I~~I~a0 (D(DtDo0 (DI~a0~~ f~NVM a0a0 V hIf7 0Df0WO VN h 0D ~ 1f7O7I~c0 ~ ~ ~ O ~

I~N~W O707~tMOf~07N N O01(Dt0a0OfOt0 ~ f0~ M 1~MN (O M I~a0n(1N O7W(D~
N O f~O a~VN MONa0M cDrN tDaD~N Of0M47~NVI~m47N~ h I~cDNMMO~aDM(Oa0a0rNa0 f0 a0 ~, MVM~ Mh Na0f~M MO)O OOfDN 1~WO7f~Of0OI~NcDrf~~ m V'f~MM MOMN t0O1NO
' O N ~
I

O NN ~ NO1O f~Int0 N ~ O~N v!7~MO Na0(OO InN V O OI Q7 r M(ONO MN1f1M
Q <~a0~ MMu7f~f~ma0 MNN ~NON rMM~ V7M~tD(Or~N ~ M~ M~~ ~VM t(7f~~(O
N
~

Y N
Z

~
~

O OW MOO OOmON OMO Oa0VO 4D~rM NOrO OOmm O OO OmNO mOf0O OOON
O

~ f~O cD I~ ~f7 NO (D~rO r ~ O(D Nr ~ N O
M t0 M Nf0 ~V ~ Or ~ N

M
M
I

o Q

a0(DtD07f~NM NNOa0~ (O<D t001~f7t0~~ 0101m c01~O~ (O~M O N(OOOOf~hf~c0fD
~ ~f1 ~ N ONO N~f1 N a0MO1 m f~O NOM f0 O I~O N O01MN t!7v!71nf~
a0 N IO1fOa~N~M a0aMt0N a0~Or ~(p~~ M~<~ 4DNVV rMa0~ f~Nr 47Nh OOrr n(7p~ON
~~~~ M a0cDM r M C~O I~ t0McDI~apf~ NO W(1N ~ (ONf~oDN MMNt0(O~(D1f7Of~ rfp1nM ropI~of N ~ ~
O
I

O m~ ~MN rM47r rN(D(O4DOM MN ~ Nc0NN I~f~~ M N47N~(O I~~M rMN1~
Q N

OrOof~ON I~t0NNN OON fDNapO V~I~O r(pOO roOO r ~O OOOO OOOcDOOOO
r r f~ 07f0 fOMrNW!7 a0rMO7 tONN a0a0 Vf~ O c0 O
~n rN N M N N N

O
N
~
I

Y
Q

~

fn O

VOOO ONO f~ OOW OOO a00DOO a0OOO O V VMOO O V(D(OMNO IAOO~ OOO117 O

~ NN f~ mN N ~a0 ~~ a0 MVM N a0 N
N Mr NN M rI~ r47 N

I
r Q

Y
~

N
D

Ot~O><DNNm OONOO 1~NI~~MOO OO(OO(OO(OOa0I~~ u7a0O OO O OO07O Ma0MM
~ M VMN f 0D ~ ~ I~
1~
M

L ~ hN O01~ I ~ O c0 0 aO I ~O t~(OMOa0(O
r 1~rN rOa0ra0r~ V yO~ r opM ~ N 1~cOrN

N M N

Y

r N-~ CCCC CCC CCCCC CCC CCCC CCCC CCCC CCCC C CC CCCC CCCC CCCC
C

M p, ;?mmyuu~~~ommmm~ mmm mmcg~ mmm~,o~mmm mm~m mm~c~g~~cmm cgmmm mmcgm >>5> > >5~5~ ~>> 5>>5 >>>> ~>> 5>>> 5 >> 5>5> >5>> >5>>
>

d EEEE EEE EEEEE EEE EEEE EEEE EEEE EEEE E EE EEEE EEEE EEEE
~ E

'o c W

E
L

O

Z

L
O
N

V

F-L
O

H

O
N

Z

L
O

H

N

N

H_ H

r NM~W Of~a0 MV'N(Df~epr NM~~tDf~ap 1O4747InN ~Otnr N ~Tnf7f0f~a0~ NM~In(Of~07 M r MOOM OMr NM~~ <Df~aD ~~~ ~a0a000a0N~a0a0O '''' '''' ' ~ ~ ~~ a0 OOOO OOO

XXXX XXo~a~a~w o>o~o>o~o>o~ma~a~a>o> aQQ Q~om,cn~ococomaoaoaomaoaoaoaoV~
~N NN_N_~_N_NN_N NNNN NNNU UUUU UUUN m N(VfVNN~ ~f~~ ~~~U
fVfV

LIJ11JLIJLIJILLll2 2ZS22 Z22 S22Z 222U OOUU OUOfnInfn(n(nIn(n(nfn(n(n(n(nInfn~
(n ,r ca Q
d ~I
H
M
d H

