PARG, a GTPase activating protein which interacts with PTPL1

Abstract

The invention describes nucleic acids encoding the PARG protein, including fragments and biologically functional variants thereof. Also included are polypeptides and fragments thereof encoded by such nucleic acids, and antibodies relating thereto. Methods and products for using such nucleic acids and polypeptides also are provided.

1 . An isolated nucleic acid molecule 
 (a) which hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ ID NO:1 and which codes for a GTPase-activating polypeptide,    (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to the degeneracy of the genetic code, and    (c) complements of (a) and (b).    
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated nucleic acid molecule comprises nucleotides 184-3966 of SEQ ID NO:1.  
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated nucleic acid molecule consists of SEQ ID NO:1.  
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein the isolated nucleic acid molecule comprises a molecule having a sequence which encodes amino acids 658-898 of SEQ ID NO:2.  
     
     
         5 . An isolated nucleic acid molecule selected from the group consisting of (a) a unique fragment of nucleotides 184-3966 of SEQ ID NO:1 between 12 and 3781 nucleotides in length and (b) complements of “(a)”, provided that the nucleic acid molecule excludes molecules consisting solely of nucleotide sequences selected from the group consisting of accession numbers T32345, Z28937, Z28520, AA431926, AA326126, AA342471, AA716829, L49573, Z43348, AA303722, T32495, AA330162, Z25350, AA794256, T32506, T32263, F06673, AA462548, X85558, R14952, AA870705, AA120493, AA415591, AA131400, C76597, C76601, AA870475, AA234871, C77518, and AA672012.  
     
     
         6 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of at least 14 contiguous nucleotides.  
     
     
         7 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of at least 15 contiguous nucleotides.  
     
     
         8 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of at least 16 contiguous nucleotides.  
     
     
         9 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of at least 17 contiguous nucleotides.  
     
     
         10 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of at least 18 contiguous nucleotides.  
     
     
         11 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of at least 20 contiguous nucleotides.  
     
     
         12 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of at least 22 contiguous nucleotides.  
     
     
         13 . The isolated nucleic acid molecule of  claim 5 , wherein the isolated nucleic acid molecule consists of between 12 and 32 contiguous nucleotides.  
     
     
         14 . An isolated nucleic acid molecule, comprising 
 (a) a molecule having a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10;    (b) nucleic acid molecules that differ from the nucleic acid molecules of (a) in codon sequence due to the degeneracy of the genetic code, and    (c) complements of (a) and (b).    
     
     
         15 . An expression vector comprising the isolated nucleic acid molecule of any of claims  1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 , or  14  operably linked to a promoter.  
     
     
         16 . A host cell transformed or transfected with the expression vector of  claim 15 .  
     
     
         17 . An isolated polypeptide encoded by the isolated nucleic acid molecule of any of claims  1 ,  2  or  4 , wherein the polypeptide has GTPase activating activity.  
     
     
         18 . The isolated polypeptide of  claim 17 , wherein the isolated polypeptide comprises a polypeptide having the sequence of amino acids 658-898 of SEQ ID NO:2.  
     
     
         19 . An isolated polypeptide, comprising a polypeptide having the sequence of amino acids 613-652 of SEQ ID NO:2, wherein the isolated polypeptide is a cysteine-rich domain.  
     
     
         20 . An isolated polypeptide, comprising a polypeptide having the sequence of amino acid 193-509 of SEQ ID NO:2, wherein the isolated polypeptide is a ZPH domain polypeptide.  
     
     
         21 . The isolated polypeptide of any of claims  1 ,  2 ,  4 ,  19 , or  20 , wherein the isolated polypeptide consists of a functional fragment or variant.  
     
     
         22 . An isolated polypeptide comprising a polypeptide selected from the group consisting of a polypeptide having the sequence of SEQ ID NO:7, a polypeptide having the sequence of SEQ ID NO:9, and a polypeptide having the sequence of SEQ ID NO:11.  
     
     
         23 . The isolated polypeptide of  claim 22 , wherein the polypeptide consists of a polypeptide selected from the group consisting of a polypeptide having the sequence of SEQ ID NO:7, a polypeptide having the sequence of SEQ ID NO:9, and a polypeptide having the sequence of SEQ ID NO:11.  
     
     
         24 . An isolated polypeptide which binds selectively a polypeptide encoded by the isolated nucleic acid molecule of  claim 1 ,  2 ,  4 , or  14 , provided that the isolated polypeptide is not PTPL1.  
     
     
         25 . The isolated polypeptide of  claim 24 , wherein the isolated polypeptide binds to a polypeptide comprising the sequence of amino acids 658-898 of SEQ ID NO:2.  
     
     
         26 . The isolated polypeptide of  claim 24 , wherein the isolated polypeptide binds to a polypeptide selected from the group consisting of a polypeptide comprising the sequence of SEQ ID NO:7, a polypeptide comprising the sequence of SEQ ID NO:9, and a polypeptide comprising the sequence of SEQ ID NO:11.  
     
     
         27 . The isolated polypeptide of  claim 24 , wherein the isolated polypeptide binds to a polypeptide consisting essentially of the sequence of SEQ ID NO:2.  
     
     
         28 . The isolated polypeptide of  claim 24 , wherein the isolated polypeptide is an antibody fragment selected from the group consisting of a Fab fragment, a F(ab) 2  fragment or a fragment including a CDR3 region selective for a PARG polypeptide.  
     
     
         29 . An isolated complex of polypeptides comprising: 
 a polypeptide comprising the amino acid sequence of SEQ ID NO:12 bound to a polypeptide as claimed in  claim 1 , wherein the complex has phosphatase and GTPase activating activities.    
     
