Abstract:
Disclosed are novel mammalian kinases, p54 S6K  and p85 S6K . Also disclosed are methods for identifying compounds that modulate, or which are modulated by, p54 S6K  or p85 S6K . In addition, the invention discloses methods for diagnosing or treating a cellular proliferative disease.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority from U.S. Provisional Application Ser. No. 60/106,141, filed Oct. 29, 1998. 
    
    
     STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made in part with Government funding, and the Government therefore has certain rights in the invention. 
     BACKGROUND OF THE INVENTION 
     The invention relates to the diagnosis and treatment of conditions associated with cell proliferation. 
     The control of cell proliferation is a fundamental issue in medicine. Cell proliferation is regulated by the formation and dissociation of multiple protein complexes, the components of which often use post-translational modifications as an additional control mechanism. Many diseases result from inappropriate cell proliferation, including cancer. Methods and reagents to modulate cell proliferation would have the potential to yield new treatments for cancer, major opportunistic infections, immune disorders, certain cardiovascular diseases, and inflammatory disorders. 
     SUMMARY OF THE INVENTION 
     In general, the invention features a substantially pure nucleic acid (for example, genomic DNA, cDNA, synthetic DNA, mRNA, or antisense RNA) encoding a p54 S6K  or p85 S6K  polypeptide, as defined below. 
     In a preferred embodiment, the substantially pure nucleic acid encoding a p54 S6K  or p85 S6K  polypeptide is mammalian DNA. More preferably, the substantially pure nucleic acid encoding a p54 S6K  or p85 S6K  polypeptide is human DNA. In another embodiment, the invention features a DNA sequence substantially identical to, or which hybridizes with high stringency to, the DNA sequence shown in FIG. 1A or  5  (SEQ ID NO: 1). 
     In another embodiment, the invention features DNA encoding fragments of p54 S6K  or p85 S6K  polypeptides. In preferred embodiments, the fragments include the C-terminal and N-terminal regions of p54 S6K or p 85 S6K  that are distinct from p70 S6k , the catalytic domain of p54 S6K  or p85 S6K , the linker domain, the proline rich domain, or the acidic region of p54 S6K  or p85 S6K . In preferred embodiments, the fragment contains amino acids 1-65 (N-terminal domain), 6-23 (acidic domain), 65-332 (catalytic domain), 332-397 (linker domain), 398-482 (C-terminal domain), or 446-467 (proline rich domain) of p54 S6K  (SEQ ID NOs: 4-10). In other preferred embodiments, the fragment contains amino acids 14-78 (N-terminal domain), 19-36 (acidic domain), 78-345 (catalytic domain), 345-410 (linker domain), 411-495 (C-terminal domain), or 495-480 (proline rich domain) of p85 S6K  (SEQ ID NOs: 10-15). In another embodiment, the invention features nucleic acids that bind with high stringency to SEQ ID NOs: 1 or 2. 
     In another aspect, the invention features a substantially pure polypeptide having a sequence substantially identical to SEQ ID NOs: 2 or 3. In a preferred embodiment, the substantially pure polypeptide has an acidic region at its N-terminus and a central catalytic region. In another the preferred embodiment, the substantially pure polypeptide has a post-translational modification 
     In other aspects of the invention, a substantially pure p54 S6K  or p85 S6K  polypeptide has one or more amino acids changed by natural or artificial means, the p54 S6K  or p85 S6K  polypeptide is p54 S6K  KR or p85 S6K  KR, or the p54 S6K  or p85 S6K  polypeptide is a fusion with a heterologous polypeptide, for example, glutathione-S-transferase (GST) or influenza hemagglutinin (HA), relative to the wild-type p54 S6K  or p85 S6K  sequence shown in FIG. 1A or FIG.  5 . 
     In another aspect, the invention features fragments of p54 S6K  or p85 S6K  polypeptides. In a preferred embodiment, the fragment is a peptide comprising a C-terminal sequence of p54 S6K  or p85 S6K  that is distinct from p70 S6k  or p85 S6K . In another embodiment, the fragment comprises a N-terminal sequence of p54 S6K  or p85 S6K  that is distinct from p70 S6k . In another embodiment, the fragment comprises the catalytic domain of p54 S6K  or p85 S6K . In another embodiment, the fragment comprises the acidic region of p54 S6K  or p85 S6K  In another embodiment, the fragment comprises the linker domain of p54 S6K  or p85 S6K . In another embodiment, the fragment comprises the proline rich region of p54 S6K  or p85 S6K . In another embodiment, the fragment contains a post-translational modification of p54 S6K  or p85 S6K . In yet another embodiment, the fragment is a fragment of p54 S6K  KR or p85 S6K  KR. In still another embodiment, the fragment is fused to a heterologous polypeptide. In preferred embodiments, the fragment contains amino acids 1-65 (N-terminal domain), 6-23 (acidic domain), 65-332 (catalytic domain), 332-397 (linker domain), 398-482 (C-terminal domain), or 446-467 (proline rich domain) of p54 S6K  (SEQ ID NOs: 4-9). In other preferred embodiments, the fragment contains amino acids 14-78 (N-terminal domain), 19-36 (acidic domain), 78-345 (catalytic domain), 345-410 (linker domain), 411-495 (C-terminal domain), or 495-480 (proline rich domain) of p85 S6K  (SEQ ID NOs: 10-15). 
     In another aspect, the invention features an antibody that specifically binds to a p54 S6K  or p85 S6K  polypeptide, a p54 S6K  or p85 S6K  polypeptide fragment, or a post-translational modification of a p54 S6K  or p85 S6K  polypeptide. In a preferred embodiment, the antibody is Ab#167. 
     In another aspect, the invention features a cell line having a genetically engineered null mutation in a p54 S6K  or p85 S6K  gene. In a preferred embodiment, the cell line is an embryonic stem cell line. If the cell is in a mammal, the mammal is preferably a non-human. 
     In another aspect, the invention features a cell line, genetically engineered to overexpress a p54 S6K  or p85 S6K  polypeptide. In a preferred embodiment, the cell line is a tumor cell line. 
     In another aspect, the invention features a non-human transgenic animal, or embryo thereof, with a knockout mutation in the p54 S6K  or p85 S6K  gene. In a related aspect, the invention features a non-human transgenic animal with additional copies of p54 S6K  or p85 S6K  nucleic acids added to its genome. In a preferred embodiment of these aspects, the non-human transgenic animal is a rodent, more preferably a mouse. In a preferred embodiment the animal has a knockout mutation in both genes. 
     In other aspects, the invention features methods of identifying a compound which modulates, or whose activity is modulated by, p54 S6K  or p85 S6K  biological activity involving: a) providing a cell expressing a p54 S6K  or p85 S6K  polypeptide, or a lysate from the cell expressing a p54 S6K  or p85 S6K  polypeptide, or a transgenic animal expressing a p54 S6K  or p85 S6K  polypeptide; b) exposing the cell, lysate, or transgenic animal to the test compound; c) assaying for a modulation in p54 S6K  or p85 S6K  biological activity; and d) comparing the modulation in p54 S6K  or p85 S6K  biological activity to that of a cell, lysate, or transgenic animal which did not receive the test compound, wherein a modulation of p54 S6K  or p85 S6K  biological activity identifies a test compound. In a preferred embodiment the biological activity is assayed by measuring p54 S6K  or p85 S6K  phosphorylation, p54 S6K  or p85 S6K  kinase activity, p54 S6K  or p85 S6K  polypeptide or nucleic acid levels, or cell proliferation. In another preferred embodiment, p54 or p85 S6K  biological activity is measured by using S6 or BRCA1 as a substrate. In other embodiments, the cell is induced or genetically engineered to express the p54 S6K  or p85 S6K  polypeptide. In a preferred embodiment the cell is a tumor cell and/or the p54 S6K  or p85 S6K  polypeptide is p54 S6K  KR or p85 S6K  KR. In another preferred embodiment, the test compound is BRCA1. In a related aspect, the cell may express an altered p54 S6K  or p85 S6K , as described herein before (e.g., a fusion, fragment, etc.), 
     In other embodiments, the cell is exposed to a stimulus which could include serum or insulin. In other embodiments the cell is exposed to an inhibitor which could include rapamycin or wortmannin. 
     In another aspect, the invention features a method of diagnosing an increased likelihood of a cell proliferative disease in a subject. The method includes detecting the level of p54 or p85 S6K  gene expression in the subject. In preferred embodiments of this aspect, detection of the level of p54 S6K  or p85 S6K  gene expression is done by nucleic acid hybridization, including Northern blotting, Southern blotting, Western blotting, far-Western blotting, or DNA-mRNA hybridization. In another embodiment the subject is a human. 
     In a final aspect, the invention features a therapeutic composition comprising as an active ingredient a p54 S6K  or p85 S6K  polypeptide or nucleic acid, the active ingredient being formulated in a physiologically acceptable carrier. In a preferred embodiment, the p54 S6K  or p85 S6K  nucleic acid is in the context of a vector, more preferably a gene therapy vector. 
     By “substantially pure nucleic acid” is meant nucleic acid that is free of the substances that naturally accompany it. In the case of DNA, it would mean DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. In the case of RNA, it would include mRNA free of associated proteins and nucleic acids. 
     By “p54 S6K ” is meant any gene or polypeptide, which is not p70 S6k , which hybridizes with high stringency to SEQ ID NO: 1 or and has p54 S6K  biological activity. 
     By “p85 S6K ” is meant any gene or polypeptide, which is not p70 S6k , which hybridizes with high stringency to a gene encoding the polypeptide of SEQ ID NO: 3 and has p85 S6K  biological activity. 
     By “high stringency” is meant conditions that are commonly understood in the art as stringent. An exemplary set of high stringency conditions include a temperature of 60-70° C., (preferably about 65° C.) and a salt concentration of 0.70 M to 0.80 M (preferably about 0.75 M). Further exemplary conditions include, hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 5×Denhardt&#39;s solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS. Further examples of stringent conditions can be found in Sambrook, Fritsch and Maniatis,  Molecular Cloning: A Laboratory Manual  (2 d ed .), Cold Spring Harbor Press, 1989, or Ausubel et al.,  Current Protocols in Molecular Biology , John Wiley &amp; Sons, New York, N.Y., 1994). 
     By “polypeptide” or “polypeptide fragment” is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide, or constituting a non-naturally occurring polypeptide. 
     By “substantially pure p54 S6K  polypeptide” is meant a p54 S6K  polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, p54 S6K  polypeptide. A substantially pure p54 S6K  polypeptide may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an p54 S6K  polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. 
     A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cells from which is naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure p54 S6K  polypeptides include those derived from eukaryotic organisms but synthesized in  E. coli  or other prokaryotes. 
     By “substantially pure p85 S6K  polypeptide” is meant a p85 S6K  polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, p85 S6K  polypeptide. A substantially pure p85 S6K  polypeptide may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an p85 S6K  polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. 
     A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cells from which is naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure p85 S6K  polypeptides include those derived from eukaryotic organisms but synthesized in  E. coli  or other prokaryotes. 