NNa0n47 rnMnMrnn 47a0rnOMa0u7a0rnN (Oa~t00 cDN ~n (OOn(70fDa0n cOn~N rn n M Nn~ nNt0nM ~n~N 'n - Na0(O(D(OepIA~N~rn~ ~NnM ~ M 47Ma0~ On(7 rn t0~ W f7~ M u7~~rn ~ rn~n a0~~ rnInM~C (OfDV~ ~M~ rn~OfhOna0~ON~ V~ ~

~ M (N

ND

n rn~OM ~ nN Nrn naDrn rnrnaDM rn~(OrnOo7MrnO~D n Nrnn (O(D~M ~a0O_ a0 Mt0M rnV'f0Oa0rnNn nOn~ 47G7a0rntDa000~ rn MO V Mv(7VrnOrn NNna0rnO rnM
~

~ MVO NOM rnWnNrn~M~n ~N~ aDMO~ ~O~ rn1A(D~O (ON~_O a047a0Onr-N O~M~_N
O N O i 0 ~ 0 iN ~ n N

t0rn~ (DOt0n O o7O f0(DncDMaf0tnNNN n a0V NN InMf nOf O rn I M rnrnno n rnNM n(DNO ~0NaDorn tn f0M a0f~~rnO t0Ml0a0rnMn na0 n a0ONn o Q ?OM V'Mt17V~~Nc0V'47(O~ 07MVM 1n~V'N V~a0ON~V(Dc0fO(D~ n nn~~ In(OV~ V7 V

Y N M

On(DONn OONOO OOOM InOrnO ~OOM ONrnONOOO OOOn f0nO MOOO OOcDO O
NO ~~ rn n N a0 O7 aD M n~ ~~ N ~M~ ~ M

M
M ai Y
Z

3c ND

1nN~ ON u7Ma0Mt0~Mf0N n1nM OONn rnrn~ C nVN NO u7nO~N(Dc0QOM ~Na0N rn N c0 (OVN MfDNfDa0 nnO OVa0O '-ONO MVN ~~O(On1nO(O~ rn a0n ~~OIAa0 hn t n 117 hO ~ntA I~ It17 M

0 MIn~ODrnO~nNrna0nrn~ OOna0~nn(O~_~N ~(OInC( OI~ON O etW aD(h(h~1~~ ~
N N t ~7a0n c0N(VstOu7 n fVe0 N n f0 N u7 rnO a0 a0 I NO~O ~ N 0 N N M Mn O?~M Mt0~V NWL7O NM N'-N N tD~N~ MV a0 N~N ~o~o ONOON OMa~~ OVOO a0tf1nrnOrnO OnOOo ~~OO OoO t0OnO rnOa0~ N
f0rnO rn NN MO~V I!7M ~ ~~ N n a0 NN rn t0 N

m I

a Y

~

ND

no0 ovo 00000ornoon omoo rnono o~0 00oco0 0orno 000 ornvaoMrnoo M
Ifl n aD N n NI

~ N rn ~ n a0 I M N ~M~ N

Y
Z

~

N

M~ rnOO OOMO(DOOOn OONInt0~OM OO nOnOO NrnNO OOO rnOOO (ONrna0O
n W n u7 Wn fDn u7 rnao N ~ fD rn n W An L ~ etN~ fD t!7 N ~DN ~n~n ID M O~N (D~ rn(O1N
L ~ (D 47 tn MV
~

Y
Z

ix N

N

~ CCC CCC C

a EEE EEE E~~~~ ~~3~ ~~~~ ~~~~ ~~3 ~~~~3 '~3~~~ ~~~ ~~~~ ~~~S ~

H

H
c m ~a E

Z

O
N

V

H

O

F-O
N

Z

O

tN

N

N

H

NMv ic7canao ~NMm wnnao NM~N (Dnap NMV ~t0n o0~NM~ MIDna0~OcD(D(Dt0(DcDv-NM ~tDna0n ~ ~~,v,~ vV
~~ ~

~UU UOU Uo>rnrnrnrnrnrnrn~~~~ ~~~~ aomm a7aoaoaon nrnn nr-n QQ~Q QQ
H~ ~ = ~~ ~ a OOO OOO Ov~~ ~~~~ ~ FHHH ~rnrnrnrnrnrn~ ~u. u. UUUU UUUU ,~
NNN NNN Ntn~IW ~ UUUU UUUU U UUUU UUU >>>>

n n~u7 4JN4!NNNN >>>
>>> >>> >QQQQ QQQQ 2222 S222 2S2 2222~ ~~~~ ~~~ OOOO OOOO fn w~ ;-"~~a B..,,~.:~-~;;: ~~..:'y;;.. : ~,':-~,.::.;.,; ;:';; ~' ::; .:,' ' °e .r a ~ ~., ;:;.°
-~ B ~ ,~, , sr '" ~ ~ ~ »"" ', w cn ~ ~~ n.c~~~ w'~ ~,~ °° °r ~, ~ °' m .
