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 t he receptor for Hepatocyte Growth Factor/Scatter Factor (HGF/SF). Prev ious work has shown that autophosphorylation of p190(MET) enhances its enzymatic activity and that the major phosphorylation site is Tyr(123 5), located in the catalytic domain. This residue is part of a 'three tyrosine' motif, including Tyr(1230), Tyr(1234), and Tyr(1235), conser ved in several other receptor kinases. We studied the role of these ty rosines in the positive regulation of the p190(MET) kinase by site-dir ected mutagenesis. Substitution of either Tyr(1235) Or Tyr(1234) with phenylalanine severely reduced the in vitro kinase activity toward exo genous substrates. Kinetic experiments showed that the residual activi ty of these mutants could still be enhanced by autophosphorylation. Ph osphopeptide mapping indicated that, in the absence of Tyr(1235), Tyr( 1234) is phosphorylated. Only the replacement of both Tyr(1234) and Ty r(1235) yielded a mutant which completely lost the ability to be activ ated by autophosphorylation. In stable transfectants expressing the HG F/SF receptor with single substitution of either Tyr(1234) Or Tyr(1235 ) the response to HGF/SF was impaired. The ligand did not induce tyros ine phosphorylation of the receptor nor stimulated chemotaxis. These d ata 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 lig and in intact cells.