Human protein kinase, phosphatase, and protease family members and uses thereof (04-Jul-2006)

RELATED APPLICATIONS

This application is a continuation-in-part and claims priority to U.S. application Ser. No. 09/797,039, filed Feb. 28, 2001, now U.S. Pat. No. 6,730,491 issued May 4, 2004, which claims the benefit of U.S. Provisional Application Serial No. 60/186,061, filed Feb. 29, 2000; and U.S. application Ser. No. 09/882,166, filed Jun. 15, 2001, now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/212,078, filed Jun. 15, 2000; and U.S. application Ser. No. 09/934,406, filed Aug. 21, 2001 now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/226,740, filed Aug. 21, 2000; and U.S. application Ser. No. 09/861,801, filed May 21, 2001, now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/205,508, filed May 19, 2000; and U.S. application Ser. No. 09/801,267, filed Mar. 6, 2001, now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/187,454, filed Mar. 7, 2000; and U.S. application Ser. No. 09/829,671, filed Apr. 10, 2001, now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/197,508, filed Apr. 18, 2000; and U.S. application Ser. No. 09/961,721, filed Sep. 24, 2001 now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/235,023, filed Sep. 25, 2000; and U.S. application Ser. No. 10/045,367, filed Nov. 7, 2001 now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/246,561, filed Nov. 7, 2000; and U.S. application Ser. No. 09/801,275, filed Mar. 6, 2001 now abandoned, which claims the benefit of U.S. Provisional Application Serial No. 60/187,420, filed Mar. 7, 2000, the contents of which are incorporated herein by reference.

BACKGROUND OF THE 2504, 15977, AND 14760 INVENTION

Phosphate tightly associated with protein has been known since the late nineteenth century. Since then, a variety of covalent linkages of phosphate to proteins have been found. The most common involve esterification of phosphate to serine, threonine, and tyrosine with smaller amounts being linked to lysine, arginine, histidine, aspartic acid, glutamic acid, and cysteine. The occurrence of phosphorylated proteins implies the existence of one or more protein kinases capable of phosphorylating amino acid residues on proteins, and also of protein phosphatases capable of hydrolyzing phosphorylated amino acid residues on proteins.

Protein kinases play critical roles in the regulation of biochemical and morphological changes associated with cellular growth and division (D'Urso, G. et al. (1990) Science 250: 786-791; Birchmeier. C. et al. (1993) Bioessays 15: 185-189). They serve as growth factor receptors and signal transducers and have been implicated in cellular transformation and malignancy (Hunter, T. et al. (1992) Cell 70: 375-387; Posada, J. et al. (1992) Mol. Biol. Cell 3: 583-592; Hunter, T. et al. (1994) Cell 79: 573-582). For example, protein kinases have been shown to participate in the transmission of signals from growth-factor receptors (Sturgill, T. W. et al. (1988) Nature 344: 715-718; Gomez, N. et al. (1991) Nature 353: 170-173), control of entry of cells into mitosis (Nurse, P. (1990) Nature 344: 503-508; Maller, J. L. (1991) Curr. Opin. Cell Biol. 3: 269-275) and regulation of actin bundling (Husain-Chishti, A. et al. (1988) Nature 334: 718-721). Protein kinases can be divided into two main groups based on either amino acid sequence similarity or specificity for either serine/threonine or tyrosine residues. A small number of dual-specificity kinases are structurally like the serine/threonine-specific group. Within the broad classification, kinases can be further sub-divided into families whose members share a higher degree of catalytic domain amino acid sequence identity and also have similar biochemical properties. Most protein kinase family members also share structural features outside the kinase domain that reflect their particular cellular roles. These include regulatory domains that control kinase activity or interaction with other proteins (Hanks, S. K. et al. (1988) Science 241: 42-52).

SUMMARY OF THE 2504, 15977, AND 14760 INVENTION

The present invention is based, in part, on the discovery of novel protein kinase family members, referred to herein as “2504, 15977, and 14760”. The nucleotide sequence of a cDNA encoding 2504 is shown in SEQ ID NO:1, and the amino acid sequence of a 2504 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:3. The nucleotide sequence of a cDNA encoding 15977 is shown in SEQ ID NO:4, and the amino acid sequence of a 15977 polypeptide is shown in SEQ ID NO:5. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:6. The nucleotide sequence of a cDNA encoding 14760 is shown in SEQ ID NO:7, and the amino acid sequence of a 14760 polypeptide is shown in SEQ ID NO:8. In addition, the nucleotide sequences of the coding region are depicted in SEQ ID NO:9.

Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 2504, 15977, or 14760 protein or polypeptide, e.g., a biologically active portion of the 2504, 15977, or 14760 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8. In other embodiments, the invention provides isolated 2504, 15977, or 14760 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ U) NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, wherein the nucleic acid encodes a full length 2504, 15977, or 14760 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acid constructs which include a 2504, 15977, or 14760 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 2504, 15977, or 14760 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 2504, 15977, or 14760 nucleic acid molecules and polypeptides.

In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 2504, 15977, or 14760-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules that are antisense to a 2504, 15977, or 14760 encoding nucleic acid molecule are provided.

In another aspect, the invention features, 2504, 15977, or 14760 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 2504, 15977, or 14760 mediated or related disorders. In another embodiment, the invention provides 2504, 15977, or 14760 polypeptides having a 2504, 15977, or 14760 activity. Preferred polypeptides are 2504, 15977, or 14760 proteins including at least one protein kinase domain, e.g. a serine/threonine kinase domain, and, preferably, having a 2504, 15977, or 14760 activity, e.g., a 2504, 15977, or 14760 activity as described herein.

In other embodiments, the invention provide; 2504, 15977, or 14760 polypeptides, e.g., a 2504, 15977, or 14760 polypeptide having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO: 8; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringent hybridization condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, wherein the nucleic acid encodes a full length 2504, 15977, or 14760 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acid constructs which include a 2504, 15977, or 14760 nucleic acid molecule described herein.

In a related aspect, the invention provides 2504, 15977, or 14760 polypeptides or fragments operatively linked to non-2504, 15977, or 14760 polypeptides to form fusion proteins.

In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 2504, 15977, or 14760 polypeptides.

In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 2504, 15977, or 14760 polypeptides or nucleic acids.

In still another aspect, the invention features a method of modulating (e.g., enhancing or inhibiting) the proliferation, survival, and/or differentiation of a cell, e.g., a 2504-, 15977-, or a 14760-expressing cell, e.g., a neural cell (e.g., a brain or glial cell), a cardiovascular cell (e.g., a heart or blood vessel cell, e.g., a smooth muscle cell), a liver cell, a hematopoietic cell (e.g., a bone marrow cell such as a glycophorin-positive cell). The method includes contacting the cell with an agent (e.g., a screened compound) that modulates the activity or expression of a 2504-, 15977-, or a 14760 polypeptide or nucleic acid, in an amount effective to modulate the proliferation and/or differentiation of the cell.

In a preferred embodiment, the 2504-, 15977-, or a 14760 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2, 5 or 8. In other embodiments, the 2504-, 15977-, or a 14760 polypeptide is a fragment of at least 15, 20, 50, 100, 150, or more contiguous amino acids of SEQ ID NO:2, 5 or 8.

In a preferred embodiment, the 2504-, 15977-, or a 14760 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1, 3, 4, 6, 7, or 9. In other embodiments, the 2504-, 15977-, or a 14760 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more contiguous nucleotides of SEQ ID NO:1, 3, 4, 6, 7, or 9.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein kinase activity.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 2504-, 15977-, or a 14760 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.

In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 2504-, 15977-, or a 14760 nucleic acid, or any combination thereof.

In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.

In a preferred embodiment, the cell, e.g., the 2504-, 15977-, or a 14760-expressing cell, is a neural cell (e.g., a neuronal or glial cell), a cardiovascular cell (e.g., a heart or blood vessel cell, e.g., a smooth muscle cell), a liver cell, a hematopoietic cell, e.g., a myeloid, lymphoid or erythroid cell, or a precursor cell thereof. Examples of such cells include myelocytic cells (polymorphonuclear cells), erythrocytic cells, lymphocytes, monocytes, reticular cells, plasma cells and megakaryocytes, as well as stem cells for the different lineages, and precursors for the committed progenitor cells, for example, precursors of blood cells (e.g., red blood cells, such as erythroblasts), macrophages (monoblasts), platelets (megakaryocytes), polymorphonuclear leucocytes (myeloblasts), and lymphocytes (lymphoblasts).

