Patent ID: 8067155

Claim:
A method for determining phosphorylation of a receptor tyrosine kinase (“RTK”), said method comprising: (a) employing a cell comprising (i) a first expression construct expressing a fusion of an RTK fused at its C-terminus to a first enzyme fragment, and (ii) a second expression construct expressing a fusion of a phosphotyrosine binding peptide fused to a second enzyme fragment, wherein said first and second enzyme fragments are fragments of β-galactosidase that have low affinity for each other but when brought together by the binding of said RTK to said phosphotyrosine binding peptide form an active β-galactosidase, (b) with the proviso that when said RTK does not autophosphorylate, in the absence of an endogenous active cytosolic tyrosine kinase, a third expression construct is included expressing a cytosolic tyrosine kinase to phosphorylate said RTK; (c) incubating said cell for sufficient time (i) for any phosphorylation of said fusion of an RTK fused at its C terminus to a first enzyme fragment to occur to form a phosphorylated RTK and (ii) for any binding to said phosphorylated RTK by said phosphotyrosine binding peptide fused to a second enzyme fragment to occur; (d) adding a β-galactosidase substrate to said cell, wherein said substrate forms a detectable product; and (e) detecting said detectable product as indicative of the phosphorylation of said RTK.