Novel cell signaling polypeptides and nucleic acids

Abstract

The present invention relates to an isolated SRK polypeptide, biologically-active polypeptide fragments thereof, and nucleic acids which code for it. This polypeptide has various activities in regulating cell signaling and signal transduction pathways, including, e.g., a protein kinase activity; an autophosphorylating activity; a cell survival promoting activity; a HAX-1 binding activity; an apoptosis suppression activity; a MAPKK stimulatory activity; a transcription modulatory activity, and a SRK-specific immunogenic activity. The invention relates to all aspects of SRK, or homologs thereof, including assays for modulators, activators, ligands, etc. The invention also relates to a cytolic or soluble HAX-1 which produces apoptosis when expressed in cells.

What is claimed:  
     
         1 . An isolated human SRK polypeptide, or a biologically-active polypeptide fragment thereof.  
     
     
         2 . An isolated SRK polypeptide of  claim 1 , wherein said polypeptide has protein kinase activity; HAX-1 binding activity; apoptosis suppression activity; MAPKK stimulatory activity; and SRK-specific immunogenic activity.  
     
     
         3 . An isolated human SRK polypeptide of  claim 1 , comprising amino acid 1 to amino acid 455 set forth in FIG. 2.  
     
     
         4 . An isolated human SRK polypeptide of  claim 1 , comprising amino acid 1 to amino acid 455 and having the DNA sequence set forth in FIG. 2 
     
     
         5 . An isolated biologically-active fragment of human SRK of  claim 1 , wherein said fragment comprises amino acids 1-250, 23-250, 287-322, and 430-455.  
     
     
         6 . An isolated biologically-active fragment of human SRK of  claim 1 , wherein said fragment consists essentially of amino acids 1-455 as set forth in FIG. 2.  
     
     
         7 . An isolated human SRK polypeptide of  claim 1 , having 95% sequence identity to the amino acid sequence of FIG. 2.  
     
     
         8 . An isolated SRK, or a biologically-active fragment thereof, which is coded for by the complement of a nucleic acid sequence which hybridizes under high stringency conditions to the DNA sequence set forth in FIG. 2 and which has at least 95% sequence identity to said DNA sequence.  
     
     
         9 . An isolated SRK, or a biologically-active fragment thereof, of  claim 8  which is obtainable from a natural source.  
     
     
         10 . An isolated SRK of  claim 8 , having the amino acid sequence of FIG. 2, except where lysine at amino acid position 45 is replaced by alanine; lysine at amino acid position 45 is replaced by alanine and lysine amino acid position 46 is replaced by alanine; or where aspartic acid at amino acid position 133 is replaced by an alanine.  
     
     
         11 . An isolated SRK, or a biologically-active fragment thereof, of  claim 8  which has a protein kinase activity; HAX-1 binding activity; apoptosis suppression activity; MAPKK stimulatory activity; and SRK-specific immunogenic activity.  
     
     
         12 . An isolated nucleic acid comprising a nucleotide sequence coding for a full-length human SRK polypeptide, or a biologically-active polypeptide fragment thereof.  
     
     
         13 . An isolated nucleic acid of  claim 12 , wherein said coded-for polypeptide a protein kinase activity; HAX-1 binding activity; apoptosis suppression activity; MAPKK stimulatory activity; and SRK-specific immunogenic activity.  
     
     
         14 . An isolated nucleic acid of  claim 12 , wherein said nucleic acid is coded-for by an mRNA which is 7.5 kb, 3.8 kb, or 1.6 kb.  
     
     
         15 . An isolated nucleic acid of  claim 12 , wherein the nucleotide sequence codes for amino acid 1 to amino acid 455 as set forth in FIG. 2.  
     
     
         16 . An isolated nucleic acid of  claim 12 , having the nucleotide sequence set forth in FIG. 2.  
     
     
         17 . An isolated nucleic acid of  claim 12 , wherein the nucleotide sequence is operably linked to an expression control sequence.  
     
     
         18 . An isolated nucleic acid of  claim 12 , wherein the nucleic acid codes for said polypeptide without interruption.  
     
     
         19 . An isolated nucleic acid of  claim 12 , wherein the nucleic acid further comprises a detectable label.  
     
     
         20 . A method of expressing in transformed host cells, a human SRK polypeptide coded for by a nucleic acid, comprising: 
 culturing transformed host cells containing a nucleic acid of  claim 11  under conditions effective to express the polypeptide.    
     