In a preferred embodiment, the cell, e.g., the 14760-expressing cell, is a bone marrow erythroid cell, e.g., an erythroid progenitor (e.g., a glycophorin A expressing cell) or a differentiated cell, e.g., an erythrocyte or a megakaryocyte.

In a preferred embodiment, the cell, e.g., the 2504-, 15977-, or a 14760-expressing cell, is further contacted with a protein, e.g., a cytokine or a hormone. Exemplary proteins include, but are not limited to, G-CSF, GM-CSF, stem cell factor, interleukin-3 (IL-3), IL-4, Flt-3 ligand, thrombopoietin, and erythropoietin. Most preferably, the protein is erythropoietin. The protein contacting step can occur before, at the same time, or after the agent is contacted. The protein contacting step can be effected in vitro or ex vivo. For example, the cell, e.g., the 14760-expressing cell is obtained from a subject, e.g., a patient, and contacted with the protein ex vivo. The treated cell can be re-introduced into the subject. Alternatively, the protein contacting step can occur in vivo.

In a preferred embodiment, the agent and the 2504-, 15977-, or a 14760-polypeptide or nucleic acid are contacted in vitro or ex vivo.

In a preferred embodiment, the contacting step is effected in vivo in a subject, e.g., as part of a therapeutic or prophylactic protocol. Preferably, the subject is a human, e.g., a patient with an immune, cardiovascular, proliferative, or liver disorder. In other embodiments, the subject is a non-human animal, e.g., an experimental animal.

The contacting step(s) can be repeated.

In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation of the cell, e.g., the 2504-, 15977-, or a 14760-expressing cell. Such agents can be used to treat or prevent cancers, e.g., leukemic cancers such as erythroid leukemias, or carcinomas.

In preferred embodiments, the methods involve treatment or prevention of disorder related to aberrant activity or expression of the 2504, 15977, or 14760 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular proliferation or differentiation, neural disorders, immune disorders, cardiovascular disorders, liver, skin, and skeletal muscle disorders, among others. The method includes administering to the subject an effective amount of an agent that modulates the activity or expression of a 2504, 15977, and 14760 polypeptide or nucleic acid such that the disorder is ameliorated or prevented.

In a preferred embodiment, the 2504, 15977, and 14760 polypeptide has an amino acid sequence identical to, or substantially identical to, SEQ ID NO:2, 5 or 8. In other embodiments, the 2504, 15977, and 14760 polypeptide is a fragment of at least 15, 20, 50, 100, 150, or more contiguous amino acids of SEQ ID NO:2, 5 or 8.

In a preferred embodiment, the 2504, 15977, and 14760 nucleic acid has a nucleotide sequence identical to, or substantially identical to, SEQ ID NO:1, 3, 4, 6, 7 or 9. In other embodiments, the 2504-, 15977-, or a 14760 nucleic acid is a fragment of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more contiguous nucleotides of SEQ ID NO:1, 3, 4, 6, 7 or 9.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) protein kinase activity.

In a preferred embodiment, the agent modulates (e.g., increases or decreases) expression of the 2504, 15977, and 14760 nucleic acid by, e.g., modulating transcription, mRNA stability, etc.

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof. The antibody can be conjugated to a therapeutic moiety selected from the group consisting of a cytotoxin, a cytotoxic agent and a radioactive metal ion.

In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or a 2504, 15977, and 14760 nucleic acid, or any combination thereof.

In a preferred embodiment, the agent is administered in combination with a cytotoxic agent.

In a preferred embodiment, the subject is a human, e.g., a patient with a disorder described herein. In other embodiments, the subject is a non-human animal, e.g., an experimental animal.

In a preferred embodiment, the agent decreases the proliferation and/or enhances the differentiation of a cell, e.g., a 2504, 15977, and 14760-expressing cell, e.g., a hematopoietic cell, in the subject. Such agents can be used to treat or prevent cancers, e.g., leukemic cancers such as erythroid leukemias, or carcinomas.

In a preferred embodiment, the disorder is an immune disorder, a cardiovascular disorder, a neural disorder, a liver disorder, among others.

The administration of the agent and/or protein can be repeated.

The invention also provides assays for determining the activity of or the presence or absence of 2504, 15977, or 14760 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

In further aspect the invention provides assays for determining the presence or absence of a genetic alteration in a 2504, 15977, or 14760 polypeptide or nucleic acid molecule, including for disease diagnosis.

The invention also features a method of diagnosing, or staging, a disorder, e.g., a disorder as described herein, in a subject. The method includes evaluating the expression or activity of a 2504, 15977, and 14760 nucleic acid, or a 2504, 15977, and 14760 polypeptide, such that, a difference in the level of 2504, 15977, and 14760 nucleic acid, or 2504, 15977, and 14760 polypeptide relative to a normal subject or a cohort of normal subjects is indicative of the disorder, or a stage in the disorder.

In a preferred embodiment, the subject is a human.

In a preferred embodiment, the evaluating step occurs in vitro or ex vivo. For example, a sample, e.g., a blood sample or biopsy, is obtained from the subject.

In a preferred embodiment, the evaluating step occurs in vivo. For example, by administering to the subject a detectably labeled agent that interacts with the 2504, 15977, and 14760 nucleic acid or polypeptide, such that a signal is generated relative to the level of activity or expression of the 2504, 15977, and 14760 nucleic acid or polypeptide.

In still another aspect, the invention features a method for evaluating the efficacy of a treatment of a disorder (e.g., a disorder as described herein), in a subject. The method includes treating a subject with a protocol under evaluation; assessing the expression of a 2504, 15977, or 14760 nucleic acid, or 2504, 15977, or 14760 polypeptide, such that a change in the level of the 2504, 15977, or 14760 nucleic acid, or the 2504, 15977, or 14760 polypeptide after treatment, relative to the level before treatment, is indicative of the efficacy of the treatment of the disorder.

In yet another aspect, the invention features a method for identifying an agent, e.g., a compound, which modulates the activity or expression of a 2504, 15977, and 14760 polypeptide, e.g., a 2504, 15977, and 14760 polypeptide as described herein, or a 2504, 15977, and 14760 nucleic acid, e.g., a 2504, 15977, and 14760 nucleic acid as described herein. The method includes contacting the 2504, 15977, and 14760 polypeptide or nucleic acid with a test agent (e.g., a test compound); and determining the effect of the test compound on the activity of the polypeptide or nucleic acid to thereby identify a compound which modulates the activity of the polypeptide or nucleic acid.

In a preferred embodiment, the activity of the 2504, 15977, and 14760 polypeptide is a protein kinase activity.

In a preferred embodiment, the activity of the 2504, 15977, and 14760 polypeptide is proliferation, differentiation, and/or survival of a cell, e.g., a 2504, 15977, and 14760-expressing cell, e.g., a neural cell, a cardiovascular cell, a hematopoietic cell (e.g., a bone marrow cell such as a glycophorin-positive cell, an erythroid cell, a megakaryocyte).

In preferred embodiments, the agent is a peptide, a phosphopeptide, a small molecule, e.g., a member of a combinatorial library, or an antibody, or any combination thereof.

In additional preferred embodiments, the agent is an antisense, a ribozyme, or a triple helix molecule, or an 2504, 15977, and 14760 nucleic acid, or any combination thereof.

In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 2504, 15977, and 14760 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 2504, 15977, and 14760 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 2504, 15977, and 14760 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS, 1A-1B depict the cDNA sequence (SEQ ID NO:1) and predicted amino acid sequence (SEQ ID NO:2) of human 2504. The methionine-initiated open reading frame of human 2504 (without the 5′ and 3′ untranslated regions) extends from nucleotide position 154 to position 1656 of SEQ ID NO:1 (coding sequence shown in SEQ ID NO:3).

FIG. 2 depicts a hydropathy plot of human 2504. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 2504 are indicated.