fir', !,~ w° " rn .r ' u~ ~ sr ;
Z ~a=~, ,r .I B, ~ ~,, a;,~,s"~;;.~~, ",~- ° .~ ~k,~,' h ~' ~ er ,r ~;~, t a~t~' " ,?;,has ~ 3 , , F" ", ',, x'.. ' ~, ' ." d '. 3 t'~~, ':
:.» .~~'",. ;'~ f4,~,p~y'~ ._, f '.. .x '" ,u ~ ', ~~ ~~, . a <x~°°yt-".a"~,ro-A;~_~.~'"
~, ,.,~'.."- r° ~ , ,. x"v...". , ~'"B°;~,~ :;;use- ~ "
i~ r~
'.:..9d- :ra ~. ..r ~'~°;r .. , PA "', . Y.~'e;v ~
'. .'n I' ,''~~u=' Y",. ,-f' v'~ ..°V x~"~ s ~", S. ~"F~ .."F ""~
U' ln~"ni F 2~,'~,~z' ~ b ,-,-, .,ui~a ~, ?' .,c,'~. "~,~ ' w. .,rr ",ifi.."rm z~ : awe ff ~~~~ °~°: ,ra '~3 -t ..y ~"y,nn°'~' ';S " BBB" .,,, ~,, , ~ ~ P;", ,i ~-,M" ':. ;'. 2 H
,3,. .. ;~~e, N ''-rT-:y:'a.:~~='.r V ' ' "y'~'" " , ~k._.a , ~, 'x ,3~ . ,.r'~..' ,J~ v ~.=.r", ~ ,.,x,", ..,!
's r , ,y~.j .< .~.b" o., ~.~,.yY~st$~~;J x."~_. ~~'v~Tr° .,~ ..
.. ., ~. "f,Y2.'''9:.,",,.-~.;';s,~,,,°.~ 2..", lC7",Iy,s'f.>x" F.rr;.
.b. . P ~..><«'~"'':'P.'~;,",, .", ~"v,,~~M~, ""~~,"~~~~~, ''Erg y4f.,~w~~f~y ~a°, ;°~'d'. tD v-,.., ",'e- ,~ x"r I...
V : °"~~:D,~GJ ; rf r~ldt2.~~',.~ C"',; ta~ ': '' 9,.%~ '".,~~."[~' ~
f0 13 ~:.
z ~ ~~ ~r~ ~ a5 "x ', '; .~_. ~ , U
~ °s N :'~:=.~ ' .
,.v, .,H.~-r~.~:_ ': " ''' ~" : ~, -, ~ .,xi. ~~, ~"",.;, "r'~~.e; yyt '° '~z~~ ~;?;x :.;B;
V ....~; "' .;;;'-;';" , B
-"Y,, ..,;;" ",. 5 "
r '~, ,~ ~,,~l ' .,' x r-x,. ~'.,r.' ~s ~~~~ ~' ~ d vn '. ''' g, ' ;, '::'.
& Y a"
" 'a i ~.°,,~~.w -~s°.'~» "..mg. g y,~'' e~j~.,r. ;v~;,rf,y; ~""~a,~~,~' ~B.'~~ ; v~.'~'' ,~~ ' ,Ya, N ~ >s,",~ ; Iw 07 ~r ; , '~~ , ~'',~. , .,".,," ", ' ' "C7,: N 'i B%,a;~r., ';' rs .. ~u ~ ~M.e o , ':,, ~ ,~ oc w- ~ '~ M err "M .~, '",.: °~y "~, o o °'°;
e' .<~>;,.~ ~,."fs . ~, ~.. ~.,';cv, ,.;; r ~ ,"~,nv~" m,;'.~:;
',,;,~:, ~_. .