     
         30 . The isolated complex of polypeptides of  claim 29 , wherein the polypeptides consist of the polypeptide of SEQ ID NO:12 and the polypeptide of SEQ ID NO:2.  
     
     
         31 . A method for reducing Rho family signal transduction in a mammalian cell, comprising 
 administering to the mammalian cell an amount of the isolated polypeptide of  claim 17  effective to reduce Rho family signal transduction in the mammalian cell.    
     
     
         32 . A method for reducing Rho family signal transduction in a mammalian cell, comprising 
 administering to the mammalian cell an amount of the isolated polypeptide complex of  claim 29  effective to reduce Rho family signal transduction in the mammalian cell.    
     
     
         33 . A method for reducing the proliferation of a cancer cell, the proliferation of which is increased by Rho family protein signal transduction, comprising 
 administering to the cancer cell an amount of a polypeptide comprising a polypeptide encoded by the nucleic acid of SEQ ID NO:1 effective to reduce proliferation of the cancer cell.    
     
     
         34 . A method of increasing Rho family protein signal transduction in a cell comprising 
 administering to the cell an amount of a dominant-negative variant of the polypeptide of SEQ ID NO:2 effective to increase the Rho family protein signal transduction in the cell.    
     
     
         35 . The method of  claim 34 , wherein the dominant negative polypeptide has an inactivated GTPase-activating domain.  
     
     
         36 . The method of  claim 35 , wherein the inactivated GTPase-activating domain is deleted.  
     
     
         37 . The method of  claim 35 , wherein the inactivated GTPase-activating domain contains at least one inactivating point mutation.  
     
     
         38 . A method of reducing binding of a protein which includes a PDZ4 domain to a protein which includes a PDZ4 domain binding site in a mammalian cell, comprising 
 contacting the mammalian cell with an agent which binds to the PDZ4 domain for a time effective to reduce binding of the protein which includes a PDZ4 domain to the protein which includes a PDZ4 domain binding site.    
     
     
         39 . The method of  claim 38 , wherein the agent is a peptide comprising [at its carboxyl terminus] the amino acid sequence of SEQ ID NO:7, or conservative amino acid substitutions thereof excepting the terminal valine.  
     
     
         40 . The method of  claim 39 , wherein the agent is a peptide selected from the group consisting of a peptide having the amino acid sequence of SEQ ID NO:7, a peptide having the amino acid sequence of SEQ ID NO:9 and a peptide having the amino acid sequence of SEQ ID NO:11.  
     
     
         41 . The method of  claim 38 , wherein the agent is an antibody which binds to the PDZ4 domain.  
     
     
         42  The method of  claim 41 , wherein the antibody is a monoclonal antibody.  
     
     
         43 . A method of modulating mast cell secretion in a subject, comprising: 
 administering to the subject in need of such treatment an amount of a modulator of PARG GTPase activating activity effective to modulate mast cell secretion.    
     
     
         44 . A composition comprising: 
 the polypeptide of  claim 17 , and    a pharmaceutically acceptable carrier.    
     
     
         45 . A composition comprising: 
 the polypeptide complex of  claim 29 , and    a pharmaceutically acceptable carrier.    
     
     
         46 . A composition comprising: 
 the agent of  claim 39 , and    a pharmaceutically acceptable carrier.    
     
     
         47 . A method for decreasing PARG GTPase activity in a subject comprising 
 administering to a subject in need of such treatment an agent that selectively binds to an isolated nucleic acid molecule of  claim 1  or an expression product thereof, in an amount effective to decrease PARG GTPase activity in the subject.    
     
     
         48 . The method of  claim 47 , wherein the agent is an antisense nucleic acid.  
     
     
         49 . The method of  claim 47 , wherein the agent is a polypeptide.  
     
     
         50 . A method for identifying lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated with PARG GTPase activating activity, comprising 
 forming a mixture comprising a PARG polypeptide or unique fragment thereof containing a PARG GTPase activating domain, a protein containing a GTPase specifically activatable by the PARG GTPase activating domain, and a candidate pharmacological agent,    incubating the mixture under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of specific activation of the GTPase by the PARG GTPase activating domain, and    detecting a test amount of the specific activation of the GTPase by the PARG GTPase activating domain, wherein reduction of the test amount of specific activation relative to the first amount of specific activation indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which disrupts the GTPase activation activity of PARG, and wherein increase of the test amount of specific activation relative to the first amount of specific activation indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which enhances the GTPase activation activity of PARG.    
     
     
         51 . The method of  claim 50 , wherein the GTPase specifically activatable by the PARG GTPase activating domain is Rho.  
     
     
         52 . A method for identifying lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated with PARG binding to a protein containing a PDZ4 domain, comprising 
 forming a mixture comprising a PARG polypeptide or unique fragment thereof containing a PDZ4 domain binding site, a protein containing a PDZ4 domain which selectively binds the PARG PDZ4 domain binding site, and a candidate pharmacological agent,    incubating the mixture under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of selective binding of the protein containing a PDZ4 domain by the PDZ4 domain binding site, and    detecting a test amount of selective binding of the protein containing a PDZ4 domain by the PDZ4 domain binding site, wherein reduction of the test amount of selective binding relative to the first amount of selective binding indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which disrupts selective binding of a protein containing a PDZ4 domain by a PARG polypeptide containing a PDZ4 domain binding site and wherein increase of the test amount of selective binding relative to the first amount of selective binding indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which enhances selective binding of a protein containing a PDZ4 domain by a PARG polypeptide containing a PDZ4 domain binding site.    
     
     
         53 . The method of  claim 52  wherein the protein containing a PDZ4 domain is PTPL1.