     
         21 . A method of  claim 20 , wherein said host cells are mammalian.  
     
     
         22 . A method of  claim 20 , wherein said host cells are yeast.  
     
     
         23 . A method of  claim 20 , further comprising isolating said human SRK.  
     
     
         24 . An isolated human SRK polypeptide produced by a method of  claim 20 .  
     
     
         25 . A transformed host cell containing a nucleic acid of  claim 12 .  
     
     
         26 . A vector comprising a nucleic acid of  claim 12 .  
     
     
         27 . An isolated nucleic acid which hybridizes under high stringency conditions to the nucleic sequence set forth in FIG. 2, or a complement thereto, and which has at least 95% sequence identity to said nucleic sequence, or its complement.  
     
     
         28 . An isolated nucleic acid of  claim 27 , which sequence codes for a a protein kinase activity; HAX-1 binding activity; apoptosis suppression activity; MAPKK stimulatory activity; and SRK-specific immunogenic activity  
     
     
         29 . An isolated nucleic acid consisting essentially of any continuous sequence of 12-100 base pairs, or a complement thereto, selected from the nucleotide sequence set forth in FIG. 2.  
     
     
         30 . An isolated nucleic acid sequence of  claim 29 , further comprising a detectable label.  
     
     
         31 . An isolated nucleic acid of  claim 29 , having at least one but not more than five, nucleotide substitutions in said sequence, and which hybridizes under high stringency conditions to it, or a complement thereto.  
     
     
         32 . A method of detecting a protein kinase activity in a human SRK polypeptide, or a biologically-active polypeptide fragment thereof, comprising: 
 reacting a human SRK polypeptide, or a biologically-active polypeptide fragment thereof, and a substrate under conditions effective said SRK polypeptide to phosphorylate said substrate; and    detecting said phosphorylation of said substrate.    
     
     
         33 . A method of  claim 32 , wherein said substrate is MBP.  
     
     
         34 . A method of identifying agents which modulate a MAPKK stimulatory activity of of a human SRK polypeptide, or a biologically-active polypeptide fragment thereof, comprising, 
 administering a test agent to a cell expressing (1) a human SRK polypeptide, or a biologically-active polypeptide fragment thereof, and (2) an MAPKK polypeptide, under conditions effective for said SRK polypeptide to stimulate protein kinase activity of said MAPKK polypeptide;    detecting said protein kinase activity; and    identifying whether the test agent modulates said stimulatory activity of said SRK polypeptide by comparing the amount of kinase activity in the presence and absence of the test agent.    
     
     
         35 . A method of  claim 34 , wherein said administering is to a cell into which nucleic acids coding for said SRK polypeptide and said MAPKK polypeptide have been introduced and expressed.  
     
     
         36 . A method of  claim 35 , wherein said MAPKK polypeptide further comprises a polypeptide epitope.  
     
     
         37 . A method of  claim 36 , further comprising: 
 lysing said cells comprising said expressed SRK polypeptide and MAPKK polypeptide;    contacting said lysate with an anti-polypeptide epitope antibody, under conditions effective for said antibody to binds to said epitope to form a complex;    isolating said complex; and    detecting kinase activity in said isolated complex.    
     
     
         38 . A method of  claim 37 , wherein said MAPKK polypeptide is MEK and said polypeptide epitope is a myc, KT3, Glu, or hemaglutinin polypeptide sequence fused in-frame with said MEK polypeptide.  
     
     
         39 . A method of identifying agents which modulate cellular tranformation mediated by Ras and SRK, comprising: 
 administering a test agent to a cell expressing a RasV12 polypeptide and a SRK polypeptide lacking kinase activity, or a biologically-active fragment thereof, under conditions effective for said RasV12 to cause said cells to form foci, wherein said RasV12 polypeptide has a foci-forming activity and said SRK polypeptide suppresses said RasV12 foci-forming activity    detecting foci formed by said cells; and    identifying whether the test agent modulates the ability of said SRK polypeptide to suppress said RasV12 foci-forming activity by comparing the number and size of foci in the presence or absence of said test compound; and    
     
     
         40 . A method of  claim 39 , wherein said cell is a mouse NIH3T3 cell or a human 293 cell.  
     
     
         41 . A method of  claim 39 , wherein said administering is to a cell into which nucleic acids coding for said RasV12 and said SRK polypeptide have been introduced and expressed.  
     