~,~, n,,,.
~~'''' ,r:-f-::.;~": '.~%
~~"., , ~;
:;-.'B: ~ ', ?w,~., w ~'fa "' °~',c:,"
.s ; s;,,;.
,;,.t~ .
..P
rea,.~' ',;
,,",~'rr' ' ,., "~".a=;:
,: r'~M ". p , ,i~
~..L_ ~, ' :,~.""
~~' ''~. _ "a'~ s ,m.,ra .~;.
','~r", a~, "";ir,"~;.
,~f.:;,°,",~"~"~,"", e",-,N»;d,~.;~ .. a x~'~
"us "
d ",d? W %.. ';t..~"
7 ' h. ' ', .:: " M .
,.:. sl'f~'~.t;.' .=, c'~ a.. ~" r ;',~',°
w ,-;°.M"~~,c~~ ~~~f° .~
O) ". ,~ r, ".;, ; N ~ rt <T M'~', ' ,~ ~'6 V ' .-"c . . -~°~','.%h."s;, ~°,~, ;t'' . z;=
s,s,~ .
GI ;, t'..'a;; .x ,''~. ,s ~'~,; 'i,~.,~'x°''5~~. , ' .," ..~~;Y~
, .
"~ ~~.~ 'a'.°.~;~,x~r.a°~~~ .,e::~-s . °°~;' a'~;
~=..-_;r ", ' (/~ ~ ex '~d~~z- a ~~.~,~~, »' _. ~:~.x,r..'"'~""""' ,, " a.
~i~.,.; '~ E
al ..m '°'sxr~%;ff"'Y,~~'~~.'.'S' d,~'1 '. s ~~ ~~ s x. iv.
s~ , °,:~;y",',";
r :c".n'" ..,.. :~ ~" ! .«~~ ~s;,, Gz a e..'~~ ~~ a'~fIlrG .","x x . , f ' '.~ .svnu ~Y rtn 9~,zf~~xc~,' ~.&n' >~ ,b,....<. ..,~ : .»..,.~.,<., ,w ,f, at~°~rn-,.~
e, 01 r:~."f?,ri'~" e'~'#r"~ r';' g,~<°, ~. ~ .":.3:"~' -~ ..,--z ~........, ~,R.~n ;a~~.<:,~t,:- , ~ t.
r t~sm' S .~~° ~, ~',>r ~ i'B~ "tMx";xu, ~~ ~;~", .,. ~ ~ ~ '.. r,"a rt as.
~~,~~'~ .»s?'~~'., , ~~, xi '" ' ,.....;t ~'~' "~_"~;:- ,, ~°~'rt~t",«""~~b~.,°"n ~ xY~,~, ~ B a~~ 'c "
a.~ ttxs:=x~xt~
O '.;;a.;_ ~~~°'~<~<~ ~n"."" x;"-; ~~-~',. "~~
w _ x'~..:."~'~,~ ,..M;.~x°,.ra!;f9'~'~~''e.~' ~ ~;,:~a .."~r ~ ~n:~r. z' ~ '',~~',. ~~,,%yi;,, "t ", ,_ ~. !:" ,~,~~~ r "t r" t? ;~i,,u;
T~ ~ S r Z .,.~ ~~ ~ ~ ~, .,wxw- T; s ~ ' ," ' "~ ra ~, d ,~. .~c; "mss 4 rg , '~ ~ ' :~ F,s~,, _~_" ''t a, ~~=;~ , ~ :~ ,.,, "."., .,"",.. °.a.
,~3,:,°, 4~,"
N , , ",~,~, a : x .. f~""m , r x;
.,~cy,~b, ,.;;>_:
O (.»
"i.'t: ~'"
v m i m?,~~~r""v a t,;.,:~:
H~ H~., ';"~
.~ ~ ' . ~~w N
'~"'W.' »:r ~;~:, ~ ~,~,:°'"
r'~~.A,r.
.~ '';.
H .. :-'~~~~,.<~ "''~,~w:'.,,~.;; ~"~~'t . =~,~.~~, d ~_: ~':_" .~mr.. " " n", »;,:;a~: ; w ~ ~. °:~ ..w , ~ .:,'.u r,~ y .,.~,-m ;rn ~'~, .