     By “substantially identical” is meant a polypeptide or nucleic acid exhibiting at least 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid sequence (for example, the amino acid sequence described herein) or nucleic acid sequence (for example, the nucleic acid sequence described herein). For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 60 nucleotides, preferably at least 75 nucleotides, and more preferably 110 nucleotides. 
     Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs). These software programs match identical or similar sequences by assigning degrees of identity to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. 
     By “specifically binds” is meant an antibody which recognizes and binds a protein, but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes protein. 
     By “post-translational modification” is meant any changes to the polypeptide during or after synthesis. These modifications may include phosphorylation and glycosylation. Post-translational modifications may be naturally occurring (such as during synthesis within a cell), or artificially generated (such as by recombinant or chemical means). 
     By “null cell line” is meant a cell line that does not express a p54 S6K  or does not express a p85 S6K  polypeptide or which expresses a p54 S6K  or p85 S6K  polypeptide fragment having decreased biological activity. Preferably the cell line lacks detectable biological activity for said polypeptide. The cell line may be created by genetic engineering such that there is a mutation that makes a p54 S6K  or p85 S6K  polypeptide that has no detectable biological activity, or prevents the cell from making a p54 S6K  or p85 S6K  polypeptide. 
     By “overexpressing” is meant a cell line that expresses a p54 S6K  or p85 S6K  polypeptide or a p54 S6K  or p85 S6K  polypeptide fragment at a level at least 10% higher than the level of endogenous expression. This would include cells that express p54 S6K  or p85 S6K  either transiently, or stably incorporated into their genome. It would also include both prokaryotic and eukaryotic cells. 
     By “test compound” is meant a chemical, be it naturally-occurring or artificially-derived, that is surveyed for its ability to modulate p54 S6K  or p85 S6K  biological activity, by employing one of the assay methods described herein. Compounds may include, for example, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof. 
     By “modulates” is meant changes, either by increase or decrease. 
     By “p54 S6K  biological activity” is meant any activity of p54 S6K  found in cells. This includes the levels of p54 S6K  nucleic acids or polypeptides, p54 S6K  phosphorylation, p54 S6K  kinase activity towards a variety of substrates, the inhibition of p54 S6K  activity by natural or artificial compounds, and the effect of p54 S6K  on substrates. This also includes the effect of p54 S6K  on cell proliferation and cell cycle. 
     By “p85 S6K  biological activity” is meant any activity of p85 S6K  found in cells. This includes the levels f p85 S6K  nucleic acids or polypeptides, p85 S6K  phosphorylation, p85 S6K  kinase activity towards a variety of substrates, the inhibition of p85 S6K  activity by natural or artificial compounds, and the effect of p85 S6K  on substrates. This also includes the effect of p85 S6K  on cell proliferation and cell cycle. 
     By “exposing” is meant allowing contact between an animal, cell, lysate or extract derived from a cell, or molecule derived from a cell, and a compound. 
     By “transgene” is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. 
     By “transgenic” is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic mammals (e.g., rodents such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome. 
     By “knockout mutation” is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift mutation, or a missense mutation. Preferably, the mutation is an insertion or deletion, or is a frameshift mutation that creates a stop codon. 
     By “assaying” is meant analyzing the effect of a treatment or exposure, be it chemical or physical, administered to whole animals or cells. The material being analyzed may be an animal, a cell, a lysate or extract derived from cell, or a molecule derived from a cell. The analysis may be, for example, cell proliferation, cell morphology, or tumor formation. Techniques used in the analysis may include SDS polyacrylamide gel electrophoresis, in vitro kinase reactions, immunoprecipitation, etc. 
     By “substrate” is meant a molecule whose activity is modulated by p54 S6K  or p85 S6K  in vivo or in vitro. Substrates may include S6 and BRCA1. Preferably, a substrate is a molecule that has a phosphorylation state which is modified by p54 S6K  or p85 S6K  protein. 
     By “antisense,” as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, a p54 S6K  or p85 S6K  gene. 
     By “detecting the level of p54 S6K  gene expression” is meant detecting the levels of p54 S6K  polypeptides or nucleic acids. The detection would be relative to normal samples from patients without the cell proliferative disease. 
     By “detecting the level of p85 S6K  gene expression” is meant detecting the levels of p85 S6K  polypeptides or nucleic acids. The detection would be relative to normal samples from patients without the cell proliferative disease. 
     The invention provides methods and reagents for the diagnosis and treatment of diseases caused by inappropriate cell proliferation. These disorders can be treated, using the methods described herein, in a variety of ways including small molecules, gene therapy, antisense oligonucleotides and protein replacement. Additionally, they can have potential diagnostic/disease management applications as prognostic markers or predisposition indicators of certain cancers. Other features and advantages of the invention will be apparent from the detailed description of the invention, and from the claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1A shows the nucleotide sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) f p54 S6K . The 1732 nucleotides of p54 S6K  contains one open reading frame that encodes a protein of 482 amino acids (SEQ ID NO: 2). Multiple sequencing of clones isolated from the library as well as the examination of p54 S6K  EST clones show that seven of the clones have T at the nucleotide position 1294 (encoding valine at amino acid position 420), whereas four contain C at the position 1294 (encoding alanine at 420). Three clones isolated from Jurkat library (including the two full-length clones) have A at the nucleotide position at 1203 (encoding methionine at amino acid position 390), whereas one partial clone isolated from Jurkat library and four EST entries had G at the nucleotide position at 1203 (encoding valine at 390). 
     FIG. 1B is a schematic representation of the structural similarities between p54 S6K  and p70 S6k .Different domains of the proteins as well as the conserved amino acid sequences are shown. The degree of homology between the amino acid sequences of the two proteins are also shown. 
     FIG. 2 shows an image of a Northern blot of p54 S6K . Poly(A)+ mRNAs from multiple human tissues, probed with a 0.7 kb fragment of p54 S6K  cDNA labeled with [α- 32 P]dCTP. The RNA size markers are shown on the left. 
     FIG. 3A shows an image of a Western blot of lysates from human Jurkat T cells, 293 cells, and 293 cells transiently transfected with HA-p54 S6K  and immunoblotted with Ab#167, Ab#167 incubated with the immunizing peptide, and the preimmune sera. Arrows on the left show the bands corresponding to the indicated proteins. The protein size markers are shown on the right. 
     FIG. 3B shows a graphical representation of the intrinsic kinase activity of p54 S6K . p54 S6K  does not undergo an electrophoretic mobility shift upon serum stimulation. HA-p54 S6K  or HA-p54 S6K  KR were transfected into 293 cells, stimulated with serum, and the lysates were immunoprecipitated with anti-HA Ab. In vitro kinase assay of the anti-HA Ab immunoprecipitates using GST-S6 as the substrate is shown as a graph. A portion of the lysates were immunoblotted with anti-HA Ab to show the expression level of HA-p54 S6K  and HA-p54 S6K  KR as well as their lack of mobility shift upon serum stimulation (bottom left panel). The bottom right panel shows an image of an endogenous p70 S6k  immunoblot of lysates from quiescent or stimulated 293 cells in order to show the electrophoretic mobility shift of p70 S6K  upon serum stimulation. 
     FIG. 3C shows an image of Western blot of endogenous p54 S6K  kinase activity. 293 cells were treated with serum for 20 minutes, lysed, and the lysates were immunoprecipitated with 167 immune or preimmune serum. In vitro kinase assay of the immunoprecipitates using GST-S6 as the substrate is shown. 
     FIG. 3D shows a graph demonstrating that p54 S6K  kinase activity is stimulated by serum and insulin, and to some extent by EGF. 293 cells were treated with serum (10%), EGF (250 ng/ml), or insulin (200 nM) for 20 minutes, lysed, and the lysates were immunoprecipitated with 167 anti-p54 S6K  antiserum or with N2 anti-p70 S6k  antiserum. Phosphorimager™ counts from an in vitro kinase assay of the immunoprecipitates using GST-S6 as the substrate are shown. 
     FIG. 3E is a graphical illustration of the maximal activation of p54 S6K  and p70 S6k , which occurs at 30 minutes. 293 cells were treated with serum (10%) for 0, 5, 15, 30, 60, and 180 minutes, lysed, and the lysates were immunoprecipitated with either 167 anti-p54 S6K  antiserum or with N2 anti-p70 S6k  antiserum. Phosphorimager counts from an in vitro kinase assay of the immunoprecipitates using GST-S6 as the substrate are shown. 
     FIG. 4A is a graph demonstrating that rapamycin and wortmannin inhibit p54 S6K  kinase activity. 293 cells transfected with HA-p54 S6K  were treated with rapamycin (20 ng/ml) or wortmannin (100 nM) for 20 minutes prior to serum stimulation (10%, 20 minutes). The cells were lysed, and the lysates were immunoprecipitated with anti-HA Ab. Phosphorimager counts from an in vitro kinase assay of the immunoprecipitates using GST-S6 as the substrate are shown. 
     FIG. 4B is a graph showing that p110* increases both the basal and serum-stimulated activity of p54 S6K . 293 cells were transfected with the DNA for p54 S6K  alone or for p54 S6K  and two different amounts of p110* DNA as indicated. Equal expression of proteins was verified by immunoblots. The cells were stimulated with serum (10%, 20 minutes), lysed, and the lysates were immunoprecipitated with anti-HA Ab. Phosphorimager™ counts from an in vitro kinase assay of the immunoprecipitates using GST-S6 as the substrate are shown. 
     FIG. 4C is a graph illustrating that the kinase activities of p54 S6K  and p70 S6k  are differentially regulated by PDK1. 293 cells were transfected with DNA for p54 S6K  alone; for p54 S6K  alone; for p54 S6K  and two different concentrations of DNA for PDK1 (top panel); or for p70 S6k  and two different concentrations of DNA for PDK1 as indicated (bottom graph). Equal expression of the proteins was verified by immunoblots. The cells were stimulated with serum, lysed, and the lysates were immunoprecipitated with anti-HA Ab. Phosphorimager counts from an in vitro kinase assay of the immunoprecipitates using GST-S6 as the substrate are shown. 
     FIG. 5 shows a comparison between the deduced amino acid sequence of p70 S6K  (top line; SEQ ID NO: 16) and p85 S6K  (bottom line; SEQ ID NO: 3). p85 S6K  contains one open reading frame that encodes a protein of 495 amino acids. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     We have discovered new mammalian protein kinases, p54 S6K  and p85 S6K , involved in cell proliferation. p54 S6K  activity is regulated by mitogens and phosphatidylinositol 3-kinase and inhibited by wortmannin and the inmunosuppressant rapamycin. We provide the amino acid sequences for p54 S6K  and p85 S6K  in FIGS. 1A and 5. p54 S6K , which has a molecular weight of 54 kDa, is ubiquitously expressed in human tissue. Characterization of the p54 S6K  protein, by the construction of kinase-dead mutants as well as other techniques, shows that it has intrinsic protein kinase activity, as well as being phosphorylated itself. p85 S6K  is a novel isoform of p54 S6K . 
