Method for quantifying phosphokinase activity on proteins

Abstract

The invention involves a method for measuring phosphorylation of proteins at specific sites and, as such, is an indicator of the protein kinase activity of enzymes capable of phosphorylating those sites. The method involves the in vitro or in vivo phosphorylation of a target protein at a specific serine, threonine or tyrosine residue, subjecting that protein (non-phosphorylated) to reaction mixture containing all reagents, including phosphokinase which allow the creation of a phosphorylated form of protein. The phosphorylated protein is measured by contacting it with an antibody specific for the phosphorylation site(s). The invention includes antibodies useful in practicing the methods of the invention. The invention particularly relates to all proteins modified by phosphorylation and dephosphorylation as illustrated by Tau, Rb and EGFR proteins and antibodies specific for the site of phosphorylation of the Tau, Rb or EGFR proteins.

What is claimed:  
     
         1 . A method for measuring phosphokinase activity on a protein comprising: 
 (a) subjecting a protein to a phosphokinase to phosphorylate a phosphorylation site on the protein    (b) providing an antibody specific to a phosphorylated phosphorylation site on the protein;    (c) contacting the protein from (a) with the antibody of (b); and    (d) detecting the antibody bound to the phosphorylation site.    
     
     
         2 . A method according to  claim 1  wherein the protein is the Tau protein.  
     
     
         3 . A method according to  claim 2  wherein the phosphokinase is GSK-3β, PKA, PKC, CDK-5, MARK, JNK, p38MAPK, or casein kinase II.  
     
     
         4 . The method according to  claim 2  wherein the antibody is specific to a phosphorylated site at a specific location in the Tau protein, selected from the sites shown in Table II.  
     
     
         5 . A method according to  claim 1  wherein the protein is selected from the proteins in Table 1.  
     
     
         6 . The method of  claim 1  wherein the protein is EGFR.  
     
     
         7 . The method of  claim 1  wherein the protein is Rb.  
     
     
         8 . An antibody prepared from a polypeptide immunogen having a phosphorylated serine.  
     
     
         9 . An antibody raised to a polypeptide immunogen having a phosphorylated threonine.  
     
     
         10 . An antibody raised to a polypeptide immunogen having a phosphorylated tyrosine.  
     
     
         11 . An antibody specific for sequence ID #1.  
     
     
         12 . An antibody specific for sequence ID #2.  
     
     
         13 . An antibody to EGFR phosphorylated tyrosine 1173 or tyrosine 845 site.  
     
     
         14 . An antibody to phosphorylated threonine 821 site.  
     
     
         15 . A kit for the measurement of phosphokinase activity on a protein comprising 
 (a) a first pan antibody specific and that binds to both phosphorylated and non-phosphorylated forms of the protein;    (b) a second pan antibody that binds to an independent site on the protein from the first pan antibody, wherein the second pan antibody is labeled or detected by a labeled reagent;    (c) non-phosphorylated and phosphorylated protein standards;    (d) a phosphorylation site-specific antibody (PSSA) which binds to the protein only when the target site on the protein is phosphorylated and wherein the PSSA antibody is labeled or detected by a labeled reagent; and    (e) buffers.    
     
     
         16 . A kit according to  claim 11  where the protein is Tau.  
     
     
         17 . A kit according to  claim 11  where the protein is selected from the proteins listed in Table I.  
     
     
         18 . A kit for the measurement of different kinase activities on a protein by quantitating phosphorylation site profiles comprising: 
 (a) a first pan antibody that binds to both phosphorylated and non-phosphorylated forms of the protein;    (b) a second pan antibody that binds to an independent site on the protein from the first pan antibody and wherein the second pan antibody is labeled or detected by a labeled reagent;    (c) protein standards for the non-phosphorylated and phosphorylated forms of the protein;    (d) two or more PSSAs which bind to the protein only when the target sites on the protein which are phosphorylated wherein antibody is labeled or detected by a labeled reagent;    (e) buffers.    
     
     
         19 . A kit according to  claim 14  where the protein is Tau.  
     
     
         20 . A kit according to  claim 14  where the proteins are as defined in Table I.