».~'~ , . ,~:
_ ~' .~n-~~ ,.:: v~
''.:~... _.~".. =~:...._ ;,-~" T,, "~;;~;;"~~tt~. %;~~':;:;;;-=;.°-° -~~< :..° "a.:;'c-~=;:..~' > ""..
l ~; ~~ ~ ~~ ~ d t rri% "fa B ~.:
I ,x.
ro~ _., s H
1"
:.,.:~ "~.
,~%~~w,,",~
~ °.' ~
~.,t:__~~.~ "b a,",~:~'.6 <,..;w:'C'~~"~.~~.~'t;~~;:' M:,',.,;ii~;~"':~Y',;".x:s".~ ~~.,~.
.~ t..-".t. _.
'd~..,7 .." "'~w~ ,fx°
. 9~~ , saJ _,.....
°."', ~ .. ~~y~, '_~~'~~~ ~.wx.~,Ss=v " '°"s.~:,k2" _.~~~~....~
...< ~' .w~.."ar d~;'""'..:."... '~..?~., tA. ~ "~'sdL:E;,r~s°o'-'~:Fi'...~:~~
~,~.~x,~,.". ;.,.~W.,'%~.., ,°~, ..",° .,.....",..,..<<
~:;;~~.'';:;~~~~ .,f.
Q ;a"y ,_ ,~.;~.:. .,._. i ,s~a.-.:";t-r,<?E~:='",::;'-.zsuz .r;-s=s:.r.,.p i..,~~'t~"~,r s 3','~~~~~~'xA~ ,fir r:.~?x~,.s~-" '~.;=r;x "r~ 3~ ~' ~' x --'~ , ~ ' , n~, ~.
d ',~, .~ ' ' r.:.if"~,~,.'a~ "... x, a, s~F.' ~'~ m ,r,,. ~~.r~;~.~x:.~ i.~i::~"~' ~~ ~,.~s~s'.-S;~,e ,~fj~', m ~.~~,'.~~'d a. ~~~~~: ~3 '' ~~~' ~: ,t~.'."...
p,°~:;~,.»=mgr;- ~. ..,.a"~ , wx;~; ' r ~" .,y'~,'Y'',.d's" ° _ r;.d: ,-'.~'-.°=::.:~..."'~~"..:;~.,.,.~'=.».;'3~ .:, °~.,w~ ~ , , -.:w"",:°~.~~ ',,.>r ':;':.wa~,.",'~y ~:,'~ -.~-~=;.,:~wM..:'~~ :=' z ;,yr,n"
a "
~'r~ . .er"
,~.», ",~; w";,.; ..,.~r,' ~ ;::'"
,.~;rr'~g?~,y~',.,."rn'~" .:x,rr.~" ~~
'' Z r .°~. ~r...'~~w_':~~"~~' , ; a:'':.A~""~""'~,."~'. , .« ~;,.n. v ,w." ''' x °% H; '; i (/l ~~-~"~~..~~ .;.... .,~mr,."y, "xn "..
<" : H ~ "
z;;."
Q '.
r .". "°i";,:. ::. ';owe',':~..
~;:-~--'.' ", ~,> ,'-:~;'':, , ;x O ~~'' ~ :.art ~ ~ ~. ' '~ .°° ,~;°~=~~;==,.
' L ._...,__' . _ . < fr_. ._ . "_ a _ ..,~.__.... ~ ~ '"
H ~,:.d,.:---~:a- .... ~.,. --.~~:.y~~"~-'~,~".____~~:.::,_-~»».;:ax;°::~ ~~:,;;."""".,.~..~,--~ :~,~.. ,w- ~~
s p d~;p N .9' ttrs.;.ruul W 'r'~"N:~,. ,t ice' sti' ":vlz~, .~,t.7" . M
r 2~ ~x," , a,a'~s''t'°°~''''"° a .~~~.~"~~".
a'.;,s~~~~~L;-O" ,°.x''..~..'~"~-'~~~~~~'.....M:.M,.~~.;-'N r. ...:~
.:r ,,. ~._ ~~.,r~., ~,-~~: ;~x>a:~r~..~.-n,<;.~o-,". m V) '"~' - "~ a.'"s::';.. , ~;;r,",' ~.H'r''.~ ~.arC°°-,~; H. ~~LCF:, ~("FiY(L'a:(.K ~. .., , LK~f,tif"" ;~ LY' (~~~''~~~':,,~N ,~~,~';;UI:,CO ~ '~s ''.. ~ tt In tn '~~'~~ 2 ~~,N'~,,"
'~tll':~ft? ':,1 ~"3~ .., N
,., ; " ,~ ~;;;~°s; ,~",~, , z ~ ..,;<.; °~-s._..~"°d ..~. ~'.s;'.i,:~ g~ ri,.,., m ;, ~' ;;,a:NgM N: ,;" ~ r,r<,<~ ', ~~,~, ,~,"awv.; %ai~a",.,."