     p54 S6K  and p85 S6K  Protein Expression 
     p54 S6K  and p85 S6K  genes may be expressed in both prokaryotic and eukaryotic cell types. Using the techniques described herein, cell lines overexpressing p54 S6K  or p85 S6K  proteins may be made. Under some circumstances, it may be desirable to express the protein under control of an inducible promoter for the purposes of protein production. 
     In general, p54 S6K  or p85 S6K  proteins according to the invention may be produced by transformation of a suitable host cell with all or part of a p54 S6K -or p85 S6K , encoding cDNA fragment, respectively, in a suitable expression vehicle. 
     Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. The p54 S6K  or p85 S6K  protein may be produced in a prokaryotic host (e.g.,  E. coli ) or in a eukaryotic host (e.g.,  Saccharomyces cerevisiae , insect cells, e.g., Sf21 cells, or mammalian cells, e.g., HEK 293, COS 1, NIH 3T3, or HeLa cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al.,  Current Protocols in Molecular Biology , John Wiley &amp; Sons, New York, 1994). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al., (supra); expression vehicles may be chosen from those provided, e.g., in  Cloning Vectors: A Laboratory Manual  (P. H. Pouwels et al., 1985, Supp. 1987). 
     Alternatively, a p54 S6K  or p85 S6K  protein is produced by a stably-transfected mammalian cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra); methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. (supra). In one example, cDNA encoding the protein is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, the protein-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 μM methotrexate in the cell culture medium (as described in Ausubel et al., supra). This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra); such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al., supra). Any of the host cells described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR −  cells, ATCC Accession No. CRL 9096) are among the host cells preferred for DHFR selection of a stably-transfected cell line or DHFR-mediated gene amplification. In addition, tumor cell lines overexpressing p54 S6K  or p85 S6K  proteins, or fragments of p54 S6K  or p85 S6K  proteins, may also be made. 
     Once the recombinant p54 S6K  or p85 S6K  protein is expressed, it is isolated, e.g., using affinity chromatography. In one example, an anti-p54 S6K  or p85 S6K  protein antibody (e.g., produced as described herein) may be attached to a column and used to isolate the p54 S6K  or p85 S6K  protein, respectively. Lysis and fractionation of p54 S6K  or p85 S6K  protein-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). 
     Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher,  Laboratory Techniques In Biochemistry And Molecular Biology , eds., Work and Burdon, Elsevier, 1980) or other protein purification methods. 
     Polypeptides of the invention, particularly short p54 S6K  or p85 S6K  protein fragments, can also be produced by chemical synthesis (e.g., by the methods described in  Solid Phase Peptide Synthesis , 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). 
     These general techniques of polypeptide expression and purification can also be used to produce and isolate useful p54 S6K  or p85 S6K  fragments or analogs. 
     Anti-p54 S6K  or p85 S6K  Antibodies 
     To generate p54 S6K  or p85 S6K  specific antibodies, a p54 S6K  or p85 S6K  coding sequence can be expressed as a C-terminal fusion with glutathione S-transferase (GST) (Smith et al., Gene 67:31-40, 1988). The fusion protein can be purified on glutathione-Sepharose beads, eluted with glutathione, cleaved with thrombin or another protease (at the engineered cleavage site), and purified to the degree necessary for immunization of rabbits. Primary immunizations can be carried out with Freund&#39;s complete adjuvant and subsequent immunizations with Freund&#39;s incomplete adjuvant. Antibody titers are monitored by Western blot and immunoprecipitation analyses, using the thrombin-cleaved p54 S6K  or p85 S6K  protein fragment of the GST-p54 S6K  or p85 S6K  fusion protein. Antisera are affinity purified using CNBr-Sepharose-coupled p54 S6K  or p85 S6K  protein or by other standard methods. Antiserum specificity is determined using a panel of unrelated GST proteins. 
     As an alternate or adjunct immunogen to GST fusion proteins, peptides corresponding to relatively unique regions of p54 S6K  or p85 S6K  may be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced C-terminal lysine. Antiserum to each of these peptides is similarly affinity purified on peptides conjugated to BSA, and specificity tested in ELISA and Western blots using peptide conjugates, and by Western blot and immunoprecipitation using p54 S6K  or p85 S6K  expressed as a GST fusion protein. 
     Alternatively, monoclonal antibodies may be prepared using the p54 S6K  or p85 S6K  proteins described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981; Ausubel et al., supra). Once produced, monoclonal antibodies are also tested for specific p54 S6K  or p85 S6K  recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra). Antibodies which specifically recognize p54 S6K  or p85 S6K  are considered to be useful in the invention; such antibodies may be used, e.g., in an immunoassay to monitor the level of p54 S6K  or p85 S6K  produced by a mammal (for example, to determine the amount or subcellular location of p54 S6K  or p85 S6K ). 
     Preferably, antibodies of the invention are produced using fragments of either the p54 S6K  or p85 S6K  proteins which lie outside regions highly conserved between p70 S6k , and p54 S6K  or p85 S6K , for example Ab#167, and appear likely to be antigenic, by criteria such as those provided by the Peptidestructure program of the Genetics Computer Group Sequence Analysis Package (Program Manual for the GCG Package, Version 7, 1991) using the algorithm of Jameson and Wolf (CABIOS 4:181 1988)). In one specific example, such fragments are generated by standard techniques of PCR and cloned into the pGEX expression vector (Ausubel et al., supra). Fusion proteins are expressed in  E. coli  and purified using a glutathione agarose affinity matrix as described in Ausubel et al. (supra). To attempt to minimize the potential problems of low affinity or specificity of antisera, two or three such fusions are generated for each protein, and each fusion is injected into at least two rabbits. Antisera are raised by injections in a series, preferably including at least three booster injections. 
     Construction of a Non-human Transgenic Animal 
     Characterization of p54 S6K  or p85 S6K  can provide information that allows for the development of a p54 S6K  or p85 S6K  knockout mouse models by homologous recombination (or a p54 S6K  or p85 S6K  overproducing mouse, which may contain additional copies of the p54 S6K  or p85 S6K  gene, respectively, by other means of integration). The instant invention therefore also features a transgenic non-human animal containing a transgene which encodes a p54 S6K  or p85 S6K  polypeptide that is expressed in or delivered to tissue normally susceptible to unregulated proliferation. 
     A replacement type targeting vector can be constructed using an isogenic genomic clone from a mouse strain, e.g., 129/Sv (Stratagene La Jolla, Calif.). The targeting vector can be introduced into a J1 line of embryonic stem (ES) cells by electroporation to generate ES cell lines that carry a profoundly truncated form of a p54 S6K  or p85 S6K .To generate chimeric founder mice, the targeted cell lines will be injected into a mouse blastula stage embryo. Heterozygote offspring will be interbred to homozygosity. Knockout mice may be constructed as a means of screening in vivo for therapeutic compounds which modulate cell proliferation. Other non-human mammals such as rats, goats, or sheep may also be used for the expression of transgenic p54 S6K  or p85 S6K . 
     Cell lines that lack functional p54 S6K  or p85 S6K  or overexpress p54 S6K  or p85 S6K  are also a part of this invention. 
     Identification of Molecules that Modulate p54 S6K  or p85 S6K  Biological Activity 
     Isolation of the p54 S6K  or p85 S6K  cDNA (as described herein) may also facilitate the identification of molecules which increase or decrease p54 56 K or p85 S6K  biological activity. Similarly, molecules whose activity is modulated by p54 S6K  or p85 S6K  biological activity may also be identified. According to one approach, candidate molecules are added at varying concentrations to the culture medium of cells expressing p54 S6K  or p85 S6K  mRNA. p54 S6K  or p85 S6K  biological activity is then measured using standard techniques. The measurement of biological activity can include the measurement of p54 S6K  or p85 S6K  protein and nucleic acid levels, p54 S6K  or p85 S6K  phosphorylation, p54 S6K  or p85 S6K  kinase activity, or the effect of p54 S6K  or p85 S6K  on cell proliferation. Additionally, the measurement of biological activity can be done by using S6 or BRCA1 as substrates for p54 S6K  or p85 S6K  biological activity. For example, standard Northern blot analysis (Ausubel et al., supra) using a p54 S6K  or p85 S6K  cDNA (or cDNA fragment) as a hybridization probe can be used to measure levels of p54 S6K  or p85 S6K  expression, respectively. The level of p54 S6K  or p85 S6K  expression in the presence of the candidate molecule is compared to the level measured for the same cells in the same culture medium but in the absence of the candidate molecule. 
     If desired, the effect of candidate modulators on expression may, in the alternative, be measured at the level of p54 S6K  or p85 S6K  protein production using the same general approach and standard immunological detection techniques, such as Western blotting or immunoprecipitation with a p54 S6K  or p85 S6K -specific antibody (for example, the p54 S6K  antibody #167 described herein). 
     Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells; Ausubel et al., supra). In a mixed compound assay, p54 S6K  or p85 S6K  expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, e.g., HPLC or FPLC) until a single compound or minimal compound mixture is demonstrated to modulate p54 S6K  or p85 S6K  expression, respectively. 
     Alternatively, or in addition, candidate compounds may be screened for those which modulate p54 S6K  or p85 S6K  cell proliferation inhibiting activity. In this approach, the degree of cell proliferation in the presence of a candidate compound is compared to the degree of cell proliferation in its absence, under equivalent conditions. Again, such a screen may begin with a pool of candidate compounds, from which one or more useful modulator compounds are isolated in a step-wise fashion. Cell proliferation activity may be measured by any standard assay, such as the mixed tumor transplantation (MTT) assay. 
     These assays may be done in a variety of ways that would be known to one of ordinary skill in the art. These include using p54 S6K  or p85 S6K  variants such as a kinase-dead mutant of p54 S6K  or p85 S6K , p54 S6K  KR or p85 S6K  KR; using fragments of p54 S6K  or p85 S6K , such as those described herein; or subjecting the cells to stimulation by the addition of serum or insulin, etc. 
     Test Compounds 
     Candidate p54 S6K  or p85 S6K  modulators include peptides, such as BRCA1, as well as non-peptide molecules (e.g., peptide or non-peptide molecules found, e.g., in a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured). In general, novel drugs for prevention or treatment of cellular proliferation are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceanographic Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods. 
     In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their therapeutic activities for cell proliferation disorders should be employed whenever possible. 