FIG. 3A depicts an alignment of the eukaryotic protein kinase domain of human 2504 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:10), while the lower amino acid sequence corresponds to amino acids 37 to 286 of SEQ ID NO:2.

FIG. 3B depicts an alignment of the serine/threonine kinase domain of human 2504 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:11), while the lower amino acid sequence corresponds to amino acids 24 to 286 of SEQ ID NO:2.

FIGS. 4A-4C depict the cDNA sequence (SEQ ID NO:4) and predicted amino acid sequence (SEQ ID NO:5) of human 15977. The methionine-initiated open reading frame of human 15977 (without the 5′ and 3′ untranslated regions) extends from nucleotide position 337 to position 1713 of SEQ ID NO:4 (coding sequence shown in SEQ ID NO:6).

FIG. 5 depicts a hydropathy plot of human 15977. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 15977 are indicated.

FIG. 6A depicts an alignment of the eukaryotic protein kinase domain of human 15977 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:12), while the lower amino acid sequence corresponds to amino acids 44 to 276 of SEQ ID NO:5.

FIG. 6B depicts an alignment of the serine/threonine kinase domain of human 15977 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:11), while the lower amino acid sequence corresponds to amino acids 44 to 329 of SEQ ID NO:5.

FIGS. 7A-7B depict the cDNA sequence (SEQ ID NO:7) and predicted amino acid sequence (SEQ ID NO:8) of human 14760. The methionine-initiated open reading frame of human 14760 (without the 5′ and 3′ untranslated regions) extends from nucleotide position 119 to position 1906 of SEQ ID NO:7 (coding sequence shown in SEQ ID NO:9).

FIG. 8 depicts a hydropathy plot of human 14760. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 14760 are indicated.

FIG. 9A depicts an alignment of the eukaryotic protein kinase domain of human 14760 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:13), while the lower amino acid sequence corresponds to amino acids 285 to 540 of SEQ ID NO:8.

FIG. 9B depicts an alignment of the serine/threonine kinase domain of human 14760 with a consensus amino acid sequence derived from a hidden Markov model. The upper sequence is the consensus amino acid sequence (SEQ ID NO:11), while the lower amino acid sequence corresponds to amino acids 285 to 540 of SEQ ID NO:8.

FIG. 10 is a bar graph depicting relative 2504 mRNA expression as determined by TaqMan assays on mRNA derived from the following tissues: MK (monkey) cortex; MK dorsal root ganglion; MK spinal cord; MK sciatic nerve; MK kidney; MK hairy skin; MK heart left ventricle; MK gastro muscle; MK liver; human brain; human spinal cord; human heart; human kidney; human liver; and human lung. The highest 2504 mRNA expression was observed in MK cortex, human brain, and MK and human spinal cord.

FIG. 11 is a bar graph depicting relative 15977 mRNA expression as determined by TaqMan assays on mRNA derived from the following human tissues. Columns are numbered at five-column intervals at the bottom of the Figure (i.e., columns 1-46), and correspond to the following: (1) Aorta/normal; (2) Fetal heart/normal; (3) Heart normal; (4) Heart/congestive heart failure (CHF); (5) Vein/Normal; (6) Smooth muscle cells (SMC) (Aortic); (7) Spinal cord/Normal; (8) Brain cortex/Normal; (9) Brain hypothalamus/Normal; (10) Glial cells (Astrocytes); (11) Brain/Glioblastoma; (12) Breast/Normal; (13) Breast tumor/(invasive carcinoma (IDC); (14) Ovary/Normal; (15) Ovary/Tumor; (16) Pancreas; (17) Prostate/Normal; (18) Prostate/Tumor; (19) Colon/normal; (20) Colon/tumor; (21) Colon/IBD; (22) Kidney/normal; (23) Liver/normal; (24) Liver fibrosis; (25) Fetal Liver/normal; (26) Lung/normal; (27) Lung/tumor; (28) Lung/COPD; (29) Spleen/normal; (30) Tonsil/normal; (31) Lymph node/normal; (32) Thymus/normal; (33) Epithelial Cells (prostate); (34) Endothelial Cells (aortic); (35) Skeletal Muscle/Normal; (36) Fibroblasts (Dermal); (37) Skin/normal; (38) Adipose/Normal; (39) Osteoblasts (primary); (40) Osteoblasts (undifferentiated); (41) Osteoblasts (Diff); (42) Osteoclasts; (43) Aortic smooth muscle cells (SMC) Early; (44) Aortic SMC Late; (45) Shear human umbilical vein endothelial cells (HUVEC); and (46) Static HUVEC. Elevated 15977 mRNA expression was observed in epithelial cells, astrocytes (glial cells), normal brain (e.g., cortex and hypothalamus), HUVEC, and normal fetal liver.

FIG. 12A is a bar graph depicting relative 14760 mRNA expression as determined by TaqMan assays on mRNA derived from the following human tissues. Columns are numbered at five-column intervals at the bottom of the Figure (i.e., columns 1-42), and correspond to the following: (1) Aorta/Normal; (2) Fetal Heart/Normal; (3) Heart/Normal; (4) Heart/CHF; (5) Vein/Normal; (6) SMC/aortic; (7) Nerve; (8) Spinal Cord/Normal; (9) Brain Cortex/Normal; (10) Brain hypothalamus; (11) Glial Cells (astrocytes); (12) Glioblastoma; (13) Breast/Normal; (14) Breast/IDC; (15) Ovary/Normal; (16) Ovary/Tumor; (17) Pancreas; (18) Prostate/Normal; (19) Prostate/tumor adenocarcinoma; (20) Colon/Normal; (21) Colon/Tumor; (22) Colon/IBD; (23) Kidney/Normal; (24) Liver/Normal; (25) Liver/Fibrosis; (26) Fetal Liver/Normal; (27) Lung/Normal; (28) COPD; (29) Spleen/Normal; (30) Tonsil/Normal; (31) Lymph Node/Normal; (32) Thymus/Normal; (33) Epithelial Cells; (34) Endothelial cells; (35) Skeletal Muscle/Normal; (36) Fibroblasts; (37) Skin/Normal; (38) Adipose/normal; (39) Osteoblast/Primary; (40) Osteoblast/undifferentiated; (41) Osteoblast/differentiated; and (42) Osteoclasts. Elevated 14760 mRNA expression was observed in normal brain (e.g., cortex and hypothalamus), and normal fetal liver and fetal heart.

FIG. 12B is a bar graph depicting relative 14760 mRNA expression as determined by TaqMan assays on mRNA derived from the following tissues and cell lines. Columns are numbered at five-column intervals at the bottom of the Figure (i.e., columns 1-46), and correspond to the following: (1) Heart; (2) Lung; (3) Kidney; (4) Fetal Liver; (5) Spleen; (6) Granulocytes.; (7) NHDF mock; (8) NHLF mock; (9) NHLF TGF; (10) HepG2 Mock; (11) HepG2 TGF; (12) Pass Stell; (13) Liver Pool; (14) Control liver; (15) LF/NDR 191; (16) LF/NDR 193; (17) LF/NDR 079; (18) LN NDR 173; (19) Tonsil; (20) TH1 24 hr. MP39; (21) TH2 24 hr. MP39; (22) TH1 24 hr. MP21; (23) TH2 24 hr. MP21; (24) CD4; (25) CD8; (26) CD19; (27) CD3 MP42 rest; (28) CD14; (29) PBMC MOCK; (30) Bone marrow mononuclear cells (BM MNC); (31) CD34-positive cells (MPB CD34+); (32) Bone marrow glycophorin-positive cells (BM GPA+); (33) Cord Blood; (34) Erythroid; (35) Megakaryocytes; (36) Neutrophils (Neut) after 14 days in culture (d14); (37) CD14−/CD15+; (38) MBM CD11b; (39) HepG2; (40) HepG2.2.15; (41) MAI 01; (42) HL60; (43) K562; (44) Molt 4; (45) Hep3B Normoxia; and (46) Hep3B Hypoxia. Elevated 14760 mRNA expression was observed in pass stell, bone marrow glycophorin-positive cell lines, MOLT-4 cell lines and fetal liver.

