Dsp-10 dual-specificity phosphatase

Abstract

Compositions and methods are provided for the treatment of conditions associated with cell proliferation, cell differentiation and cell survival. In particular, the dual-specificity phosphatase DSP-10, and polypeptide variants thereof that stimulate dephosphorylation of DSP-10 substrates, are provided. The polypeptides may be used, for example, to identify antibodies and other agents that inhibit DSP-10 activity. The polypeptides and agents may be used to modulate cell proliferation, differentiation, and survival.

1 . An isolated antibody, or antigen-binding fragment thereof, that specifically binds to a dual specificity phosphatase-10 (DSP-10) polypeptide comprising the amino acid sequence set forth SEQ ID NO:2. 
     
     
         2 . The antibody or antigen-binding fragment thereof according to  claim 1 , wherein the antibody is a monoclonal antibody. 
     
     
         3 . A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to  claim 1  in combination with a physiologically acceptable carrier. 
     
     
         4 . A method for detecting a dual specificity phosphatase-10 (DSP-10) polypeptide expression in a sample, comprising:
 (a) contacting a sample with the antibody, or the antigen-binding fragment thereof, according to  claim 1 , under conditions and for a time sufficient to allow formation of an antibody/DSP-10 complex; and   (b) detecting the level of antibody/DSP-10 complex, and therefrom detecting the presence of DSP-10 in a sample.   
     
     
         5 . The method according to  claim 4 , wherein the antibody, or the antigen-binding fragment thereof, is linked to a support material. 
     
     
         6 . The method according to  claim 4 , wherein the antibody, or the antigen-binding fragment thereof, is linked to a detectable marker. 
     
     
         7 . The method according to  claim 4 , wherein the sample is a biological sample obtained from a patient. 
     
     
         8 . A method for detecting dual specificity phosphatase-10 (DSP-10) expression in a sample, comprising:
 (a) contacting a sample with an antisense polynucleotide that comprises
 (i) a nucleotide sequence complementary to a polynucleotide that encodes a DSP-10 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2; 
 (ii) a nucleotide sequence complementary to a polynucleotide that encodes a DSP-10 polypeptide variant, wherein the variant differs from the amino acid sequence set forth in SEQ ID NO:2 in one or more amino acid deletions, additions, insertions or substitutions at no more than 25% of the residues in SEQ ID NO:2, and wherein the DSP-10 variant comprises the peptide sequence LLIHCQAGVSRSATIV (SEQ ID NO:3), such that the polypeptide retains the ability to dephosphorylate an activated MAP-kinase; or 
 (iii) a nucleotide sequence complementary to a polynucleotide that comprises the sequence set forth in SEQ ID NO:1; and 
   (b) detecting in the sample an amount of DSP-10 polynucleotide that hybridizes to the antisense polynucleotide, and therefrom detecting DSP-10 expression in the sample.   
     
     
         9 . The method according to  claim 8 , wherein the amount of DSP-10 polynucleotide that hybridizes to the antisense polynucleotide is determined using polymerase chain reaction. 
     
     
         10 . The method according to  claim 8 , wherein the amount of DSP-10 polynucleotide that hybridizes to the antisense polynucleotide is determined using a hybridization assay. 
     
     
         11 . The method according to  claim 8 , wherein the sample comprises an RNA or cDNA preparation. 
     
     
         12 . A method for screening for an agent that modulates DSP-10 activity, comprising the steps of:
 (a) contacting a candidate agent with a polypeptide that comprises
 (i) the amino acid sequence set forth in SEQ ID NO:2; 
 (ii) an amino acid sequence set forth in SEQ ID NO:2 that has one or more amino acid deletions, additions, insertions, or substitutions at no more than 25% of the residues in SEQ ID NO:2, and wherein the polypeptide comprises the peptide sequence LLIHCQAGVSRSATIV (SEQ ID NO:3), such that the polypeptide retains the ability to dephosphorylate an activated MAP-kinase; 
 (iii) an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence at least 90% identical to SEQ ID NO:1, wherein the polypeptide is capable of dephosphorylating an activated MAP-kinase, and wherein the polypeptide comprises the peptide sequence LLIHCQAGVSRSATIV (SEQ ID NO:3); or 
 (iv) an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence at least 80% identical to SEQ ID NO:1, wherein the polypeptide is capable of dephosphorylating an activated MAP-kinase, and wherein the polypeptide comprises the peptide sequence LLIHCQAGVSRSATIV (SEQ ID NO:3), 
   under conditions and for a time sufficient to permit interaction between the polypeptide and candidate agent; and   (b) subsequently evaluating the ability of the polypeptide to dephosphorylate a DSP-10 substrate, relative to a predetermined ability of the polypeptide to dephosphorylate the DSP-10 substrate in the absence of candidate agent, and therefrom identifying an agent that modulates DSP-10 activity.   
     