     When a crude extract is found to regulate cellular proliferation, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having cell proliferation, -preventative, or -palliative activities. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using a mammalian cell proliferation model. 
     Modulators found to be effective at the level of p54 S6K  or p85 S6K  expression or activity may be confirmed as useful in animal models and, if successful, may be used as anti-cancer therapeutics for either the inhibition or the enhancement of cell proliferation, as appropriate. 
     Diagnosis of a Condition Involving Altered Cell Proliferation 
     p54 S6K  or p85 S6K  polypeptides and nucleic acid sequences may find diagnostic use in the detection or monitoring of conditions involving aberrant levels of cell proliferation. For example, altered expression of p54 S6K  or p85 S6K  may be correlated with enhanced cell proliferation in humans. Accordingly, a decrease or increase in the level of p54 S6K  or p85 S6K  production may provide an indication of a deleterious condition. Levels of expression may be assayed by any standard technique. For example, p54 S6K  or p85 S6K  expression in a biological sample (e.g., a biopsy) may be monitored by standard Northern blot analysis or may be aided by PCR (see, e.g., Ausubel et al., supra;  PCR Technology: Principles and Applications for DNA Amplification , ed., H. A. Ehrlich, Stockton Press, NY; and Yap and McGee, Nucl. Acids. Res. 19:4294, 1991). 
     Alternatively, a patient sample may be analyzed for one or more mutations in the p54 S6K  or p85 S6K  sequences using a mismatch detection approach. Generally, these techniques involve PCR amplification of nucleic acid from the patient sample, followed by identification of the mutation (i.e., mismatch) by either altered hybridization, aberrant electrophoretic gel migration, binding or cleavage mediated by mismatch binding proteins, or direct nucleic acid sequencing. Any of these techniques may be used to facilitate mutant p54 S6K  or p85 S6K  detection, and each is well known in the art; examples of particular techniques are described, without limitation, in Orita et al. (Proc. Natl. Acad. Sci. USA 86:2766-2770, 1989); and Sheffield et al.(Proc. Natl. Acad. Sci. USA 86:232-236, 1989). 
     In yet another approach, immunoassays are used to detect or monitor p54 S6K  or p85 S6K  proteins in a biological sample. p54 S6K  or p85 S6K  specific polyclonal or monoclonal antibodies (produced as described above) may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure p54 S6K  or p85 S6K  polypeptide levels; again comparison is to wild-type p54 S6K  or p85 S6K  levels, respectively, and an alteration in p54 S6K  or p85 S6K  production is indicative of a condition involving increased cell proliferation. Examples of immunoassays are described, e.g., in Ausubel et al., supra. Immunohistochemical techniques may also be utilized for detection. For example, a tissue sample may be obtained from a patient, and a section stained for the presence of p54 S6K  or p8 5   S6K  using an anti-p54 S6K  or p85 S6K  antibody and any standard detection system (e.g., one which includes a secondary antibody conjugated to horseradish peroxidase). General guidance regarding such techniques can be found in, e.g., Bancroft and Stevens ( Theory and Practice of Histological Techniques , Churchill Livingstone, 1982) and Ausubel et al. (supra). 
     In one preferred example, a combined diagnostic method may be employed that begins with an evaluation of protein production, for example, by immunological techniques or the protein truncation test (Hogerrorst, F. B. L., et al., Nature Genetics 10:208-212, 1995) and also includes a nucleic acid-based detection technique designed to identify more subtle mutations (for example, point mutations). As described above, a number of mismatch detection assays are available to those skilled in the art, and any preferred technique may be used (see above). By this approach, mutations in p54 S6K  or p85 S6K  may be detected that either result in loss of p54 S6K  or p85 S6K  expression or loss of p54 S6K  or p85 S6K  biological activity, respectively. In a variation of this combined diagnostic method, biological activity is measured as protease activity using any appropriate protease assay system (for example, those described above). 
     Mismatch detection assays also provide the opportunity to diagnose a mediated predisposition to diseases of cell proliferation. For example, a patient heterozygous for a p54 S6K  or p85 S6K  mutation may show no clinical symptoms and yet possess a higher than normal probability of developing one or more types of cancer or cell proliferative diseases. Given this diagnosis, a patient may take precautions to minimize their exposure to adverse environmental factors (for example, UV exposure or chemical mutagens) and to carefully monitor their medical condition (for example, through frequent physical examinations). This type of diagnostic approach may also be used to detect mutations in prenatal screens. 
     The diagnostic assays described above may be carried out using any biological sample (for example, any biopsy sample or bodily fluid or tissue) in which p54 S6K  or p85 S6K  is normally expressed (for example, the inhibition of cell proliferation). Identification of a mutant p54 S6K  or p85 S6K  gene may also be assayed using these sources for test samples. Alternatively, a p54 S6K  or p85 S6K  mutation, particularly as part of a diagnosis for predisposition to p54 S6K  or p85 S6K -associated proliferative disease, may be tested using a DNA sample from any cell, for example, by mismatch detection techniques; preferably, the DNA sample is subjected to PCR amplification prior to analysis. 
     p54 S6K  or p85 S6K  Therapy 
     Because expression levels of p54 S6K  or p85 S6K  genes may correlate with the levels of cell proliferation, the p54 S6K  or p85 S6K  gene can also be used in anti-cell proliferation gene therapy. In particular, to inhibit neoplastic cells, either a functional or a dominant-negative p54 S6K  or p85 S6K  gene may be introduced into cells at the sites predicted to undergo undesirable cell proliferation. 
     Retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or other viral vectors with the appropriate tropism for cells likely to be involved in cell proliferation may be used as a gene transfer delivery system for a therapeutic p54 S6K  or p85 S6K  gene construct. Numerous vectors useful for this purpose are generally known (Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis and Anderson, BioTechniques 6:608-614, 1988; Tolstoshev and Anderson, Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; and Miller and Rosman, Biotechniques 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). 
     Non-viral approaches may also be employed for the introduction of therapeutic DNA into cells otherwise predicted to undergo cell proliferation. For example, p54 S6K  or p85 S6K  may be introduced into a neoplastic cell by the techniques of lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Lett 117:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger and Papahadjopoulos, Meth. Enz. 101:512, 1983); asialorosonucoid-polylysine conjugation (Wu and Wu, J. Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem. 264:16985, 1989); or, less preferably, microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). 
     For any of the above approaches, the therapeutic p54 S6K  or p85 S6K  DNA construct is preferably applied to the site of the predicted cell proliferation event (for example, by injection), but may also be applied to tissue in the vicinity of the predicted cell proliferation event or even to a blood vessel supplying the cells predicted to undergo cell proliferation. 
     In the gene therapy constructs, p54 S6K  or p85 S6K  cDNA expression is directed from any suitable promoter (e.g., the human cytomegalovirus, simian virus 40, or metallothionein promoters), and its production is regulated by any desired mammalian regulatory element. For example, if desired, enhancers known to direct preferential gene expression in neural cells or T-cells may be used to direct p54 S6K  or p85 S6K  expression. Such enhancers include, without limitation, those enhancers which are characterized as tissue or cell specific in their expression. 
     Alternatively, if a p54 S6K  or p85 S6K  genomic clone is utilized as a therapeutic construct (for example, following its isolation by hybridization with the p54 S6K  or p85 S6K  cDNA), p54 S6K  or p85 S6K  expression is regulated by its cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, e.g., any of the promoters or regulatory elements described above. 
     Less preferably, p54 S6K  or p85 S6K  gene therapy is accomplished by direct administration of the p54 S6K  or p85 S6K  mRNA to a cell predicted to undergo cell proliferation. This mRNA may be produced and isolated by any standard technique, but is most readily produced by in vitro transcription using a p54 S6K  or p85 S6K  cDNA under the control of a high efficiency promoter (e.g., the T7 promoter). Administration of p54 S6K  or p85 S6K  mRNA to malignant cells is carried out by any of the methods for direct nucleic acid administration described above. 
     Ideally, the production of p54 S6K  or p85 S6K  proteins by any gene therapy approach described above results in a cellular level of p54 S6K  or p85 S6K , respectively, that is at least equivalent to the normal, cellular level of p54 S6K  or p85 S6K  in an unaffected individual. Treatment by any p54 S6K  or p85 S6K -mediated gene therapy approach may be combined with more traditional therapies. 
     Another therapeutic approach included within the invention involves direct administration of recombinant p54 S6K  or p85 S6K  proteins, either to the site of a predicted cell proliferation event (for example, by injection) or systemically by any conventional recombinant protein administration technique. The actual dosage of p54 S6K  or p85 S6K  depends on a number of factors, including the size and health of the individual patient, but, generally, between 0.1 mg and 100 mg inclusive are administered per day to an adult in any pharmaceutically-acceptable formulation. 
     In a patient diagnosed to be heterozygous for a p54 S6K  or p85 S6K  mutation or to be susceptible to p54 S6K  or p85 S6K  mutations (even if those mutations do not yet result in alteration or loss of biological activity), or a patent diagnosed as HIV positive, any of the above therapies may be administered before the occurrence of the disease phenotype. For example, the therapies may be provided to a patient who is HIV positive but does not yet show a diminished T-cell count or other signs of full-blown AIDS. In particular, compounds shown to increase expression or biological activity may be administered by any standard dosage and route of administration (see above). Alternatively, gene therapy using a p54 S6K  or p85 S6K  expression construct may be undertaken to reverse or prevent the cell defect prior to the development of the proliferative disease. 
     The methods of the instant invention may be used to reduce or diagnose the disorders described herein in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated or diagnosed, the polypeptide, nucleic acid, or antibody employed is preferably specific for that species. 
     Administration of p54 S6K  or p85 S6K  Polypeptides, p54 S6K  or p85 S6K  Genes, or Modulators of Synthesis or Function 
     A p54 S6K  or p85 S6K  protein, gene, or modulator may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer p54 S6K  or p85 S6K  to patients suffering from or presymptomatic for a p54 S6K  or p85 S6K -associated cell proliferative disease. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. 
     Methods well known in the art for making formulations are found in, for example, “Remington&#39;s Pharmaceutical Sciences” (18 th  edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. 