FIG. 12C is a bar graph (cardiovascular organ panel) depicting relative 14760 mRNA expression as determined by TaqMan assays on mRNA derived from the following cardiovascular tissues: normal atria; normal left ventricle; diseased right ventricle; diseased left ventricle; kidney; liver; and skeletal muscle. Elevated 14760 mRNA expression was observed in skeletal muscle and cardiovascular tissues.

FIG. 13 depicts a hydropathy plot of human 53070. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 53070 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 63 to 73, from about 86 to 102, and from about 199 to 216 of SEQ ID NO:15; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 103 to 119, from about 226 to 247, and from about 301 to 329 of SEQ ID NO:15.

FIG. 14 depicts an alignment of the protein kinase domain of human 53070 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:17), while the lower amino acid sequence corresponds to amino acids 12 to 272 of SEQ ID NO:15.

FIG. 15 depicts an alignment of the serine/threonine protein kinase domain of human 53070 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:18), while the lower amino acid sequence corresponds to amino acids 12 to 272 of SEQ ID NO:15.

FIG. 16 depicts a hydropathy plot of human 15985. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 15985 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 83 to 91, from about 465 to 472, and from about 568 to 585 of SEQ ID NO:21; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 8 to 20, from about 592 to 600, and from about 652 to 672 of SEQ ID NO:21; a sequence which includes a Cys, or a glycosylation site.

FIG. 17 depicts an alignment of the protein kinase domain of human 15985 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:23), while the lower amino acid sequence corresponds to amino acids 394 to 651 of SEQ ID NO:21.

FIGS. 18A-18B depicts an alignment of the doublecortin repeats of human 15985 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from SMART. A. The upper sequence is the consensus amino acid sequence (SEQ ID NO:24), while the lower amino acid sequence corresponds to the first doublecortin repeat of human 15985, amino acids 67 to 158 of SEQ ID NO:21. B. The upper sequence is the consensus amino acid sequence (SEQ ID NO:24), while the lower amino acid sequence corresponds to the second doublecortin repeat of human 15985, amino acids 192 to 280 of SEQ ID NO:21.

FIG. 19 depicts an alignment of the protein kinase domain of human 15985 with a consensus amino acid sequence for serine/threonine protein kinases derived from a hidden Markov model (HMM) from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:25), while the lower amino acid sequence corresponds to the protein kinase domain of human 15985, amino acids 394 to 651 of SEQ ID NO:21.

FIG. 20 depicts an alignment of the doublecortin repeats of human 15985 with a consensus amino acid sequence derived from a ProDom family PD024506 (ProDomain Release 2000.1; http://www.toulouse.inra.fr/). The lower sequence is the consensus amino acid sequence (SEQ ID NO:26), while the upper amino acid sequence corresponds to the doublecortin repeats of human 15985, amino acids 42 to 291 of SEQ ID NO:21.

FIG. 21 depicts a hydropathy plot of human 50365. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 50365 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence of from about amino acid residue 365 to about amino acid residue 380, or from about amino acid residue 645 to about amino acid residue 655, of SEQ ID NO:28; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid residue 98 to about amino acid residue 120, or from about amino acid residue 715 to about amino acid residue 745 of SEQ ID NO:28. The two hexokinase domains are indicated.

FIGS. 22A-22D depict an alignment of the two hexokinase domains of 50365 with a consensus amino acid sequence derived from a hidden Markov model (PFAM Accession PF00349). The upper sequence is the consensus amino acid sequence (SEQ ID NO:30), while the lower amino acid sequence corresponds to amino acids 16 to 463 (FIGS. 22A and 22B) and amino acids 464 to 910 of SEQ ID NO:28 (FIGS. 22C and 22D).

FIGS. 23A-23B depicts a cDNA sequence (SEQ ID NO:32) and predicted amino acid sequence (SEQ ID NO:33) of human 26583. The methionine-initiated open reading frame of human 26583 (without the 5′ and 3′ untranslated regions) starts at nucleotide 462 and ends at nucleotide 2075 of SEQ ID NO:32 (shown also as coding sequence (SEQ ID NO:34)).

FIG. 24 depicts a hydropathy plot of human 26583. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 26583 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence of 262-279; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of 60-70; a sequence which includes a Cys, or a glycosylation site.

FIGS. 25A-25B depict alignments of human 26583 amino acid sequence with a consensus amino acid sequence derived from protein phosphatase 2C (PP2C) (FIG. 25A) and protein phosphatase 2C_—4 (PP2C_—4) (FIG. 25B). In FIG. 25A, the upper sequence is the consensus amino acid sequence (SEQ ID NO:35) for PP2C, while the lower amino acid sequence corresponds to amino acids 173 to 461 of SEQ ID NO:33. In FIG. 25B, the upper sequence is the consensus amino acid sequence (SEQ ID NO:36) for PP2C_—4, while the lower amino acid sequence corresponds to amino acids 99 to 522 of SEQ ID NO:33.

FIG. 26 shows a bar graph depicting relative 26583 mRNA expression as determined by TaqMan assays on mRNA derived from the following tissue samples. Columns are numbered at five-column intervals at the bottom of the Figure (i.e., columns 1-42), and correspond to the following: columns 1-3, normal breast; columns 4-10, breast tumor; columns 11-13, normal lung; columns 14-20, lung tumor; columns 21-23, normal colon; columns 24-31, colon tumor; columns 32-35, colon metastases; columns 36-37, normal liver; columns 38-39, normal brain; columns 40-42, brain tumor.

FIG. 27 depicts a hydropathy plot of human 21953. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 21953 are indicated.

FIG. 28 depicts an alignment of the prolyl oligopeptidase domain of human 21953 with a consensus amino acid sequence derived from a hidden Markov model for prolyl oligopeptidase domains. The upper sequence is the consensus amino acid sequence (SEQ ID NO:40), while the lower amino acid sequence corresponds to amino acids 672 to 744 of SEQ ID NO:38.

FIGS. 29A-29B depict an alignment of human dipeptidyl peptidase IV (Accession Number P48147) (upper line, SEQ ID NO:41), to the 21953 amino acid sequence. The * symbol indicates identities, and the: or. symbols indicate similarities. The alignment was generated by ClustalW (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680).

FIG. 30 depicts a hydropathy plot of human m32404. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human m32404 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 320 to 340, and from about 450-470, of SEQ ID NO:43; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence from about amino acid 30 to 60 of SEQ ID NO:43; a sequence which includes a Cys, or a glycosylation site.

FIGS. 31A-31B depict alignments of the trypsin domains of human m32404 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:45), while the lower amino acid sequence corresponds to amino acids 45 to 268 of SEQ ID NO:43 (FIG. 31A) or upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower amino acid sequence corresponds to amino acids 311 to 520 of SEQ ID NO:43 (FIG. 31B).

FIGS. 32A-32B depict alignments of the trypsin domains of human m32404 with a consensus amino acid sequence for a model trypsin domain from SMART. The upper sequence is the consensus amino acid sequence (SEQ ID NO:47), while the lower amino acid sequence corresponds to amino acids 38 to 268 of SEQ ID NO:43 (FIG. 32A) or to amino acids 300 to 520 of SEQ ID NO:43 (FIG. 32B).

FIG. 33 depicts a hydropathy plot of human 14089. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Cysteine (cys) residues are noted by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 14089 are indicated. Polypeptides of the invention include fragments that include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 35 to 55, from about 58 to 70, and from about 175 to 184 of SEQ ID NO:52; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 71 to 79, from about 161 to 171, and from about 185 to 192 of SEQ ID NO:52; a sequence which includes a Cys, or a glycosylation site.

FIGS. 34A-34B depict alignments of the trypsin domain of human 14089 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM (3A) and SMART (3B). The upper sequences are the consensus amino acid sequences (SEQ ID NO:54 and SEQ ID NO:55), while the lower amino acid sequence corresponds to amino acids 41 to 234 of SEQ ID NO:52 and amino acids 24 to 234 of SEQ ID NO:52 (FIGS. 34A and 34B, respectively).

