Peptide substrates phosphorylated by p21-activaged protein kinase statement regarding federally sponsored reasearch or development

Abstract

The determinants for phosphorylation of substrates by γ-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides. The recognition sequence for γ-PAK contains two basic amino acids in the −2 and −3 positions, as represented by (K/R)RXS, in which the −2 position is an arginine, the −3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the −1 or −4 position improves the rate of phosphorylation. Alternatively, an acidic amino acid in the −1 position increases the rate (2.5-fold), as does an acidic residue in the −4 position, although to a lower extent (1.6-fold). Proline in the −1 or +1 position has a deleterious effect and inhibits phosphorylation by γ-PAK. The substrate requirements of other protein kinases, such as cAMP-dependent protein kinase (PKA) and Ca 2+ /phospholipid-dependent protein kinase (PKC), have been compared with γ-PAK using the same peptides. An acidic residue in the −1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used in assays for γ-PAK activity.

I/we claim:  
     
         1 . A peptide that is a selective substrate for PAK, comprising the amino acid sequence: 
       X 1 X 2 X 3 R 4 X 5 X 6 X 7 X 8   
       wherein 
 X 1  and X 8  together equal 0 to 4 amino acids, each of which is independently selected;  
 X 2 , X 5  or both X 2  and X 5  is an acidic amino acid;  
 X 3  is arginine or lysine;  
 R 4  is arginine;  
 X 5  and X 7  are not proline; and  
 X 6  is serine or threonine.  
 
     
     
         2 . The peptide of  claim 1 , wherein at least one of X 2  and X 5  is selected from the group consisting of aspartic acid, glutamic acid, phosphoserine, and phosphothreonine.  
     
     
         3 . A peptide according to  claim 2  selected from the group consisting of AKRESAA and EKRASAA.  
     
     
         4 . A peptide that is a substrate for a protein kinase, consisting essentially of the amino acid sequence: 
       X 1 X 2 X 3 R 4 X 5 X 6 X 7 X 8   
       wherein 
 X 1 , X 2 , X 7 , and X 8  together equal 1 to 6 amino acids, each of which is independently selected;  
 at least one of X 2  and X 5  is an acidic or basic amino acid;  
 X 3  is arginine or lysine;  
 R 4  is arginine;  
 X 5  is an independently selected amino acid; and  
 X 6  is serine or threonine.  
 
     
     
         5 . A peptide selected from the group consisting of AKRKSAA, KKRASAA, ARRASAA, KKRKSAA, AKRASAA, PKRASSA, KKRKSGL, YNKRSTTI, GVKRISGL, APKRGSGK, and CLRRDSHK.  
     
     
         6 . An assay for PAK activity comprising the steps of: 
 a) providing a reaction mixture comprising the peptide of  claim 1 , ATP, and a sample, wherein the sample is believed to contain PAK;    b) allowing the reaction to continue so that the peptide is phosphorylated by any PAK present in the sample; and    c) detecting the phosphorylated peptide.    
     
     
         7 . A protein kinase assay comprising the steps of: 
 a) providing a reaction mixture comprising the peptide of  claim 4  or  claim 5 , ATP, and a sample, wherein the sample is believed to contain a protein kinase;    b) allowing the reaction to continue so that the peptide is phosphorylated by any protein kinase present in the sample; and    c) detecting the phosphorylated peptide.    
     
     
         8 . The assay of  claim 7 , wherein the sample comprises PAK.  
     
     
         9 . The assay of  claim 8  wherein the PAK is selected from the group consisting of α-,β-, and γ-PAK.  
     
     
         10 . The assay of  claim 8 , wherein the sample is pre-treatedto activate inactive forms of PAK.  
     
     
         11 . An assay according to  claim 8 , wherein the protein kinase is selected from the group consisting of PAK, PKC, and PKA.  
     
     
         12 . A mixture for use in an assay for PAK comprising the peptide of  claim 4 , ATP, and divalent magnesium.  
     
     
         13 . A kit for use in an assay for PAK comprising: 
 a. a first container comprising the peptide of  claim 4;  and    b. a second container comprising ATP.    
     
     
         14 . The kit of  claim 13 , wherein the second container further comprises divalent magnesium.  
     
     
         15 . The kit of  claim 13  further comprising a third container, the third container comprising a divalent magnesium salt.  
     
     
         16 . A method of purifying γ-PAK, comprising the steps of; 
 a) preparing a cell extract;  
 b) separating the cell extract into fractions; and  
 c) testing the fractions for γ-PAK activity using the assay of  claim 6.