     If desired, treatment with a p54 S6K  or p85 S6K  protein, gene, or modulatory compound may be combined with more traditional therapies for the disease such as, for example, surgery, radiation, or chemotherapy for cancers; surgery, steroid therapy, and chemotherapy for autoimmune diseases; antiviral therapies for AIDS; and antibiotics for opportunistic infections. 
     The following examples are to illustrate the invention. They are not meant to limit the invention in any way. 
     EXAMPLE 1 
     Cloning of the p54 S6K  Gene 
     Our search for human genes potentially involved in cellular proliferation resulted in the identification of a clone, isolated from human brain, in the expressed sequence tag (EST) database. This clone had extensive but dispersed homology to p70 S6k , a serine/threonine kinase involved in cellular proliferation. 
     Using the EST sequence information we probed a human Jurkat T cell lambda cDNA library (Stratagene) and obtained eight clones, two of which contained complete open reading frames. Sequencing of the two clones showed they were identical. We have named the clone p54 S6K . FIG. 1A shows the DNA and amino acid sequences of of p54 S6K . Multiple sequencing of the of clones isolated from the library as well as the examination of p54 S6K  EST clones show that seven of the clones have T at the nucleotide position 1294 (encoding valine at amino acid position 420), whereas four contain C at the position 1294 (encoding alanine at 420). Three clones isolated from the Jurkat library (including the two full-length clones) have A at the nucleotide position at 1203 (encoding methionine at amino acid position 390), whereas one partial clone isolated from the Jurkat library, as well as four EST entries, had G at the nucleotide position at 1203 (encoding valine at amino acid position 390). 
     The p54 S6K  cDNA clones, as well as oligonucleotides derived from the cDNA sequence, together with available genomic DNA libraries can be used to further delineate the structure and genomic organization of p54 S6K . Strategies for this are well known in the art and may include sequencing, restriction mapping, Southern blot analysis and inverse PCR techniques. These techniques may also be used to map homologs and additional genes. 
     EXAMPLE 2 
     Northern Blot Analysis of the p54 S6K  Transcript 
     A human multiple tissue northern (MTN II, Clontech, Palo Alto, Calif.) was probed with a 32P-labeled 0.7 kb fragment of p54 S6K  using Clontech protocols (Clontech). FIG. 2 shows the Northern Blot analysis of p54 S6K  using a human multiple tissue poly(A)+ mRNA blot. p54 S6K  does not have alternatively-spliced variants; only one mRNA species of approximately 1.9 kb is detected. Northern blot analysis of mRNA isolated from the Jurkat cell line also shows one transcript of approximately 1.9 kb. Multiple tissue northern shows that p54 S6K  is ubiquitously expressed, with a higher level of expression in prostate, spleen, and peripheral blood leukocytes. Although we had isolated p54 S6K  based on the sequence of one EST clone, several more EST clones have been posted since the cloning of p54 S6K . Some of these ESTs were isolated from various tissues such as brain, colon, and parathyroid gland, and in transformed tissues such as colon tumor, breast tumor, and Wilms&#39; tumor. 
     EXAMPLE 3 
     Characterization of p54 S6K  Protein 
     The predicted molecular weight of p54 S6K  is 53.5 kDa, and in vitro transcription/translation of the two full-length clones of p54 S6K  shows that the apparent molecular weight of the protein in SDS-PAGE is 54 kDa. The molecular weight of p54 S6K  was also verified using an antiserum generated, using standard techniques, against a C-terminal peptide (FIG.  3 A). 
     Antibody (Ab.) #167, raised against a peptide containing a C-terminal sequence of p54 S6K , distinct from p70 S6k , immunoblotted a 54 kDa protein in the lysates of Jurkat cells and HEK 293 cells (FIG.  3 A). Lysates from the 293 cells transfected with hemagglutinin (HA)-tagged p54 S6K  showed the endogenous protein at 54 kDa as well as the 56 kDa tagged protein. Immunoblotting with the preimmune serum or with the antiserum incubated with the immunizing peptide showed that the antisera was specific to the 54 kDa band (FIG.  3 A). 
     Amino acid sequence comparison between p54 S6K  and p70 S6k  shows extensive homology (FIG.  1 B). The catalytic domain is most similar with amino acid sequence homology of 84%, whereas the N-terminal and C-terminal domains are 43% and 59% homologous, respectively. The acidic region found on the N-terminus of p70 S6k , which is thought to be important for interaction with the basic pseudosubstrate domain for kinase activity inhibition, is also found in p54 S6K . Many amino acid residues in p70 S6k  that have been shown to be important for its regulation are also conserved on p54 S6K . With the exception of T421, the proline-directed phosphorylation sites in the C-terminal pseudosubstrate domain of p70 S6k , S404, S411, and S424, are conserved. The recently identified S371, which is important for the function of p70 S6k , is also conserved on p54 S6K . T229, S411, T389, and S404, the p70 S6k  residues whose phosphorylation was shown to be regulated downstream of mTOR and PI 3-kinase, are conserved. 
     EXAMPLE 4 
     Activation of p54 S6K  Kinase Activity 
     We studied the regulation of p54 S6K  activity by using both transient transfection of epitope-tagged proteins as well as antibodies raised against the endogenous protein. 
     For transient transfections HEK 293 cells were cultured in standard (DMEM complete) media and transfected with the indicated DNA using a CaPO 4  precipitation method. Alternative methods of transfection, such as by electroporation, DEAE-Dextran treatment, or mediated by liposomes, may also be used. The experiments may also be done using cell lines, created by stable transfection techniques and expressing various forms of p54 S6K . 
     Antibodies were made using standard techniques, such as those described in Harlow and Lane (Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York. 1988). The cells were starved and then stimulated with serum, EGF, or insulin, or left unstimulated. Rapamycin or wortmannin were also incubated with the cells prior to serum stimulation when indicated. 
     A p54 S6K  mutant was generated by changing the catalytic lysine to arginine at position 99. This mutant was called p54 S6K  KR. HEK 293 cells were transfected with the DNA for HA-tagged p54 S6K  or p54 S6K  KR, stimulated with serum, and the proteins were immunoprecipitated from cell lysates for an in vitro kinase assay, using standard techniques. Glutathione-S-transferase (GST)-tagged S6 was used as a substrate since screening of different substrates in an in vitro kinase assay showed that p54 S6K  can phosphorylate both S6 and histone H2B. The graph in FIG. 3B shows that p54 S6K  kinase activity towards S6 was stimulated by serum treatment of the cells. The KR mutation of p54 S6K  abolished the kinase activity in an in vitro kinase assay, indicating that the kinase activity belongs to p54 S6K  itself and not to an associated kinase(s). Serum also increased the activity of endogenous p54 S6K  when endogenous p54 S6K  was immunoprecipitated using 167 antiserum and an in vitro kinase assay was carried out (FIG.  3 C). 
     p54 S6K  does not undergo a dramatic electrophoretic mobility shift on SDS-PAGE when it becomes phosphorylated upon stimulation (FIG.  3 B). Lysates from 293 cells transfected with HA-p54 S6K  or HA-p54 S6K  KR were immunoblotted with anti-HA Ab. (FIG. 3B bottom left panel). The immunoblots show no mobility shift for p54 S6K  upon serum stimulation, whereas p70 S6k  shows a distinct shift (FIG. 3B bottom right panel). This is of interest since a majority of the p70 S6k  phosphorylation sites are conserved in p54 S6K  (FIG.  1 B). HEK 293 cells were also treated with various mitogens and p54 S6K  activity assessed. p54 S6K  was stimulated by serum and insulin, and to a small degree by EGF in 293 cells (FIG.  3 D). Similarly, p70 S6k  was strongly activated by serum and insulin but only mildly activated by EGF. Finally, the activation kinetics of p54 S6K  and p70 S6k  were compared. FIG. 3E shows that both kinases were maximally activated by 30 minutes following stimulation by serum. 
     EXAMPLE 5 
     Regulation of p54 S6K  Activity 
     In order to assess the signaling pathways involved in regulating p54 S6K  activity, pharmacological agents and mutant forms of various signaling proteins were used. Increase in p54 S6K  kinase activity by serum was inhibited by rapamycin and wortmannin (FIG.  4 A), suggesting that both P13-kinase and mTOR regulate p54 S6K  kinase activation. When 293 cells were transiently transfected with p54 S6K  and p110*, an active form of the p110 catalytic subunit of PI 3-kinase, the basal as well as the serum-stimulated p54 S6K  kinase activity was increased (FIG.  4 B). This is similar to p70 S6k  activity regulation in that active p110, when co-transfected with p70 S6k , enhances p70 S6k  activity. 
     It has recently been shown that PDK1 is a direct upstream activator of p70 S6k . PDK1 is a serine-threonine kinase that contains a pleckstrin homology domain. It phosphorylates Akt/PKB at S308 in the activation loop, and it has been shown to phosphorylate the equivalent site T229 on p70 S6k . The role of PDK1 in p54 S6K  activation was assessed. Co-transfection of PDK1 and p70 S6k  showed that PDK1 enhances the basal activity of p70 S6k  as well as the serum-stimulated kinase activity of p70 S6k  (FIG.  4 C). At the same level of expression that enhances p70 S6k  activity, PDK1 failed to enhance the basal activity level of p54 S6K .However, the presence of PDK1 enhanced the serum-stimulated p54 S6K  activity level. Therefore overexpression of PDK1 alone without further stimulation by serum is sufficient to activate p70 S6k  but not p54 S6K . 
     EXAMPLE 6 
     The p85 S6K  Polypeptide 
     An alternative splice variant of the gene encoding p54 S6K  has also been discovered. This alternative splicing event results in the translation of a polypeptide homologous to p70 S6K  and p54 S6K , called p85 S6K  This polypeptide has a molecular weight of approximately 85 kDa. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In other embodiments, the invention includes any protein which is substantially identical to the p54 S6K  or p85 S6K  polypeptides shown in FIGS. 1 or  5 ; such homologs include other substantially pure naturally-occurring mammalian proteins as well as allelic variants; natural mutants; induced mutants; DNA sequences which encode p54 S6K  or p85 S6K  proteins and also hybridize to p54 S6K  or p85 S6K  DNA sequences under high stringency conditions or, less preferably, under low stringency conditions (e.g., washing at 2X SSC at 40° C. with a probe length of at least 40 nucleotides); and proteins specifically bound by antisera directed to a p54 S6K  or p85 S6K  polypeptide. The term also includes chimeric polypeptides that include a portion of a p54 S6K  or p85 S6K  polypeptide. 
     The invention further includes analogs of any of the naturally-occurring polypeptides described herein. Analogs can differ from the naturally-occurring protein by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino acid sequence. The length of sequence comparison is at least 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptide by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, supra, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids. 
     In addition to full-length polypeptides, the invention also includes polypeptide fragments. As used herein, the term “fragment,” means at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids. Fragments of polypeptides can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events). 
     Preferable fragments or analogs according to the invention are those which facilitate specific detection of a p54 S6K  or p85 S6K  nucleic acid or amino acid sequence in a sample to be diagnosed. 
     All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. 
     
