Two mutations affecting conserved residues in the Met receptor operate via different mechanisms

We have investigated the mechanism by which two oncogenic mutations (M1268T and D1246H/N; Amino-acids are numbered according to Schmidt et al., 1999) affecting conserved residues in the catalytic domain of the Met receptor, activate its transforming potential. Both mutations were previously found in tumorigenic forms of the Ret and Kit receptors, respectively. The mutated residues are located either in the P+1 loop (M) or within the activation loop (A-loop) (D), which in a number of receptor tyrosine kinases harbors a pair of tandem tyrosines (Y1252-1253 in Met). Ligand-induced dimerization promotes their phosphorylation, and locks the A-loop into an open conformation. When unphosphorylated, the tandem tyrosines inhibit enzymatic activity by blocking the active site. Upon Y-->F mutation of Y1252-1253, neither ligand binding nor Tpr-mediated dimerization can release this block. Here we show that the M1268T mutation partially rescues the kinase activity (and the transforming ability) of the Y1252-1253F Tpr-Met mutant, but is completely dependent on dimerization for its effect. In contrast, the two D1246H/N mutants strictly depend on Y1252-1253 for activity. Surprisingly, however, they constitutively activate the isolated cytoplasmic TK domain of Met (Cyto-Met). These data indicate that the two mutations operate via distinct mechanisms.