Probes for imaging protein phosphorylation and dephosphorylation and method for detecting and determining protein phosphorylation and dephosphorylation using the same

Abstract

As a versatile method of detecting and assaying intracellular protein phosphorylation and dephosphorylation that enables nondestructive monitoring as well as spatial and temporal analysis for living cells, animal bodies, plant bodies and the like, a probe for imaging protein phosphorylation and dephosphorylation, which comprises a tandem fusion unit composed of a substrate domain that contains a phosphorylation and dephosphorylation site, a linker sequence and a phosphorylation recognition domain, interposed between a donor chromophore and an acceptor chromophore that cause fluorescence resonance energy transfer, is used.

1 - 12 . (canceled)  
     
     
         13 . A method for assaying a substance that causes protein dephosphorylation, which comprises 
 introducing a probe for imaging protein phosphorylation and dephosphorylation into cells, which comprises a substrate domain that contains a site that can be phosphorylated and a phosphorylation recognition domain, bound together by a linker sequence interposed between a donor chromophore and an acceptor chromophore that cause fluorescence resonance energy transfer (FRET) to occur, wherein the substrate domain has been phosphorylated,    introducing a candidate substance that causes protein dephosphorylation into the cells, and    measuring the change in efficiency of FRET before and after addition of the candidate substance.    
     
     
         14 . The method of  claim 13 , wherein the donor chromophore and the acceptor chromophore that cause fluorescence resonance energy transfer to occur, are fluorescent proteins each having different fluorescence wavelengths.  
     
     
         15 . The method of  claim 13 , wherein the donor chromophore and the acceptor chromophore that cause fluorescence resonance energy transfer to occur, are different color mutants of a green fluorescent protein.  
     
     
         16 . The method of  claim 15 , wherein the mutants of the green fluorescent protein are a cyan fluorescent protein and a yellow fluorescent protein.  
     
     
         17 . The method of  claim 13 , wherein the site that can be phosphorylated in the substrate domain contains an amino acid residue selected from tyrosine, serine and threonine.  
     
     
         18 . The method of  claim 13 , wherein the phosphorylation recognition domain is an endogenous domain selected from SH2 domain, phosphotyrosine binding domain and WW domain.  
     
     
         19 . The method of  claim 13 , wherein the phosphorylation recognition domain is a single chain antibody obtained using the phosphorylated substrate domain as an antigen.  
     
     
         20 . The method of  claim 13 , wherein the probe further comprises a localization sequence at the terminal end.