       
         
           
             16 
           
           
             1 
             1732 
             DNA 
             Homo sapiens 
           
            1
ggcacgagcg acgggcccgc ggggccggcg ccgccatggc ggccgtgttt gatttggatt     60
tggagacgga ggaaggcagc gagggcgagg gcgagccaga gctcagcccc gcggacgcat    120
gtccccttgc cgagttgagg gcagctggcc tagagcctgt gggacactat gaagaggtgg    180
agctgactga gaccagcgtg aacgttggcc cagagcgcat cgggccccac tgctttgagc    240
tgctgcgtgt gctgggcaag gggggctatg gcaaggtgtt ccaggtgcga aaggtgcaag    300
gcaccaactt gggcaaaata tatgccatga aagtcctaag gaaggccaaa attgtgcgca    360
atgccaagga cacagcacac acacgggctg agcggaacat tctagagtca gtgaagcacc    420
cctttattgt ggaactggcc tatgccttcc agactggtgg caaactctac ctcatccttg    480
agtgcctcag tggtggcgag ctcttcacgc atctggagcg agagggcatc ttcctggaag    540
atacggcctg cttctacctg gctgagatca cgctggccct gggccatctc cactcccagg    600
gcatcatcta ccgggacctc aagcccgaga acatcatgct cagcagccag ggccacatca    660
aactgaccga ctttggactc tgcaaggagt ctatccatga gggcgccgtc actcacacct    720
tctgcggcac cattgagtac atggcccctg agattctggt gcgcagtggc cacaaccggg    780
ctgtggactg gtggagcctg ggggccctga tgtacgacat gctcactgga tcgccgccct    840
ttaccgcaga gaaccggaag aaaaccatgg ataagatcat caggggcaag ctggcactgc    900
ccccctacct caccccagat gcccgggacc ttgtcaaaaa gtttctgaaa cggaatccca    960
gccagcggat tgggggtggc ccaggggatg ctgctgatgt gcagagacat ccctttttcc   1020
ggcacatgaa ttgggacgac cttctggcct ggcgtgtgga cccccctttc aggccctgtc   1080
tgcagtcaga ggaggacgtg agccagtttg atacccgctt cacacggcag acgccggtgg   1140
acagtcctga tgacacagcc ctcagcgaga gtgccaacca ggccttcctg ggcttcacat   1200
acgtggcgcc gtctgtcctg gacagcatca aggagggctt ctccttccag cccaagctgc   1260
gctcacccag gcgcctcaac agtagccccc gggtccccgt cagccccctc aagttctccc   1320
cttttgaggg gtttcggccc agccccagcc tgccggagcc cacggagcta cctctacctc   1380
cactcctgcc accgccgccg ccctcgacca ccgcccctct ccccatccgt cccccctcag   1440
ggaccaagaa gtccaagagg ggccgtgggc gtccagggcg ctaggaagcc gggtgggggt   1500
gagggtagcc cttgagccct gtccctgcgg ctgtgagagc agcaggaccc tgggccagtt   1560
ccagagacct gggggtgtgt ctgggggtgg ggtgtgagtg cgtatgaaag tgtgtgtctg   1620
ctggggcagc tgtgcccctg aatcatgggc acggagggcc gcccgccaca ccccgcgctc   1680
aactgctccc gtggaagatt aaagggctga atcatgaaaa aaaaaaaaaa aa           1732
 
           
             2 
             482 
             PRT 
             Homo sapiens 
           
            2
Met Ala Ala Val Phe Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser Glu
 1               5                  10                  15
Gly Glu Gly Glu Pro Glu Leu Ser Pro Ala Asp Ala Cys Pro Leu Ala
            20                  25                  30
Glu Leu Arg Ala Ala Gly Leu Glu Pro Val Gly His Tyr Glu Glu Val
        35                  40                  45
Glu Leu Thr Glu Thr Ser Val Asn Val Gly Pro Glu Arg Ile Gly Pro
    50                  55                  60
His Cys Phe Glu Leu Leu Arg Val Leu Gly Lys Gly Gly Tyr Gly Lys
65                  70                  75                  80
Val Phe Gln Val Arg Lys Val Gln Gly Thr Asn Leu Gly Lys Ile Tyr
                85                  90                  95
Ala Met Lys Val Leu Arg Lys Ala Lys Ile Val Arg Asn Ala Lys Asp
            100                 105                 110
Thr Ala His Thr Arg Ala Glu Arg Asn Ile Leu Glu Ser Val Lys His
        115                 120                 125
Pro Phe Ile Val Glu Leu Ala Tyr Ala Phe Gln Thr Gly Gly Lys Leu
    130                 135                 140
Tyr Leu Ile Leu Glu Cys Leu Ser Gly Gly Glu Leu Phe Thr His Leu
145                 150                 155                 160
Glu Arg Glu Gly Ile Phe Leu Glu Asp Thr Ala Cys Phe Tyr Leu Ala
                165                 170                 175
Glu Ile Thr Leu Ala Leu Gly His Leu His Ser Gln Gly Ile Ile Tyr
            180                 185                 190
Arg Asp Leu Lys Pro Glu Asn Ile Met Leu Ser Ser Gln Gly His Ile
        195                 200                 205
Lys Leu Thr Asp Phe Gly Leu Cys Lys Glu Ser Ile His Glu Gly Ala
    210                 215                 220
Val Thr His Thr Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu Ile
225                 230                 235                 240
Leu Val Arg Ser Gly His Asn Arg Ala Val Asp Trp Trp Ser Leu Gly
                245                 250                 255
Ala Leu Met Tyr Asp Met Leu Thr Gly Ser Pro Pro Phe Thr Ala Glu
            260                 265                 270
Asn Arg Lys Lys Thr Met Asp Lys Ile Ile Arg Gly Lys Leu Ala Leu
        275                 280                 285
Pro Pro Tyr Leu Thr Pro Asp Ala Arg Asp Leu Val Lys Lys Phe Leu
    290                 295                 300
Lys Arg Asn Pro Ser Gln Arg Ile Gly Gly Gly Pro Gly Asp Ala Ala
305                 310                 315                 320
Asp Val Gln Arg His Pro Phe Phe Arg His Met Asn Trp Asp Asp Leu
                325                 330                 335
Leu Ala Trp Arg Val Asp Pro Pro Phe Arg Pro Cys Leu Gln Ser Glu
            340                 345                 350
Glu Asp Val Ser Gln Phe Asp Thr Arg Phe Thr Arg Gln Thr Pro Val
        355                 360                 365
Asp Ser Pro Asp Asp Thr Ala Leu Ser Glu Ser Ala Asn Gln Ala Phe
    370                 375                 380
Leu Gly Phe Thr Tyr Val Ala Pro Ser Val Leu Asp Ser Ile Lys Glu
385                 390                 395                 400
Gly Phe Ser Phe Gln Pro Lys Leu Arg Ser Pro Arg Arg Leu Asn Ser
                405                 410                 415
Ser Pro Arg Val Pro Val Ser Pro Leu Lys Phe Ser Pro Phe Glu Gly
            420                 425                 430
Phe Arg Pro Ser Pro Ser Leu Pro Glu Pro Thr Glu Leu Pro Leu Pro
        435                 440                 445
Pro Leu Leu Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro Leu Pro Ile
    450                 455                 460
Arg Pro Pro Ser Gly Thr Lys Lys Ser Lys Arg Gly Arg Gly Arg Pro
465                 470                 475                 480
Gly Arg
 
