TYROSINES(1234-1235) ARE CRITICAL FOR ACTIVATION OF THE TYROSINE KINASE ENCODED BY THE MET PROTOONCOGENE (HGF RECEPTOR)

The tyrosine kinase encoded by the MET proto-oncogene (p190(MET)) is the receptor for Hepatocyte Growth Factor/Scatter Factor (HGF/SF). Previous work has shown that autophosphorylation of p190(MET) enhances its enzymatic activity and that the major phosphorylation site is Tyr(1235), located in the catalytic domain. This residue is part of a 'three tyrosine' motif, including Tyr(1230), Tyr(1234), and Tyr(1235), conserved in several other receptor kinases. We studied the role of these tyrosines in the positive regulation of the p190(MET) kinase by site-directed mutagenesis. Substitution of either Tyr(1235) Or Tyr(1234) with phenylalanine severely reduced the in vitro kinase activity toward exogenous substrates. Kinetic experiments showed that the residual activity of these mutants could still be enhanced by autophosphorylation. Phosphopeptide mapping indicated that, in the absence of Tyr(1235), Tyr(1234) is phosphorylated. Only the replacement of both Tyr(1234) and Tyr(1235) yielded a mutant which completely lost the ability to be activated by autophosphorylation. In stable transfectants expressing the HGF/SF receptor with single substitution of either Tyr(1234) Or Tyr(1235) the response to HGF/SF was impaired. The ligand did not induce tyrosine phosphorylation of the receptor nor stimulated chemotaxis. These data show that Tyr(1234) and Tyr(1235) are critical for the activation of the HGF/SF receptor kinase both in vitro and in response to the ligand in intact cells.