FIGS. 35A-35B depict a BLAST alignment of the serine protease zymogen domain of human 14089 with a consensus amino acid sequence derived from ProDomain No. 46 (Release 1999.2; see also ProDom family PD00000046 (ProDomain Release 2000.1); http://www.toulouse.inra.fr/prodom.html). FIG. 35A: The lower sequence is the consensus amino acid sequence (SEQ ID NO:56), while the upper amino acid sequence corresponds to the serine protease zymogen domain of human 14089, about amino acids 72 to 234 of SEQ ID NO:52. FIG. 35B: The lower sequence is the consensus amino acid sequence (SEQ ID NO:57), while the upper amino acid sequence corresponds to the serine protease zymogen domain of human 14089, about amino acids 41 to 109 of SEQ ID NO:52.

FIGS. 36A-36B depicts a cDNA sequence (SEQ ID NO:58) and predicted amino acid sequence (SEQ ID NO:59) of human 23436. The methionine-initiated open reading frame of human 23436 (without the 5′ and 3′ untranslated regions) until the end of SEQ ID NO:58 is shown also as coding sequence SEQ ID NO:60.

FIG. 37 depicts a hydropathy plot of human 23436. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 23436 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 103 to 114, from about 285 to 297, and from about 413 to 420 of SEQ ID NO:59; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 76 to 87, from about 138 to 143, and from about 458 to 478 of SEQ ID NO:59; a sequence which includes a Cys, or a glycosylation site.

FIGS. 38A-38B depict alignment of the ubiquitin carboxy-terminal hydrolase (family 2) domain of human 23436 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM. The consensus sequence for the ubiquitin carboxy-terminal hydrolase (family 2) domain comprises two non-contiguous segments, UCH-1 and UCH-2. FIG. 38A depicts the alignment of human 23436 with the UCH-1 segment of the ubiquitin carboxy-terminal hydrolase (family 2) domain. The upper sequence is the consensus amino acid sequence (SEQ ID NO:61), while the lower amino acid sequence corresponds to amino acids 89 to 120 of SEQ ID NO:59. FIG. 38B depicts the alignment of human 23436 with the UCH-2 segment of the ubiquitin carboxy-terminal hydrolase (family 2) domain. The upper sequence is the consensus amino acid sequence (SEQ ID NO:62), while the lower amino acid sequence corresponds to amino acids 332 to 420 of SEQ ID NO:59.

FIG. 39 is a bar graph depicting relative 23436 mRNA expression as determined by TaqMan assays on mRNA derived from human hematological cell lines treated for various times with transforming growth factor-β (TGF-β) and VPA. Erythroid lineage precursors have elevated 23436 expression levels. Expression is reduced by TGF-β treatment.

FIG. 40 is a bar graph depicting relative 23436 mRNA expression as determined by TaqMan assays on mRNA derived from human hematological cells including neutrophils, platelets, blood forming units (BFU), and TGFβ-treated hematopoietic precursors. BFUs treated with erythropoietin (EPO) have elevated 23436 expression levels.

FIG. 41 is a bar graph depicting relative 23436 mRNA expression as determined by TaqMan assays on mRNA derived from the following cell types: (1) lung; (2) kidney; (3) fetal liver; (4) grans.; (5) NHDF mock; (6) NHDF TGF; (7) NHLF mock; (8) NHLF TGF; (9) NC Heps; (10) Pass Stell; (11) Liver CHT 339; (12) Liver NDR 191; (13) LF NDR 079; (14) Lymph Node; (15) Th0 046 6h; (16) Th1 046 6h; (17) Th2 046 6h; (18) CD8; (19) CD14; (20) PBMC Rest; (21) MBM MNC; (22) MPB CD34; (23) ABM CD34; (24) Cord Blood; (25) Erythroid cells; (26) Megakaryocytes; (27) Neutrophil d14; (28) CD15+/CD14-cells; (29) MBM CD11b−; (30) BM GPA; (31) VZV mock; (32) VZV 18h; (33) VZV 72h; (34) K562; (35) NTC; (36) HL60; (37) Molt4; (38) Hep3b Normal; and (39) Hep3b Hyp. Erythroid K562 cells (34), erythroid cells (26), and fetal liver cells (3) have elevated 23436 mRNA expression levels.

FIG. 42 is a bar graph depicting relative 23436 mRNA expression as determined by TaqMan assays on mRNA derived from the following cell types: (1) Lung; (2) Colon 60; (3) Kidney 58; (4) Liver NDR 200; (5) Fetal Liver 425; (6) Skeletal Muscle 167; (7) mBone Marrow MNC LP139; (8) mBone Marrow CD34+ LP92; (9) mBone Marrow CD34+ LP143; (10) mPB CD34+ LF70; (11) mPB CD34+ LP152; (12) Bone Marrow CD34+ LF68; (13) Bone Marrow CD34+ LF154; (14) Cord Blood CD34+ LP121; (15) Bone Marrow GPA+; (16) Bone Marrow GPA+ LP34-1; (17) Bone Marrow GPA Lo LP69; (18) Bone Marrow GPA Lo LP82; (19) Bone Marrow CD41+ CD14− LP78; (20) mBone Marrow CD15+ LP15; (21) mBone Marrow CD15+ CD11b− LP7-4; (22) mBone Marrow CD15+ CD11b+ LP15-2; (23) Bone Marrow CD15+ CD11b− LP23-2; (24) Bone Marrow CD15+ CD34− LP27-2; (25) Bone Marrow CD15+ CD34− LP41-1; (26) Erythroid 24hr LF90; (27) Erythroid 48hr LF73; (28) Erythroid 48hr LF76; (29) Erythroid 48hr LF90; (30) Erythroid d6 LP31-1; (31) Erythroid d7 LF24-5; (32) Erythroid d10 LP25-4; (33) Erythroid d1 2 LF23-8; (34) Erythroid d12 LF24-10; (35) Erythroid d14 GPA+ LP31-4; (36) Meg 48hr LF76; (37) Meg 48hr LF790; (38) Meg d7 LP31-2; (39) Meg d12 LF102; (40) Meg d12 LF35; and (41) Meg d14 LP31-5. Fetal Liver (5) and day 12 erythroid cells (33) and (34) have elevated 23436 mRNA expression levels.

FIG. 43 is a bar graph depicting 23436 expression in human prostate, hypothalamus, lung, bone marrow, differentiated osteoblasts, and aorta cells as assessed by TaqMan analysis. Elevated expression is observed in some prostate, hypothalamus, and bone marrow cells. Relative expression levels were determined by normalizing against a trachea control.

FIG. 44 is a bar graph depicting 23436 expression in human liver, several hepatoma cell lines (HepG2) and ganglia, as assessed by TaqMan analysis. Elevated expression is observed in hepatoma cells (HepG2 cell line). Relative expression levels were determined by normalizing against a trachea control.

FIG. 45 is a bar graph depicting 23436 expression as determined by TaqMan assays on mRNA derived from the following cell types: (1) brain; (2) brain cortex; (3) breast; (4) colon tumor; (5) heart; (6) kidney; (7) liver norm; (8) liver fib; (9) lung tumor; (10) ovary; (11) fetal liver; (12) mBM CD34+ LP92; (13) mBM CD34+ LP143; (14) mPB CD34+ LF70; (15) mPB CD34+ LF162; (16) BM CD34+ LF93; (17) BM CD34+ LP154; (18) Cord Blood CD34+ LF101; (19) GPA+ High LP34-1; (20) GPA+ High 69; (21) GPA+ High 74; (22) Gpa+ Low LP69; (23) GPA+ Low LP82; (24) Ery 24hr LF102; (25) Ery 48h LF87; (26) Ery 48h LF102; (27) Ery 48h LF72; (28) Ery d6 LP31-1; (29) Ery d6 LF113; (30) Ery d7 LF24-5; (31) Ery d8 LF113; (32) Ery d10 LP24-4; (33) Ery d12 LF23-8; (34) Ery d12 LF24-10; (35) Ery d12 LF113; (36) Ery d14 GPA+ LP31-4; (37) BFU d7 LP79; (38) BFU d7 LP95; (39) BFU d7+3 Epo LP81; and (40) BFU d7+3 Epo LP104.