           
             3 
             495 
             PRT 
             Homo sapiens 
           
            3
Met Ala Arg Gly Arg Arg Ala Arg Gly Ala Gly Ala Ala Met Ala Ala
 1               5                  10                  15
Val Phe Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser Glu Gly Glu Gly
            20                  25                  30
Glu Pro Glu Leu Ser Pro Ala Asp Ala Cys Pro Leu Ala Glu Leu Arg
        35                  40                  45
Ala Ala Gly Leu Glu Pro Val Gly His Tyr Glu Glu Val Glu Leu Thr
    50                  55                  60
Glu Thr Ser Val Asn Val Gly Pro Glu Arg Ile Gly Pro His Cys Phe
65                  70                  75                  80
Glu Leu Leu Arg Val Leu Gly Lys Gly Gly Tyr Gly Lys Val Phe Gln
                85                  90                  95
Val Arg Lys Val Gln Gly Thr Asn Leu Gly Lys Ile Tyr Ala Met Lys
            100                 105                 110
Val Leu Arg Lys Ala Lys Ile Val Arg Asn Ala Lys Asp Thr Ala His
        115                 120                 125
Thr Arg Ala Glu Arg Asn Ile Leu Glu Ser Val Lys His Pro Phe Ile
    130                 135                 140
Val Glu Leu Ala Tyr Ala Phe Gln Thr Gly Gly Lys Leu Tyr Leu Ile
145                 150                 155                 160
Leu Glu Cys Leu Ser Gly Gly Glu Leu Phe Thr His Leu Glu Arg Glu
                165                 170                 175
Gly Ile Phe Leu Glu Asp Thr Ala Cys Phe Tyr Leu Ala Glu Ile Thr
            180                 185                 190
Leu Ala Leu Gly His Leu His Ser Gln Gly Ile Ile Tyr Arg Asp Leu
        195                 200                 205
Lys Pro Glu Asn Ile Met Leu Ser Ser Gln Gly His Ile Lys Leu Thr
    210                 215                 220
Asp Phe Gly Leu Cys Lys Glu Ser Ile His Glu Gly Ala Val Thr His
225                 230                 235                 240
Thr Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu Ile Leu Val Arg
                245                 250                 255
Ser Gly His Asn Arg Ala Val Asp Trp Trp Ser Leu Gly Ala Leu Met
            260                 265                 270
Tyr Asp Met Leu Thr Gly Ser Pro Pro Phe Thr Ala Glu Asn Arg Lys
        275                 280                 285
Lys Thr Met Asp Lys Ile Ile Arg Gly Lys Leu Ala Leu Pro Pro Tyr
    290                 295                 300
Leu Thr Pro Asp Ala Arg Asp Leu Val Lys Lys Phe Leu Lys Arg Asn
305                 310                 315                 320
Pro Ser Gln Arg Ile Gly Gly Gly Pro Gly Asp Ala Ala Asp Val Gln
                325                 330                 335
Arg His Pro Phe Phe Arg His Met Asn Trp Asp Asp Leu Leu Ala Trp
            340                 345                 350
Arg Val Asp Pro Pro Phe Arg Pro Cys Leu Gln Ser Glu Glu Asp Val
        355                 360                 365
Ser Gln Phe Asp Thr Arg Phe Thr Arg Gln Thr Pro Val Asp Ser Pro
    370                 375                 380
Asp Asp Thr Ala Leu Ser Glu Ser Ala Asn Gln Ala Phe Leu Gly Phe
385                 390                 395                 400
Thr Tyr Val Ala Pro Ser Val Leu Asp Ser Ile Lys Glu Gly Phe Ser
                405                 410                 415
Phe Gln Pro Lys Leu Arg Ser Pro Arg Arg Leu Asn Ser Ser Pro Arg
            420                 425                 430
Val Pro Val Ser Pro Leu Lys Phe Ser Pro Phe Glu Gly Phe Arg Pro
        435                 440                 445
Ser Pro Ser Leu Pro Glu Pro Thr Glu Leu Pro Leu Pro Pro Leu Leu
    450                 455                 460
Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro Leu Pro Ile Arg Pro Pro
465                 470                 475                 480
Ser Gly Thr Lys Lys Ser Lys Arg Gly Arg Gly Arg Pro Gly Arg
                485                 490                 495
 
           
             4 
             65 
             PRT 
             Homo sapiens 
           
            4
Met Ala Ala Val Phe Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser Glu
 1               5                  10                  15
Gly Glu Gly Glu Pro Glu Leu Ser Pro Ala Asp Ala Cys Pro Leu Ala
            20                  25                  30
Glu Leu Arg Ala Ala Gly Leu Glu Pro Val Gly His Tyr Glu Glu Val
        35                  40                  45
Glu Leu Thr Glu Thr Ser Val Asn Val Gly Pro Glu Arg Ile Gly Pro
    50                  55                  60
His
65
 
           
             5 
             18 
             PRT 
             Homo sapiens 
           
            5
Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser Glu Gly Glu Gly Glu Pro
 1               5                  10                  15
Glu Leu
 
           
             6 
             258 
             PRT 
             Homo sapiens 
           
            6
His Cys Phe Glu Leu Leu Arg Val Leu Gly Lys Gly Gly Tyr Gly Lys
 1               5                  10                  15
Val Phe Gln Val Arg Lys Val Gln Gly Thr Asn Leu Gly Lys Ile Tyr
            20                  25                  30
Ala Met Lys Val Leu Arg Lys Ala Lys Ile Val Arg Asn Ala Lys Asp
        35                  40                  45
Thr Ala His Thr Arg Ala Glu Arg Asn Ile Leu Glu Ser Val Lys His
    50                  55                  60
Pro Phe Ile Val Glu Leu Ala Tyr Ala Phe Gln Thr Gly Gly Lys Leu
65                  70                  75                  80
Tyr Leu Ile Leu Glu Cys Leu Ser Gly Gly Glu Leu Phe Thr His Leu
                85                  90                  95
Glu Arg Glu Gly Ile Phe Leu Glu Asp Thr Ala Cys Phe Tyr Leu Ala
            100                 105                 110
Glu Ile Thr Leu Ala Leu Gly His Leu His Ser Gln Gly Ile Ile Tyr
        115                 120                 125
Arg Asp Leu Lys Pro Glu Asn Ile Met Leu Ser Ser Gln Gly His Ile
    130                 135                 140
Lys Leu Thr Asp Phe Gly Leu Cys Lys Glu Ser Ile His Glu Gly Ala
145                 150                 155                 160
Val Thr His Thr Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu Ile
                165                 170                 175
Leu Val Arg Ser Gly His Asn Arg Ala Val Asp Trp Trp Ser Leu Gly
            180                 185                 190
Ala Leu Met Tyr Asp Met Leu Thr Gly Ser Pro Pro Phe Thr Ala Glu
        195                 200                 205
Asn Arg Lys Lys Thr Met Asp Lys Ile Ile Arg Gly Lys Leu Ala Leu
    210                 215                 220
Pro Pro Tyr Leu Thr Pro Asp Ala Arg Asp Leu Val Lys Lys Phe Leu
225                 230                 235                 240
Lys Arg Asn Pro Ser Gln Arg Ile Gly Gly Gly Pro Gly Asp Ala Ala
                245                 250                 255
Asp Val
 
           
             7 
             66 
             PRT 
             Homo sapiens 
           
            7
Asn Trp Asp Asp Leu Leu Ala Trp Arg Val Asp Pro Pro Phe Arg Pro
 1               5                  10                  15
Cys Leu Gln Ser Glu Glu Asp Val Ser Gln Phe Asp Thr Arg Phe Thr
            20                  25                  30
Arg Gln Thr Pro Val Asp Ser Pro Asp Asp Thr Ala Leu Ser Glu Ser
        35                  40                  45
Ala Asn Gln Ala Phe Leu Gly Phe Thr Tyr Val Ala Pro Ser Val Leu
    50                  55                  60
Asp Ser
65
 
           
             8 
             85 
             PRT 
             Homo sapiens 
           
            8
Ile Lys Glu Gly Phe Ser Phe Gln Pro Lys Leu Arg Ser Pro Arg Arg
 1               5                  10                  15
Leu Asn Ser Ser Pro Arg Val Pro Val Ser Pro Leu Lys Phe Ser Pro
            20                  25                  30
Phe Glu Gly Phe Arg Pro Ser Pro Ser Leu Pro Glu Pro Thr Glu Leu
        35                  40                  45
Pro Leu Pro Pro Leu Leu Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro
    50                  55                  60
Leu Pro Ile Arg Pro Pro Ser Gly Thr Lys Lys Ser Lys Arg Gly Arg
65                  70                  75                  80
Gly Arg Pro Gly Arg
                85
 