>...,tC'zl~~..C7 V ;'~a"~",«"i~,~~' ~ '~ ~ai0""i;f"~°si~~ r~ ' T=~~'t i~~~ "", f, Y t C'r.~ N,,,,,, ~ ~q~ "',~,N~,icV ~
"rE,~'t ., , ~. ~~ ~~Y4<~~-r. ,.".., ",...,~ ...aa:r.t..~~~ ~ n '",se. s.
<,r~. ~' f: .... ~~~,xd~~t z~,S",..zs....ur~.'. .a-. ~ . r(,m~ttts..l_r..Y~.S
D
V

V
_d V
Z
N
R
d ~ li ra~mo~ao v NwIn aovaoofaornov oon ccor~vovvo 000~ oaoo aoao0o mo0 N0D 1~~I~~t0 NWIf1h N cD01M m N N Nt0 I~V M t0 1f (O(O I ~ e! f0~
O ~f~(OM (O~~ (D~O(OfhNIf7('~WaD~N I() N ~ f0N M(~7 O7c0 N VI~
~ ~

a~
I

Q
Y

N
D

Wl7NM Vf~N 10VMM 1~Mt0N t0Mof1(fN~hN NI~OfIf7071~OO ~~a0 t0f~ON N~ I~et'-V
N00 I~ONt0OOf~(Oi~W ON m (OM ON~ ON01 M OI~O tD ~

N (Dc7~ NO~ Owf1I()MOVNN ~M01IAIT)~I~f~~~N a0f~~Na0~Vt0I~Va0NO a0NO(DW(h1 m~ ~~ - ~ ~ N~ aD V M O

nOit0NVO a0O)VO NW N (D t0OOIAM W f7f0 ('~f~l1i1 OInM~~M aDhN~ MM
t0I~N O7M N In Mhf0O01WI700It)V~ t0V~ ~M~V 1f7~ V'Nc0 ~~IntoInInvMvtoIn~ao0 Y
Z

ak N
D

000 000 0000 000on o000 0000 oNo rooNo ovno 0oNo~vono 00 I~ N

Mc0 M ON 01N Osf ~
N ~01N 117 (DM N N O7f~

M

Y

Z

(9 ix D

I~W0101 MO~ M~ O1Ma0I~O 1~~MN OI~O NOO OI~OO07O Ina0O~OOO OMOOfMO
~ ~

N NN~N mNf~NI~a0 V IDO~aONhOfcD M O O IDm fW1!7N OfIAO I~ c0 M ~ ~M n~N O~1'OO ~~OoW10ID~Nsf N N a0 N I~N m(7a0 '-~N M 1 N m N

O ONON nnM j~VM rOyIi N
I

Q

ND

OOOM OOO u7OIAO OONOO OOOO OOOO OOO OOOOO OOOO OOOO OOOO OOO

M N NCM,~t0 W

<O
N
I

a Y
Z

~

b7 D

mowo aooaoo vN moMro oNo0 000o ao0 00000 0oMv Inocor o<oNo Mo0 f~ O O W O~ M O f0m (7 ~N~ N N ~ ~M NN ~ N
I

r a Y
~

N

f~OON OOO N t0O u7OM OOOIAOOO(DOo7O 47O~NO a~(O07f~Va0t0N O(DOIDONO

f~O1 aD O~NO t047a0M O OI N ~O ~ha0~ OM101l7 N f~ IA
L ~ NI M1~- M ~ M 1 N

~ A e ~ W7 VfD a01~a0~f~OV st t~ V7 I

Y

!( Z

C7 it ~D

M

M a d C

W

t6 O

Z

L
O
N

t00~O N f0a0O NVf007O1 Mt f000ON ~tOa0 ~ ao~o'oaoo>~mm ~m InW Ino ocoto<ocoaomo~o~o~00 000 3 '-NN NNN
V
H

L
O

r O
~n Z

L
O

N

d N
t 7 N NJ, ~ ~

O1O(nmOfmOOm cp N Q Z

m 01p707m WaDa0a0 aoaoaomm m o00 ~ ~ ~~ Q ~ ~~o NM~117(OhGO~NM~ IA(O1~OCq LLLo qctt N(OpN ~ Ip CQ Z~ ~Q ,Q-L L LO~N 7 N ~OIG?c0a0~~" ~ ~ Vr ~ T d ~v O~mm07Of01Of~a0a0aDa0a~opop~ ~~a~ ~gga _=2 ncml1 Q ~V7N Z I~
MMMM MMM MMMM MMMM J J ' mm0N~ ~F- r I- ~ ~
III(f ItIf N

()n)I ))In --- YO J SU Ofndp Ir_Ir_I>_J xu.ti uIuJmwtowu1mw UUU ZZti~U ~Um~ v~~m OUQU H-IL

In (nIn!nfn(n(n(n~~~~ ~~~~Z xx2x Sx2x ZZZ t4(nV)(nU DxUr-HUrY 2Q~~ ~~v DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

~~ TTENANT LES PAGES 1 A 176 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:

Claims (28)

What is claimed is:

1. An isolated, enriched or purified nucleic acid molecule encoding a kinase polypeptide, wherein said nucleic acid molecule comprises a nucleotide sequence that:
(a) encodes a polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID
NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ
ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ
ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:112, SEQ ID NO:113, and SEQ ID NO:114;
(b) is the complement of the nucleotide sequence of (a);
(c) hybridizes under stringent conditions to the nucleotide molecule of (a) and encodes a naturally occurring kinase polypeptide;
(d) encodes a polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID
NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ
ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ
ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID

NO:112, SEQ ID NO:113, and SEQ ID NO:114, except that it lacks one or more, but not all, of an N-terminal domain, a C-terminal catalytic domain, a catalytic domain, a C-terminal domain, a coiled-coil structure region, a proline-rich region, a spacer region and a C-terminal tail; or (e) is the complement of the nucleotide sequence of (d).

2. The nucleic acid molecule of claim 1, further comprising a vector or promoter effective to initiate transcription in a host cell.

3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is isolated, enriched, or purified from a mammal.

4. The nucleic acid molecule of claim 3, wherein said mammal is a human.

5. The nucleic acid probe of claim 1 used for the detection of nucleic acid encoding a kinase polypeptide in a sample, wherein said kinase polypeptide is selected from the group consisting of a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ
ID
NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ
ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ
ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID
NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID NO:114.

6. A recombinant cell comprising the nucleic acid molecule of claim 1 encoding a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ
ID
NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID
NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID
NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ
ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID
NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID
NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:112, SEQ ID NO:113, and SEQ ID NO:114.

7. An isolated, enriched, or purified kinase polypeptide, wherein said polypeptide comprises an amino acid sequence having (a) an amino acid sequence selected from the group consisting of those set forth in SEQ ID
NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ
ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID
NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ
ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID
NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ
ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, and SEQ ID NO:114, respectively;
(b) an amino acid sequence selected from the group consisting of those set forth in SEQ ID
NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ

ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID
NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ
ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID
NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ
ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, and SEQ ID NO:114, respectively, except that it lacks one or more, but not all, of the domains selected from the group consisting of an N-terminal domain, a C-terminal catalytic domain, a catalytic domain, a C-terminal domain, a coiled-coil structure region, a proline-rich region, a spacer region, and a C-terminal tail.

8. The kinase polypeptide of claim 7, wherein said polypeptide is isolated, purified, or enriched from a mammal.

9. The kinase polypeptide of claim 8, wherein said mammal is a human.

10. An antibody or antibody fragment having specific binding affinity to a kinase polypeptide or to a domain of said polypeptide, wherein said polypeptide is a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID
NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ
ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID
NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ
ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID
NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ

ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, and SEQ ID NO:114.

11. A hybridoma which produces an antibody having specific binding affinity to a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ
ID
NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID
NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID
NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ
ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID
NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID
NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:112, SEQ ID NO:113, and SEQ ID NO:114.

12. A kit comprising an antibody which binds to a polypeptide of claim 7 or 8 and negative control antibody

13. A method for identifying a substance that modulates the activity of a kinase polypeptide comprising the steps of:
(a) contacting the kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID
NO:60, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:
66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID
NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ

ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ
ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ
ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID NO:114 with a test substance;
(b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide.