DETAILED DESCRIPTION OF THE 2504, 15977, AND 14760 INVENTION

Human 2504

The human 2504 sequence (FIGS. 1A-1B; SEQ ID NO:1), which is approximately 2297 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1503 nucleotides (nucleotides 154-1656 of SEQ ID NO:1; SEQ ID NO:3). The coding sequence encodes a 501 amino acid protein (SEQ ID NO:2).

This mature protein form is approximately 501 amino acid residues in length (from about amino acid 1 to amino acid 501 of SEQ ID NO:2). Human 2504 contains the following regions or other structural features (FIGS. 3A and 3B): a eukaryotic protein kinase domain (PFAM Accession PF00069) located at about amino acid residues 37 to 286 of SEQ ID NO:2; and a serine/threonine kinase domain located at about amino acid residues 24 to 286 of SEQ ID NO:2.

The 2504 protein also includes the following domains: 12 predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 21 to 23, 46-48, 51-53, 91-93, 103-105, 118-120, 138-140, 292-294, 422-424, 482-484, and 495-497 of SEQ ID NO:2; 10 predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino 7-10, 91-94, 103-106, 118-121, 276-279, 341-344, 364-367, 470-473, 483-486, and 495-498 of SEQ ID NO:2; two predicted tyrosine kinase phosphorylation sites (PS00007) located at about amino acids 127-135 and 484-491 of SEQ ID NO:2; two predicted N-myristoylation sites (PS00008) located at about amino acids 288-293 and 349-354 of SEQ ID NO:2; and one predicted amidation site located at about amino acids 59-62 of SEQ ID NO:2.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

Human 15977

The human 15977 sequence (FIGS. 4A-4C; SEQ ID NO:4), which is approximately 4417 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1377 nucleotides (nucleotides 337-1713 of SEQ ID NO:4; SEQ ID NO:6). The coding sequence encodes a 459 amino acid protein (SEQ ID NO:5).

This mature protein form is approximately 459 amino acid residues in length (from about amino acid 1 to amino acid 459 of SEQ ID NO:5). Human 15977 contains the following regions or other structural features (FIGS. 6A and 6B): a eukaryotic protein kinase domain (PFAM Accession PF00069) located at about amino acid residues 44 to 276 of SEQ ID NO:5; and a serine/threonine kinase domain located at about amino acid residues 44 to 329 of SEQ ID NO:5.

The 15977 protein also includes the following domains: two predicted N-glycosylation sites (PS00001) located at about amino acids 370-373 and 388-391 of SEQ ID NO:5; two cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acids 270-273 and 451-454 SEQ ID NO:5; nine predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 14-16, 137-139, 199-201, 214-216, 229-231, 258-260, 269-271, 355-357, and 373-375 of SEQ ID NO:5; eight predicted Casein Kinase II sites (PS00006) located at about amino 96-99, 124-127, 150-153, 229-232, 258-261, 273-276, 355-358, and 411-414 of SEQ ID NO:5; two predicted N-myristoylation sites (PS00008) located at about amino 30-35 and 422-427 of SEQ ID NO:2; one predicted amidation site (PS00009) located at about amino acids 46-49 of SEQ ID NO:5; and a Serine/Threonine protein kinase active-site signature (PS 00108) located at about amino acids 160-172 of SEQ ID NO:5.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

Human 14760

The human 14760 sequence (FIGS. 7A-7B; SEQ ID NO:7), which is approximately 2046 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1788 nucleotides (nucleotides 119-1906 of SEQ ID NO:7; SEQ ID NO:9). The coding sequence encodes a 596 amino acid protein (SEQ ID NO:8).

This mature protein form is approximately 596 amino acid residues in length (from about amino acid 1 to amino acid 596 of SEQ ID NO:2). Human 14760 contains the following regions or other structural features (FIGS. 9A and 9B): a eukaryotic protein kinase domain (PFAM Accession PF00069) located at about amino acid residues 285 to 540 of SEQ ID NO:8; and a serine/threonine kinase domain located at about amino acid residues 285 to 540 of SEQ ID NO:8.

The 14760 protein also includes the following domains: two predicted N-glycosylation sites (PS00001) located at about amino acids 278-281 and 416-419 of SEQ ID NO:8; three cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acids 140-143, 317-320, and 583-586 SEQ ID NO:8; 11 predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 17-19, 49-51, 59-61, 107-109, 159-161, 203-205, 224-226, 235-237, 247-249, 320-322, and 460-462 of SEQ ID NO:8; eight predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino 157-160, 184-187, 203-206, 247-250, 301-304, 320-323, 351-354, and 379-382 of SEQ ID NO:8; one predicted tyrosine kinase phosphorylation sites (PS00007) located at about amino acids 370-376 of SEQ ID NO:8; nine predicted N-myristoylation sites (PS00008) located at about amino acids 83-88, 116-121, 135-140, 178-183, 241-246, 277-282, 293-298, 308-313, and 589-59 ID NO:8; one predicted amidation site (PS00009) located at about amino acids 128-131 of SEQ ID NO:8; a protein kinases ATP-binding region signature located at about amino acids 291-299 of SEQ ID NO:8; and a Serine/Threonine protein kinase active-site signature (PS 00108) located at about amino acids 402-414 of SEQ ID NO:8.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

TABLE 1
Summary of Sequence Information for 2504,
15977, and 14760

					ATCC
					Accession
Gene	cDNA	ORF	Polypeptide	Figure	Number

2504	SEQ ID	SEQ ID	SEQ ID NO	FIG.
	NO:1	NO:3	NO:2	1A-B
15977	SEQ ID	SEQ ID	SEQ ID	FIG.
	NO:4	NO:6	NO:5	4A-C
14760	SEQ ID	SEQ ID	SEQ ID	FIG.
	NO:7	NO:9	NO:8	7A-B

TABLE 2
Summary of Domains of 2504, 15977,
and 14760

		Serine/Threonine Kinase
Protein	Protein Kinase Domain	Domain

2504	About amino acids 37-286	About amino acids 24-286
	of SEQ ID NO:2	of SEQ ID NO:2
15977	About amino acids 44-276	About amino acids 44-329
	of SEQ ID NO:5	of SEQ ID NO:5
14760	About amino acids 285-540	About amino acids 285-540
	of SEQ ID NO:8	of SEQ ID NO:8

The 2504, 15977, and 14760 proteins contains a significant number of structural characteristics in common with members of the protein kinase family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

A 2504, 15977, or 14760 polypeptide can include a “protein kinase domain” or regions homologous with a “protein kinase domain”.

As used herein, the term “protein kinase” includes a protein or polypeptide which is capable of modulating its own phosphorylation state or the phosphorylation state of another protein or polypeptide. Protein kinases can have a specificity for (i.e., a specificity to phosphorylate) serine/threonine residues, tyrosine residues, or both serine/threonine and tyrosine residues, e.g., the dual specificity kinases. As referred to herein, protein kinases preferably include a catalytic domain of about 200-400 amino acid residues in length, preferably about 200-300 amino acid residues in length, or more preferably about 250-300 amino acid residues in length. Specificity of a protein kinase for phosphorylation of either tyrosine or serine/threonine can be predicted by the sequence of two of the subdomains (VIb and VIII) in which different residues are conserved in each class (as described in, for example, Hanks et al. (1988) Science 241:42-52) the contents of which are incorporated herein by reference). These subdomains are also described in further detail herein.

Protein kinases play a role in signaling pathways associated with cellular growth. For example, protein kinases are involved in the regulation of signal transmission from cellular receptors, e.g., growth-factor receptors; entry of cells into mitosis; and the regulation of cytoskeleton function, e.g., actin bundling. Thus, the molecules of the present invention may be involved in: 1) the regulation of transmission of signals from cellular receptors, e.g., cell growth factor receptors; 2) the modulation of the entry of cells, e.g., precursor cells, into mitosis; 3) the modulation of cellular differentiation; 4) the modulation of cell death; and 5) the regulation of cytoskeleton function, e.g., actin bundling.