           
             9 
             22 
             PRT 
             Homo sapiens 
           
            9
Pro Leu Pro Pro Leu Leu Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro
 1               5                  10                  15
Leu Pro Ile Arg Pro Pro
            20
 
           
             10 
             65 
             PRT 
             Homo sapiens 
           
            10
Met Ala Ala Val Phe Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser Glu
 1               5                  10                  15
Gly Glu Gly Glu Pro Glu Leu Ser Pro Ala Asp Ala Cys Pro Leu Ala
            20                  25                  30
Glu Leu Arg Ala Ala Gly Leu Glu Pro Val Gly His Tyr Glu Glu Val
        35                  40                  45
Glu Leu Thr Glu Thr Ser Val Asn Val Gly Pro Glu Arg Ile Gly Pro
    50                  55                  60
His
65
 
           
             11 
             18 
             PRT 
             Homo sapiens 
           
            11
Asp Leu Asp Leu Glu Thr Glu Glu Gly Ser Glu Gly Glu Gly Glu Pro
 1               5                  10                  15
Glu Leu
 
           
             12 
             268 
             PRT 
             Homo sapiens 
           
            12
His Cys Phe Glu Leu Leu Arg Val Leu Gly Lys Gly Gly Tyr Gly Lys
 1               5                  10                  15
Val Phe Gln Val Arg Lys Val Gln Gly Thr Asn Leu Gly Lys Ile Tyr
            20                  25                  30
Ala Met Lys Val Leu Arg Lys Ala Lys Ile Val Arg Asn Ala Lys Asp
        35                  40                  45
Thr Ala His Thr Arg Ala Glu Arg Asn Ile Leu Glu Ser Val Lys His
    50                  55                  60
Pro Phe Ile Val Glu Leu Ala Tyr Ala Phe Gln Thr Gly Gly Lys Leu
65                  70                  75                  80
Tyr Leu Ile Leu Glu Cys Leu Ser Gly Gly Glu Leu Phe Thr His Leu
                85                  90                  95
Glu Arg Glu Gly Ile Phe Leu Glu Asp Thr Ala Cys Phe Tyr Leu Ala
            100                 105                 110
Glu Ile Thr Leu Ala Leu Gly His Leu His Ser Gln Gly Ile Ile Tyr
        115                 120                 125
Arg Asp Leu Lys Pro Glu Asn Ile Met Leu Ser Ser Gln Gly His Ile
    130                 135                 140
Lys Leu Thr Asp Phe Gly Leu Cys Lys Glu Ser Ile His Glu Gly Ala
145                 150                 155                 160
Val Thr His Thr Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu Ile
                165                 170                 175
Leu Val Arg Ser Gly His Asn Arg Ala Val Asp Trp Trp Ser Leu Gly
            180                 185                 190
Ala Leu Met Tyr Asp Met Leu Thr Gly Ser Pro Pro Phe Thr Ala Glu
        195                 200                 205
Asn Arg Lys Lys Thr Met Asp Lys Ile Ile Arg Gly Lys Leu Ala Leu
    210                 215                 220
Pro Pro Tyr Leu Thr Pro Asp Ala Arg Asp Leu Val Lys Lys Phe Leu
225                 230                 235                 240
Lys Arg Asn Pro Ser Gln Arg Ile Gly Gly Gly Pro Gly Asp Ala Ala
                245                 250                 255
Asp Val Gln Arg His Pro Phe Phe Arg His Met Asn
            260                 265
 
           
             13 
             66 
             PRT 
             Homo sapiens 
           
            13
Asn Trp Asp Asp Leu Leu Ala Trp Arg Val Asp Pro Pro Phe Arg Pro
 1               5                  10                  15
Cys Leu Gln Ser Glu Glu Asp Val Ser Gln Phe Asp Thr Arg Phe Thr
            20                  25                  30
Arg Gln Thr Pro Val Asp Ser Pro Asp Asp Thr Ala Leu Ser Glu Ser
        35                  40                  45
Ala Asn Gln Ala Phe Leu Gly Phe Thr Tyr Val Ala Pro Ser Val Leu
    50                  55                  60
Asp Ser
65
 
           
             14 
             85 
             PRT 
             Homo sapiens 
           
            14
Ile Lys Glu Gly Phe Ser Phe Gln Pro Lys Leu Arg Ser Pro Arg Arg
 1               5                  10                  15
Leu Asn Ser Ser Pro Arg Val Pro Val Ser Pro Leu Lys Phe Ser Pro
            20                  25                  30
Phe Glu Gly Phe Arg Pro Ser Pro Ser Leu Pro Glu Pro Thr Glu Leu
        35                  40                  45
Pro Leu Pro Pro Leu Leu Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro
    50                  55                  60
Leu Pro Ile Arg Pro Pro Ser Gly Thr Lys Lys Ser Lys Arg Gly Arg
65                  70                  75                  80
Gly Arg Pro Gly Arg
                85
 
           
             15 
             22 
             PRT 
             Homo sapiens 
           
            15
Pro Leu Pro Pro Leu Leu Pro Pro Pro Pro Pro Ser Thr Thr Ala Pro
 1               5                  10                  15
Leu Pro Ile Arg Pro Pro
            20
 
           
             16 
             525 
             PRT 
             Homo sapiens 
           
            16
Met Arg Arg Arg Arg Arg Arg Asp Gly Phe Tyr Pro Ala Pro Asp Phe
 1               5                  10                  15
Arg Asp Arg Glu Ala Glu Asp Met Ala Gly Val Phe Asp Ile Asp Leu
            20                  25                  30
Asp Gln Pro Glu Asp Ala Gly Ser Glu Asp Glu Leu Glu Glu Gly Gly
        35                  40                  45
Gln Leu Asn Glu Ser Met Asp His Gly Gly Val Gly Pro Tyr Glu Leu
    50                  55                  60
Gly Met Glu His Cys Glu Lys Phe Glu Ile Ser Glu Thr Ser Val Asn
65                  70                  75                  80
Arg Gly Pro Glu Lys Ile Arg Pro Glu Cys Phe Glu Leu Leu Arg Val
                85                  90                  95
Leu Gly Lys Gly Gly Tyr Gly Lys Val Phe Gln Val Arg Lys Val Thr
            100                 105                 110
Gly Ala Asn Thr Gly Lys Ile Phe Ala Met Lys Val Leu Lys Lys Ala
        115                 120                 125
Met Ile Val Arg Asn Ala Lys Asp Thr Ala His Thr Lys Ala Glu Arg
    130                 135                 140
Asn Ile Leu Glu Glu Val Lys His Pro Phe Ile Val Asp Leu Ile Tyr
145                 150                 155                 160
Ala Phe Gln Thr Gly Gly Lys Leu Tyr Leu Ile Leu Glu Tyr Leu Ser
                165                 170                 175
Gly Gly Glu Leu Phe Met Gln Leu Glu Arg Glu Gly Ile Phe Met Glu
            180                 185                 190
Asp Thr Ala Cys Phe Tyr Leu Ala Glu Ile Ser Met Ala Leu Gly His
        195                 200                 205
Leu His Gln Lys Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn Ile
    210                 215                 220
Met Leu Asn His Gln Gly His Val Lys Leu Thr Asp Phe Gly Leu Cys
225                 230                 235                 240
Lys Glu Ser Ile His Asp Gly Thr Val Thr His Thr Phe Cys Gly Thr
                245                 250                 255
Ile Glu Tyr Met Ala Pro Glu Ile Leu Met Arg Ser Gly His Asn Arg
            260                 265                 270
Ala Val Asp Trp Trp Ser Leu Gly Ala Leu Met Tyr Asp Met Leu Thr
        275                 280                 285
Gly Ala Pro Pro Phe Thr Gly Glu Asn Arg Lys Lys Thr Ile Asp Lys
    290                 295                 300
Ile Leu Lys Cys Lys Leu Asn Leu Pro Pro Tyr Leu Thr Gln Glu Ala
305                 310                 315                 320
Arg Asp Leu Leu Lys Lys Leu Leu Lys Arg Asn Ala Ala Ser Arg Leu
                325                 330                 335
Gly Ala Gly Pro Gly Asp Ala Gly Glu Val Gln Ala His Pro Phe Phe
            340                 345                 350
Arg His Ile Asn Trp Glu Glu Leu Leu Ala Arg Lys Val Glu Pro Pro
        355                 360                 365
Phe Lys Pro Leu Leu Gln Ser Glu Glu Asp Val Ser Gln Phe Asp Ser
    370                 375                 380
Lys Phe Thr Arg Gln Thr Pro Val Asp Ser Pro Asp Asp Ser Thr Leu
385                 390                 395                 400
Ser Glu Ser Ala Asn Gln Val Phe Leu Gly Phe Thr Tyr Val Ala Pro
                405                 410                 415
Ser Val Leu Glu Ser Val Lys Glu Lys Phe Ser Phe Glu Pro Lys Ile
            420                 425                 430
Arg Ser Pro Arg Arg Phe Ile Gly Ser Pro Arg Thr Pro Val Ser Pro
        435                 440                 445
Val Lys Phe Ser Pro Gly Asp Phe Trp Gly Arg Gly Ala Ser Ala Ser
    450                 455                 460
Thr Ala Asn Pro Gln Thr Pro Val Glu Tyr Pro Met Glu Thr Ser Gly
465                 470                 475                 480
Ile Glu Gln Met Asp Val Thr Met Ser Gly Glu Ala Ser Ala Pro Leu
                485                 490                 495
Pro Ile Arg Gln Pro Asn Ser Gly Pro Tyr Lys Lys Gln Ala Phe Pro
            500                 505                 510
Met Ile Ser Lys Arg Pro Glu His Leu Arg Met Asn Leu
        515                 520                 525