DSP-4 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-4, and polypeptide variants thereof that stimulate dephosphorylation of DSP-4 substrates, are provided. The polypeptides may be used, for example, to identify antibodies and other agents that inhibit DSP-4 activity. The polypeptides and agents may be used to modulate cell proliferation, differentiation and survival.

1 . An isolated polypeptide having the sequence of DSP-4 recited in SEQ ID NO:2, or a variant thereof that differs in one or more amino acid deletions, additions, insertions or substitutions at no more than 50% of the residues in SEQ ID NO:2, such that the polypeptide retains the ability to dephosphorylate an activated MAP-kinase.  
     
     
         2 . An isolated antibody, or antigen binding fragment thereof, that specifically binds to a DSP-4 polypeptide having the sequence of SEQ ID NO:2.  
     
     
         3 . An antibody or fragment thereof according to  claim 2 , wherein the antibody is a monoclonal antibody.  
     
     
         4 . A pharmaceutical composition comprising an antibody or fragment thereof according to  claim 2  in combination with a physiologically acceptable carrier.  
     
     
         5 . A method for detecting DSP-4 expression in a sample, comprising: 
 (a) contacting a sample with an antibody or an antigen-binding fragment thereof according to  claim 2 , under conditions and for a time sufficient to allow formation of an antibody/DSP-4 complex; and    (b) detecting the level of antibody/DSP-4 complex, and therefrom detecting the presence of DSP-4 in a sample.    
     
     
         6 . A method according to  claim 5 , wherein the antibody is linked to a support material.  
     
     
         7 . A method according to  claim 5 , wherein the antibody is linked to a detectable marker.  
     
     
         8 . A method according to  claim 5 , wherein the sample is a biological sample obtained from a patient.  
     
     
         9 . A method for screening for an agent that modulates DSP-4 activity, comprising the steps of: 
 (a) contacting a candidate agent with a polypeptide according to  claim 1 , 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-4 substrate, relative to a predetermined ability of the polypeptide to dephosphorylate the DSP-4 substrate in the absence of candidate agent;    and therefrom identifying an agent that modulates DSP-4 activity.    
     
     
         10 . A method according to  claim 9 , wherein the DSP-4 substrate is a MAP-kinase.  
     
     
         11 . A method according to  claim 9 , wherein the candidate agent is a small molecule.  
     
     
         12 . A method according to  claim 9 , wherein the small molecule is present within a combinatorial library.  
     
     
         13 . A method for screening for an agent that modulates DSP-4 activity, comprising the steps of: 
 (a) contacting a candidate agent with a cell comprising a DSP-4 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-4 activity.    
     
     
         14 . A method according to  claim 13 , wherein the polynucleotide encodes a DSP-4 polypeptide.  
     
     
         15 . A method according to  claim 13 , wherein the polynucleotide encodes a reporter protein.  
     
     
         16 . A method for modulating a proliferative response in a cell, comprising contacting a cell with an agent that modulates DSP-4 activity.  
     
     
         17 . A method for modulating differentiation of a cell, comprising contacting a cell with an agent that modulates DSP-4 activity.  
     
     
         18 . A method for modulating survival of a cell, comprising contacting a cell with an agent that modulates DSP-4 activity.  
     
     
         19 . A method according to any one of claims  16 - 18 , wherein the agent modulates a pattern of gene expression.  
     
     
         20 . A method according to any one of claims  16 - 18 , wherein the cell displays contact inhibition of cell growth.  
     
     
         21 . A method according to any one of claims  16 - 18 , wherein the cell displays anchorage independent growth.  
     
     
         22 . A method according to any one of claims  16 - 18 , wherein the cell displays an altered intercellular adhesion property.  
     
     
         23 . A method according to  claim 18 , wherein the agent modulates apoptosis.  
     
     
         24 . A method according to  claim 18 , wherein the agent modulates the cell cycle.  
     
     
         25 . A method according to  claim 15 , wherein the cell is present within a patient.  
     
     
         26 . A method for treating a patient afflicted with a disorder associated with DSP-4 activity, comprising administering to a patient a therapeutically effective amount of an agent that modulates DSP-4 activity.  
     
     
         27 . A method according to  claim 26 , 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.  
     
     
         28 . A DSP-4 substrate trapping mutant polypeptide that differs from the sequence recited in SEQ ID NO:2 in one or more amino acid deletions, additions, insertions or substitutions at no more than 50% 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-4, and such that the ability of the polypeptide to dephosphorylate a substrate is reduced relative to DSP-4.  
     
     
         29 . A substrate trapping mutant polypeptide according to  claim 28 , wherein the polypeptide contains a substitution at position 118 or position 150 of SEQ ID NO:2.  
     
     
         30 . A method for screening a molecule for the ability to interact with DSP-4, comprising the steps of: 
 (a) contacting a candidate molecule with a polypeptide according to  claim 1  under conditions and for a time sufficient to permit the candidate molecule and polypeptide to interact; and    (b) detecting the presence or absence of binding of the candidate molecule to the polypeptide, and therefrom determining whether the candidate molecule interacts with DSP-4.    
     
     
         31 . A method according to  claim 30 , wherein the step of detecting comprises an affinity purification step.  
     
     
         32 . A method according to  claim 30 , wherein the step of detecting comprises a yeast two hybrid screen or a screen of a phage display library.