     
         42 . A method of  claim 39 , wherein said transforming activity is assayed for in soft agar.  
     
     
         43 . A method of  claim 39 , wherein said SRK polypeptide lacking kinase activity is SRK-KA.  
     
     
         44 . A method of identifying agents which modulate cellular transformation mediated by Ras and SRK, comprising: 
 administering a test agent to a cell expressing a RasV12 polypeptide under conditions effective for said RasV12 to cause said cells to form foci, wherein said RasV12 polypeptide has a foci-forming activity;    detecting foci formed by said cells;    identifying whether the test agent modulates RasV12 foci-forming activity by comparing the number and size of foci in the presence or absence of said test compound; and    determining whether said test compound modulates a protein kinase activity of human SRK polypeptide, or a biologically-active polypeptide fragment thereof, wherein said activity is a protein kinase activity; an autophosphorylating activity; a cell transforming activity; a cell growth-regulatory activity; a HAX-1 binding activity; an apoptosis suppression activity; or a MAPKK stimulatory activity.    
     
     
         45 . A method of  claim 44 , whereby said detecting kinase activity comprises: reacting a human SRK polypeptide, or a biologically-active polypeptide fragment thereof, and a substrate under conditions effective said SRK polypeptide to phosphorylate said substrate; and 
 detecting said phosphorylation of said substrate.    
     
     
         46 . A method of  claim 45 , wherein said substrate is MBP.  
     
     
         47 . A method of identifying agents that modulate the apoptosis suppression activity of human SRK polypeptide, or a biologically-active polypeptide fragment thereof, comprising: 
 administering a test agent to a cell expressing a soluble HAX-1 polypeptide and a human SRK polypeptide, or a biologically-active polypeptide fragment thereof, wherein said soluble HAX-1 polypeptide causes apoptosis in said cell;    detecting apoptosis in said cell in the presence and absence of said test compound.    
     
     
         48 . A method of  claim 47 , whereby said detecting apoptosis comprises detecting chromosomal condensation using a DNA stain.  
     
     
         49 . A method of identifying agents that modulate the apoptosis suppression activity of human SRK polypeptide, or a biologically-active polypeptide fragment thereof, comprising: 
 administering a test agent to a cell, or a progeny cell thereof, into which a gene coding for a human SRK polypeptide, or a biologically-active polypeptide fragment, has been introduced;    culturing said cell under conditions in which apoptosis occurs; and    detecting apoptosis in said cell in the presence and absence of said test compound.    
     
     
         50 . A method of  claim 49 , whereby said detecting apoptosis comprises detecting chromosomal condensation using a DNA stain.  
     
     
         51 . An isolated antibody which is specific for a polypeptide sequence of human SRK.  
     
     
         52 . An isolated antibody of  claim 51 , which binds to an amino acid sequence selected from FIG. 2.  
     
     
         53 . An isolated antibody of  claim 49 , wherein said polypeptide sequence comprises AKQNSSKTTSKRRG.  
     
     
         54 . A human cytosolic HAX-1 having an apoptotic activity.  
     
     
         55 . A human cytosolic HAX-1 of  claim 54 , comprising amino acids 104-279 as set forth in FIG. 7.  
     
     
         56 . A human cytosolic HAX-1 of  claim 54 , comprising the nucleotide sequence set forth in FIG. 7 coding for amino acids 104-279.  
     
     
         57 . A human cytosolic HAX-1 of  claim 54 , consisting essentially of amino acids 104279 as set forth in FIG. 7.  
     
     
         58 . A human cytosolic HAX-1 of  claim 57 , further comprising a polypeptide epitope fused in-frame with said HAX-1 polypeptide.  
     
     
         59 . A human cytosolic HAX-1, having an apoptotic activity, which is coded for by the complement of a nucleic acid sequence which hybridizes under high stringency conditions to the DNA sequence set forth in FIG. 7 and which has at least 95% sequence identity to said DNA sequence.  
     
     
         60 . An isolated nucleic acid comprising a nucleotide sequence coding for a human cytosolic HAX-1.  
     
     
         61 . An isolated nucleic acid of  claim 60 , wherein the nucleotide sequence is operably linked to an expression control sequence.  
     
     
         62 . An isolated human SRK of  claim 1 , further comprising a human cytosolic HAX-1 having an apoptotic activity.  
     
     
         63 . An isolated human SRK of  claim 1 , further comprising a human cytosolic HAX-1 comprising amino acids 104-279 as set forth in FIG. 7.