Tumorigenesis mediated by MET mutant M1268T is inhibited by dominant-negative Src

We recently described germline and somatic mutations in the MET gene associ ated with papillary renal carcinoma type 1, MET mutation M1268T was Located in a codon highly conserved among receptor tyrosine kinases, and homologou s to the codon mutated in multiple endocrine neoplasia type 2B, and many ca ses of sporadic medullary carcinoma of the thyroid gland (Ret M918T), Ret M 918T and MET M1268T have previously been shown to be highly active in mouse NIH3T3 transformation assays, and to change the substrate specificity of t he kinase. We studied the mechanism of transformation mediated by MET M1268 T by analysing a clone, F4, derived from NIH3T3 cells transformed by MET M1 268T, In contrast to NIH3T3 cells, F4 cells grew in suspension in tissue cu lture, and rapidly formed tumors in nude mice. We found that c-Src was cons titutively bound to MET proteins in F4 cells, and that Src kinase activity was elevated. Transfection of dominant negative Src constructs into F4 cell s eliminated the ability of F4 cells to grow in suspension culture and reta rded the growth of F4 cells in vivo. The ability of transfected dominant ne gative Src constructs to inhibit the growth of F4 cells correlated with the inhibition of phosphorylation of paxillin and focal adhesion kinase, Trans fection of dominant negative Src constructs into F4 cells had no effect on Grb2 binding or PLC gamma phosphorylation, The results suggest that c-Src p articipates in the tumorigenic phenotype induced in NIH3T3 cells by MET M12 68T by signaling through focal adhesion kinase and paxillin.