     
         13 . The method according to  claim 12  wherein the DSP-10 substrate is a MAP-kinase. 
     
     
         14 . The method according to  claim 12 , wherein the candidate agent is a small molecule. 
     
     
         15 . The method according to  claim 14 , wherein the small molecule is present within a combinatorial library. 
     
     
         16 . A method for screening for an agent that modulates dual specificity phosphatase-10 (DSP-10) activity, comprising the steps of:
 (a) contacting a candidate agent with a cell comprising a DSP-10 promoter operably linked to a polynucleotide encoding a detectable transcript or protein, under conditions and for a time sufficient to permit interaction between the promoter and candidate agent; and   (b) subsequently evaluating the expression of the polynucleotide, relative to a predetermined level of expression in the absence of candidate agent, and therefrom identifying an agent that modulates DSP-10 activity.   
     
     
         17 . The method according to  claim 16 , wherein the polynucleotide encodes a DSP-10 polypeptide. 
     
     
         18 . The method according to  claim 16 , wherein the polynucleotide encodes a reporter protein. 
     
     
         19 . A method for modulating a proliferative response in a cell, comprising contacting a cell with an agent that modulates DSP-10 activity. 
     
     
         20 . A method for modulating differentiation of a cell, comprising contacting a cell with an agent that modulates DSP-10 activity. 
     
     
         21 . A method for modulating survival of a cell, comprising contacting a cell with an agent that modulates DSP-10 activity. 
     
     
         22 . The method according to any one of  claims 19 - 21 , wherein the agent modulates a pattern of gene expression. 
     
     
         23 . The method according to any one of  claims 19 - 21 , wherein the cell displays contact inhibition of cell growth. 
     
     
         24 . The method according to any one of  claims 19 - 21 , wherein the cell displays anchorage independent growth. 
     
     
         25 . The method according to any one of  claims 19 - 21 , wherein the cell displays an altered intercellular adhesion property. 
     
     
         26 . The method according to  claim 21 , wherein the agent modulates apoptosis. 
     
     
         27 . The method according to  claim 21 , wherein the agent modulates the cell cycle. 
     
     
         28 . The method according to  claim 21 , wherein the cell is present within a patient. 
     
     
         29 . A method for treating a patient afflicted with a disorder associated with dual specificity phosphatase-10 (DSP-10) activity, comprising administering to a patient a therapeutically effective amount of an agent that modulates DSP-10 activity. 
     
     
         30 . The method according to  claim 29 , wherein the disorder is selected from the group consisting of Duchenne muscular dystrophy, cancer, graft-versus-host disease, autoimmune diseases, allergies, metabolic diseases, abnormal cell growth, abnormal cell proliferation and cell cycle abnormalities. 
     
     
         31 . A dual specificity phosphatase-10 (DSP-10) substrate trapping mutant polypeptide that differs from the sequence set forth in SEQ ID NO:2 in one or more amino acid deletions, additions, insertions or substitutions at no more than 25% of the residues in SEQ ID NO:2, such that the polypeptide binds to a substrate with an affinity that is not substantially diminished relative to DSP-10, and such that the ability of the polypeptide to dephosphorylate a substrate is reduced relative to DSP-10. 
     
     
         32 . The substrate trapping mutant polypeptide according to  claim 31 , wherein the polypeptide contains a substitution at position 377 or position 408 of SEQ ID NO:2. 
     
     
         33 . A dual specificity phosphatase-10 (DSP-10) substrate trapping mutant polypeptide comprising a sequence that differs from the sequence set forth in SEQ ID NO:2 wherein the DSP-10 substrate trapping mutant polypeptide contains a substitution at position 377 or position 408 of SEQ ID NO:2, wherein the DSP-10 substrate trapping mutant polypeptide retains the ability to bind a DSP-10 substrate, and wherein the ability of the DSP-10 substrate trapping mutant polypeptide to dephosphorylate a substrate is reduced relative to the DSP-10 polypeptide having a sequence set forth in SEQ ID NO:2.