Assays for protein kinases using fluorescent protein substrates

Abstract

This invention provides assays for protein kinase activity using fluorescent proteins engineered to include sequences that can be phosphorylated by protein kinases. The proteins exhibit different fluorescent properties in the non-phosphorylated and phosphorylated states.

What is claimed:  
     
         1 . A method for determining whether a sample contains protein kinase activity comprising; 
 contacting the sample with a phosphate donor and a fluorescent protein substrate for a protein kinase, the fluorescent protein substrate comprising a fluorescent protein moiety and a phosphorylation site for a protein kinase,    wherein the fluorescent protein substrate exhibits a different fluorescent property in the phosphorylated state than in the un-phosphorylated state    wherein the fluorescent protein moiety is a green fluorescent protein (SEQ. ID. NO: 2), and    wherein the phosphorylation site is within ten amino acids of the terminus of the fluorescent protein moiety;    exciting the protein substrate; and    measuring the amount of a fluorescent property of the fluorescent protein substrate that differs in the unphosphorylated state and phosphorylated state, whereby an amount that is consistent with the presence of the fluorescent protein substrate in its phosphorylated state indicates the presence of protein kinase activity.    
     
     
         2 . The method of  claim 1 , wherein the fluorescent protein is an Aequorea-related fluorescent protein  
     
     
         3 . The method of  claim 2 , wherein said Aequorea-related fluorescent protein comprises at least one mutation selected from the group consisting of T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T, V163A, 1167T, S175G, S205T and N212K.  
     
     
         4 . The method of  claim 2 , wherein said Aequorea-related fluorescent protein comprises a phosphorylation site within ten amino acids of the terminus of said Aequorea-related fluorescent protein.  
     
     
         5 . The method of  claim 2 , wherein said Aequorea-related fluorescent protein comprises a phosphorylation site within twenty amino acids of the terminus of said Aequorea-related fluorescent protein.  
     
     
         6 . A method for determining whether a cell exhibits protein kinase activity, comprising the steps of: 
 providing a transfected host cell comprising a recombinant nucleic acid molecule comprising expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent protein substrate for a protein kinase, the fluorescent protein substrate comprising a fluorescent protein moiety containing a phosphorylation site for a protein kinase, wherein the fluorescent protein substrate exhibits a different fluorescent property in the phosphorylated state than in the un-phosphorylated state, the cell expressing the fluorescent protein substrate; 
 wherein the fluorescent protein moiety is a green fluorescent protein (SEQ. ID. NO: 2), and  
   wherein the phosphorylation site is within ten amino acids of the terminus of the fluorescent protein moiety;    exciting the protein substrate in the cell; and 
 measuring the amount of a fluorescent property of the fluorescent protein substrate that differs in the un-phosphorylated and phosphorylated states, wherein the presence of the fluorescent property associated with the fluorescent state indicates the presence of protein kinase activity in the cell.  
   
     
     
         7 . The method of  claim 6  wherein the fluorescent protein is an Aequorea-related fluorescent protein.  
     
     
         8 . The method of  claim 7 , wherein said Aequorea-related fluorescent protein comprises at least one selected from the group consisting of T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T, V163A, 1167T, S175G, S205T and N212K.  
     
     
         9 . The method of  claim 7 , wherein said Aequorea-related fluorescent protein comprises a phosphorylation site within ten amino acids of the terminus of said Aequorea-related fluorescent protein.  
     
     
         10 . The method of  claim 7 , wherein said Aequorea-related fluorescent protein comprises a phosphorylation site within twenty amino acids of the terminus of said Aequorea-related fluorescent protein.  
     
     
         11 . A method for determining whether a compound alters the activity of a protein kinase, comprising the steps of; 
 1) contacting a sample comprising a protein kinase activity with the compound, a phosphate donor for the protein kinase and a fluorescent protein substrate,    wherein the fluorescent protein substrate comprises a fluorescent protein moiety and a phosphorylation site for a protein kinase, and    wherein the fluorescent protein substrate exhibits a different fluorescent property in the phosphorylated state than in the un-phosphorylated state;    wherein the fluorescent protein moiety is a green fluorescent protein (SEQ. ID. NO: 2), and    wherein the phosphorylation site is within ten amino acids of the terminus of the fluorescent protein moiety;    2) exciting the protein substrate;    3) measuring the amount of protein kinase activity in the sample as a function of the quantity of change or rate of change of a fluorescent property of the fluorescent protein substrate that differs in the un-phosphorylated and phosphorylated states; and    comparing the amount of activity in the sample with a standard activity for the same amount of said protein kinase, whereby a difference between the amount of protein kinase activity in the sample and the standard activity indicates that said compound has an effect on the activity of the protein kinase.    
     
     
         12 . The method of  claim 11  wherein the fluorescent protein is an Aequorea-related fluorescent protein.  
     
     
         13 . The method of  claim 11 , wherein said Aequorea-related fluorescent protein comprises at least one selected from the group consisting of T44A, F64L, V68L, S72A, F99S, Y145F, N1461, M153T, V163A, 1167T, S175G, S205T and N212K.  
     
     
         14 . The method of  claim 11 , wherein said Aequorea-related fluorescent protein comprises a phosphorylation site within ten amino acids of the terminus of said Aequorea-related fluorescent protein.  
     
     
         15 . The method of  claim 11 , wherein said Aequorea-related fluorescent protein comprises a phosphorylation site within twenty amino acids of the terminus of said Aequorea-related fluorescent protein.