Inhibition or over stimulation of the activity of protein kinases involved in signaling pathways associated with cellular growth can lead to perturbed cellular growth, which can in turn lead to cellular growth related disorders. As used herein, a “cellular growth related disorder” includes a disorder, disease, or condition characterized by a deregulation, e.g., an upregulation or a downregulation, of cellular growth. Cellular growth deregulation may be due to a deregulation of cellular proliferation, cell cycle progression, cellular differentiation and/or cellular hypertrophy. Examples of cellular growth related disorders include cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina; proliferative disorders or differentiative disorders such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma.

As used herein, the term “protein kinase domain” includes an amino acid sequence of about 150 to 400 amino acid residues in length and having a bit score for the alignment of the sequence to the protein kinase domain (HMM) of at least 50. Preferably, a protein kinase domain includes at least about 200-400 amino acids, more preferably about 200-300 amino acid residues, or about 220-270 amino acids and has a bit score for the alignment of the sequence to the protein kinase domain (HMM) of at least 120 or greater. The protein kinase domain (HMM) has been assigned the PFAM Accession PF00069 (http://genome.wustl.edu/Pfam/html). An alignment of the protein kinase domain (amino acids 37 to 286 of SEQ ID NO:2) of human 2504 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 3A. An alignment of the protein kinase domain (amino acids 44 to 276 of SEQ ID NO:5) of human 15977 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 6A. An alignment of the protein kinase domain (amino acids 285 to 540 of SEQ ID NO:8) of human 14760 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 9A.

In a preferred embodiment 2504, 15977, or 14760 polypeptide or protein has a “protein kinase domain” or a region which includes at least about 200-400 more preferably about 200-300 or 220-270 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “protein kinase domain,” e.g., the protein kinase domain of human 2504, 15977, or 14760 (e.g., residues 37-286 of SEQ ID NO:2; residues 44-276 of SEQ ID NO:5, or residues 285-540 of SEQ ID NO:8).

A 2504, 15977, or 14760 molecule can further include a “serine/threonine kinase domain.”

As used herein, the term “serine/threonine kinase domain” includes an amino acid sequence of about 150 to 400 amino acid residues in length and having a bit score for the alignment of the sequence to the protein kinase domain (HMM) of at least 15. Preferably, a serine/threonine kinase domain includes at least about 200-400 amino acids, more preferably about 200-300 amino acid residues, or about 220-270 amino acids and has a bit score for the alignment of the sequence to the serine/threonine kinase domain (HMM) of at least 50 or greater. An alignment of the serine/threonine kinase domain (amino acids 24 to 286 of SEQ ID NO:2) of human 2504 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 3B. An alignment of the serine/threonine kinase domain (amino acids 44 to 329 of SEQ ID NO:5) of human 15977 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 6B. An alignment of the serine/threonine kinase domain (amino acids 285 to 540 of SEQ ID NO:8) of human 14760 with a consensus amino acid sequence derived from a hidden Markov model is depicted in FIG. 9A.

In a preferred embodiment 2504, 15977, or 14760 polypeptide or protein has a “serine/threonine kinase domain” or a region which includes at least about 200-400 more preferably about 200-300 or 220-270 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “serine/threonine kinase domain,” e.g., the serine/threonine kinase domain of human 2504, 15977, or 14760 (e.g., residues 24-286 of SEQ ID NO:2; residues 44-329 of SEQ ID NO:5, or residues 285-540 of SEQ ID NO:8).

To identify the presence of a “protein kinase” domain or a “serine/threonine kinase” domain in a 2504, 15977, or 14760 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM—search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al.(1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

A 2504, 15977, or 14760 family member can include a protein kinase domain, e.g. a serine/threonine kinase domain.

As the 2504, 15977, or 14760 polypeptides of the invention may modulate 2504, 15977, or 14760-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 2504, 15977, or 14760-mediated or related disorders, as described below.

As used herein, a “2504, 15977, or 14760 activity”, “biological activity of 2504, 15977, or 14760” or “functional activity of 2504, 15977, or 14760”, refers to an activity exerted by a 2504, 15977, or 14760 protein, polypeptide or nucleic acid molecule on e.g., a 2504, 15977, or 14760-responsive cell or on a 2504, 15977, or 14760 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, a 2504, 15977, or 14760 activity is a direct activity, such as an association with a 2504, 15977, or 14760 target molecule. A “target molecule” or “binding partner” is a molecule with which a 2504, 15977, or 14760 protein binds or interacts in nature, e.g., a protein containing one or more serine and or threonine residues. A 2504, 15977, or 14760 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 2504, 15977, or 14760 protein with a 2504, 15977, or 14760 receptor. For example, the 2504, 15977, or 14760 proteins of the present invention can have one or more of the following activities: 1) the regulation of transmission of signals from cellular receptors, e.g., cell growth factor receptors; 2) the modulation of the entry of cells, e.g., precursor cells, into mitosis; 3) the modulation of cellular differentiation; 4) the modulation of cell death; 5) the regulation of cytoskeleton function, e.g., actin bundling; or 6) the ability to phosphorylate a substrate.

Based on the above-described sequence similarities, the 2504, 15977, and 14760 molecules of the present invention are predicted to have similar biological activities as protein kinase family members. Thus, the 2504, 15977, and 14760 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, hematopoietic disorders, cardiovascular disorders, liver disorders, viral diseases, pain or metabolic disorders.

In addition, the 2504, 15977, and 14760 molecules of the invention may modulate physiological and pathological processes in the cells or tissues where they are expressed. For example, Taq Man studies described herein show abundant expression of 2504, 15977, and 14760 mRNAs in neural tissues, including the brain cortex and hypothalamus (FIGS. 10, 11 and 12A). 15977 mRNA is also highly expressed in epithelial cells, astrocytes (glial cells), HUVEC cells, smooth muscle cells and fetal liver (FIG. 11). 14760 mRNA is also abundantly expressed in the fetal liver, endothelial cells, fetal heart, fibroblasts, bone marrow glycophorin-positive cells, hepatocytes, cardiovascular cells, and skeletal muscle. Accordingly, these molecules can act as novel diagnostic targets and therapeutic agents of disorders involving the cells or tissues where they are expressed, e.g., neural (e.g., brain or astrocytic) disorders; cardiovascular and blood vessel (smooth muscle or endothelial cell) disorders; immune disorders (e.g., disorders involving glycophorin-positive cells); hepatic or liver disorders; skin disorders; skeletal disorders, among others.

Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

Aberrant expression and/or activity of 2504, 15977, or 14760 molecules may mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by 2504, 15977, or 14760 molecules effects in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration. For example, 2504, 15977, or 14760 molecules may support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, 2504, 15977, or 14760 molecules that modulate the production of bone cells can influence bone formation and degeneration, and thus may be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

The 2504, 15977, or 14760 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders. Exemplary immune disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

Additional examples of immune disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

Examples of cardiovascular disorders include, but are not limited to, heart failure, cardiac hypertrophy, left-sided heart failure, and right-sided heart failure; ischemic heart disease, including but not limited to angina pectoris, myocardial infarction, chronic ischemic heart disease, and sudden cardiac death; hypertensive heart disease, including but not limited to, systemic (left-sided) hypertensive heart disease and pulmonary (right-sided) hypertensive heart disease; valvular heart disease, including but not limited to, valvular degeneration caused by calcification, such as calcific aortic stenosis, calcification of a congenitally bicuspid aortic valve, and mitral annular calcification, and myxomatous degeneration of the mitral valve (mitral valve prolapse), rheumatic fever and rheumatic heart disease, infective endocarditis, and noninfected vegetations, such as nonbacterial thrombotic endocarditis and endocarditis of systemic lupus erythematosus (Libman-Sacks disease), carcinoid heart disease, and complications of artificial valves; myocardial disease, including but not limited to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and myocarditis; pericardial disease, including but not limited to, pericardial effusion and hemopericardium and pericarditis, including acute pericarditis and healed pericarditis, and rheumatoid heart disease; neoplastic heart disease, including but not limited to, primary cardiac tumors, such as myxoma, lipoma, papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms; congenital heart disease, including but not limited to, left-to-right shunts—late cyanosis, such as atrial septal defect, ventricular septal defect, patent ductus arteriosus, and atrioventricular septal defect, right-to-left shunts—early cyanosis, such as tetralogy of fallot, transposition of great arteries, truncus arteriosus, tricuspid atresia, and total anomalous pulmonary venous connection, obstructive congenital anomalies, such as coarctation of aorta, pulmonary stenosis and atresia, and aortic stenosis and atresia, and disorders involving cardiac transplantation.