14. A method for identifying a substance that modulates the activity of a kinase polypeptide in a cell comprising the steps of:
(a) expressing a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID
NO:60, SEQ ID
NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:
66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID
NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ
ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ
ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ
ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID NO:l 12, SEQ ID NO:113, and SEQ ID NO:114;
(b) adding a test substance to said cell; and (c) monitoring a change in cell phenotype or the interaction between said polypeptide and a natural binding partner.

15. A method for treating a disease or disorder by administering to a patient in need of such treatment a substance that modulates the activity of a kinase having an amino acid sequence selected from the group consisting of those set forth in SEQ ID
NO:58, SEQ ID
NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ
ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID
NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ
ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID
NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ
ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID
NO:114.

16. The method of claim 15, wherein said disease or disorder is selected from the group consisting of cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders.

17. The method of claim 15, wherein said disease or disorder is selected from the group consisting of cancers of tissues; cancers of hematopoietic origin;
diseases of the central nervous system; diseases of the peripheral nervous system; Alzheimer's disease; Parkinson's disease; multiple sclerosis; amyotrophic lateral sclerosis; viral infections;
infections caused by prions; infections caused by bacteria; infections caused by fungi; and ocular diseases.

18. The method of claim 15, wherein said disease or disorder is selected from the group consisting of migraines; pain; sexual dysfunction; mood disorders;
attention disorders;
cognition disorders; hypotension; hypertension; psychotic disorders;
neurological disorders;
dyskinesias; metabolic disorders; and organ transplant rejection.

19. The method of claim 15, wherein said substance modulates kinase activity in vitro.

20. The method of claim 19, wherein said substance is a kinase inhibitor.

21. A method for detection of a kinase polypeptide in a sample as a diagnostic tool for a disease or disorder, wherein said method comprises:
(a) contacting said sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a kinase polypeptide having an amino acid sequence selected from the group consisting of those set forth in SEQ ID
NO:58, SEQ ID
NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ
ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID
NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ
ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID
NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ
ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID
NO:114, said probe comprising the nucleic acid sequence encoding said polypeptide, fragments thereof, or the complements of said sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of said disease.

22. The method of claim 21, wherein said disease or disorder is selected from the group consisting of cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders.

23. The method of claim 21, wherein said disease or disorder is selected from the group consisting of cancers of tissues; cancers of hematopoietic origin;
diseases of the central nervous system; diseases of the peripheral nervous system; Alzheimer's disease; Parkinson's disease; multiple sclerosis; amyotrophic lateral sclerosis; viral infections;
infections caused by prions; infections caused by bacteria; infections caused by fungi; and ocular diseases.

24. The method of claim 21, wherein said disease or disorder is selected from the group consisting of migraines, pain; sexual dysfunction; mood disorders;
attention disorders;
cognition disorders; hypotension; hypertension; psychotic disorders;
neurological disorders;
dyskinesias; metabolic disorders; and organ transplant rejection.

25. A method for detection of a kinase polypeptide in a sample as a diagnostic tool for a disease or disorder, wherein said method comprises:
(a) comparing a nucleic acid target region encoding said kinase polypeptide in a sample, wherein said kinase polypeptide has an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72; SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ
ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID
NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ
ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID
NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ
ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:112, SEQ ID NO:113, and SEQ ID NO:114, or one or more fragments thereof, with a control nucleic acid target region encoding said kinase polypeptide, or one or more fragments thereof; and (b) detecting differences in sequence or amount between said target region and said control target region, as an indication of said disease or disorder.

26. The method of claim 25, wherein said disease or disorder is selected from the group consisting of cancers, immune-related diseases and disorders, cardiovascular disease, brain or neuronal-associated diseases, and metabolic disorders.

27. The method of claim 25, wherein said disease or disorder is selected from the group consisting of cancers of tissues; cancers of hematopoietic origin;
diseases of the central nervous system; diseases of the peripheral nervous system; Alzheimer's disease; Parkinson's disease; multiple sclerosis; amyotrophic lateral sclerosis; viral infections;
infections caused by prions; infections caused by bacteria; infections caused by fungi; and ocular diseases.

28. The method of claim 25, wherein said disease or disorder is selected from the group consisting of migraines, pain; sexual dysfunction; mood disorders;
attention disorders;
cognition disorders; hypotension; hypertension; psychotic disorders;
neurological disorders;
dyskinesias; metabolic disorders; and organ transplant rejection.