Disorders involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease—the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis (microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis, thromboanglitis obliterans (Buerger disease), vasculitis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and aortic dissection (dissecting hematoma); disorders of veins and lymphatics, such as varicose veins, thrombophlebitis and phlebothrombosis, obstruction of superior vena cava (superior vena cava syndrome), obstruction of inferior vena cava (inferior vena cava syndrome), and lymphangitis and lymphedema; tumors, including benign tumors and tumor-like conditions, such as hemangioma, lymphangioma, glomus tumor (glomangioma), vascular ectasias, and bacillary angiomatosis, and intermediate-grade (borderline low-grade malignant) tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignant tumors, such as angiosarcoma and hemangiopericytoma; and pathology of therapeutic interventions in vascular disease, such as balloon angioplasty and related techniques and vascular replacement, such as coronary artery bypass graft surgery.

Disorders which may be treated or diagnosed by methods described herein include, but are not limited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, A1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

Additionally, 2504, 15977, or 14760 molecules may play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 2504, 15977, or 14760 activity could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 2504, 15977, or 14760 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

Additionally, 2504, 15977, or 14760 may play an important role in the regulation of metabolism or pain disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H. L. (1987) Pain, New York:McGraw-Hill); pain associated with muscoloskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; http://164.195.100.11/netacgi/nph-Parser?Sect1=PTO2&Sect2-HITOFF&p=1&u=/netahtml/search-bool.html&r=3&f=G&1=50&col=AND&d=curr&s1-millennium.ASNM.&s2=pain&OS=AN/millennium+AND+pain&RS=AN/-h3http://164.195.100.11/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=/netahtml/search-bool.html&r=3&f=G&1=50&co1=AND&d=curr&s1=millennium.ASNM.&s2=pain&OS=AN/millennium+AND+pain&RS=AN/-h5pain related to irritable bowel syndrome; or chest http://164.195.100.11/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=/netahtml/search-bool.html&r=3&f=G&1=50&co1=AND&d=curr&s1=millennium.ASNM.&s2=pain&OS=AN/millennium+AND+pain&RS=AN/-h4http://164.195.100.11/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=/netahtml/search-bool.html&r=3&f=G&1=50&co1=AND&d=curr&s1=millennium.ASNM.&s2=pain&OS=AN/millennium+AND+pain&RS=AN/-h6pain.

Disorders involving the skeletal muscle include tumors such as rhabdomyosarcoma. Diseases of the skin, include but are not limited to, disorders of pigmentation and melanocytes, including but not limited to, vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, and malignant melanoma; benign epithelial tumors, including but not limited to, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors of the dermis, including but not limited to, benign fibrous histiocytoma, dernatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of cellular immigrants to the skin, including but not limited to, histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis; disorders of epidermal maturation, including but not limited to, ichthyosis; acute inflammatory dermatoses, including but not limited to, urticaria, acute eczematous dernatitis, and erythema multiforme; chronic inflammatory dermatoses, including but not limited to, psoriasis, lichen planus, and lupus erythematosus; blistering (bullous) diseases, including but not limited to, pemphigus, bullous pemphigoid, dermatitis herpetiformis, and noninflammatory blistering diseases: epidermolysis bullosa and porphyria; disorders of epidermal appendages, including but not limited to, acne vulgaris; panniculitis, including but not limited to, erythema nodosum and erythema induratum; and infection and infestation, such as verrucae, molluscum contagiosum, impetigo, superficial fungal infections, and arthropod bites, stings, and infestations.

The 2504, 15977, or 14760 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8 thereof are collectively referred to as “polypeptides or proteins of the invention” or “2504, 15977, or 14760 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “2504, 15977, or 14760 nucleic acids.” 2504, 15977, or 14760 molecules refer to 2504, 15977, or 14760 nucleic acids, polypeptides, and antibodies.

As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

The term “isolated or purified nucleic acid molecule” includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a 2504, 15977, or 14760 protein, preferably a mammalian 2504, 15977, or 14760 protein, and can further include non-coding regulatory sequences, and introns.

An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language “substantially free” means preparation of 2504, 15977, or 14760 protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-2504, 15977, or 14760 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-2504, 15977, or 14760 chemicals. When the 2504, 15977, or 14760 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 2504, 15977, or 14760 (e.g., the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, without abolishing or more preferably, without substantially altering a biological activity, whereas an “essential” amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention, e.g., those present in the protein kinase or serine/threonine kinase domain, are predicted to be particularly unamenable to alteration.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 2504, 15977, or 14760 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 2504, 15977, or 14760 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 2504, 15977, or 14760 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4. SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

1. A method for identifying a candidate compound which modulates the ubiquitin hydrolase activity of a polypeptide selected from the group consisting of: a) a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:58, 60, or a complement thereof, b) a polypeptide comprising the amino acid sequence of SEQ ID NO:59, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:58, 60, or a complement thereof under conditions of hybridization in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes in 0.2×SSC, 1% SDS at 65° C.; c) a polypeptide comprising amino acids 89 to 420 of SEQ ID NO:59; and d) a polypeptide comprising the amino acid sequence of SEQ ID NO:59, the method comprising the steps of: i) contacting the polypeptide, or a cell expressing the polypeptide with a test compound; ii) determining the effect of the test compound on ubiquitin hydrolase activity of the polypeptide, to thereby identify a candidate compound which modulates ubiquitin hydrolase activity of the polypeptide; and iii) determining the effect of the candidate compound identified in ii) on a cellular activity selected from the group consisting of cell proliferation, cell signaling, cell death, cell motility, receptor-mediated endocytosis, organelle biogenesis, hematopoietic cell proliferation or differentiation, and cytokine-mediated signaling events.

2. The method of claim 1, wherein the test compound is labeled.

3. The method of claim 1, wherein the polypeptide is in liquid phase.

4. The method of claim 1, wherein the polypeptide is on a solid support.

5. The method of claim 1, wherein the test compound contacts a polypeptide expressed by a cell.

6. The method of claim 5, wherein the cell is selected from the group consisting of an erythroid cell, an erythroid progenitor cell, a liver cell, a prostate cell, a hypothalamus cell, a bone marrow cell, a brain cell, a kidney cell, an ovary cell, a human vascular endothelial cell, and a hematopoietic progenitor cell.

7. The method of claim 5, wherein the cell is selected from the group consisting of an erythroid cell, an erythroid progenitor cell, a liver cell, a prostate cell, and a hypothalamus cell.

8. The method of claim 1, wherein the cellular activity modulated by the test compound is cell signaling that is mediated by the 23436 protein or a protein de-ubiquitinated by 23436.

9. The method of claim 1, wherein the polypeptide is a fusion protein further comprising a polypeptide of the group consisting of glutathione-S-transferase (GST) and all or part of a serum protein.

10. The method of claim 5, wherein the cellular activity modulated by the test compound is cell proliferation or cell differentiation.

11. The method of claim 10, wherein the cell proliferation modulated by the test compound is growth factor mediated cell proliferation.

12. The method of claim 1, further comprising the step: (iv) determining the effect of the candidate compound identified in ii) on a hematopoietic disorder, an erythroid disorder, or a neoplastic disorder.

13. A method for identifying a candidate compound which modulates ubiquitin hydrolase activity of a polypeptide comprising the amino acid sequence of SEQ ID NO:59 the method comprising the steps of: a) contacting the polypeptide, or a cell expressing the polypeptide with a test compound; b) determining the effect of the test compound on ubiquitin hydrolase activity of the polypeptide, to thereby identify a candidate compound which modulates ubiquitin hydrolase activity of the polypeptide; and c) determining the effect of the candidate compound identified in b) on a cellular activity selected from the group consisting of cell proliferation, cell signaling, cell death, cell motility, receptor-mediated endocytosis, organelle biogenesis, hematopoietic cell proliferation or differentiation, and cytokine-mediated signaling events.