Two mutations affecting conserved residues in the Met receptor operate viadifferent mechanisms

We leave investigated the mechanism by which two oncogenic mutations (M1268 T and D1246H/N; Amino-acids are numbered according to Schmidt ct a]., 1999) affecting conserved residues in the catalytic domain of the Met receptor, activate its transforming potential. Both mutations were previously found i n 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 l oop (A-loop) (D), which in a number of receptor tyrosine kinases harbors a pair of tandem tyrosines (Y1252-1253 in Met), Ligand-induced dimerization p romotes their phosphorylation, and locks the A-loop into an open conformati on. When unphosphorylated, the tandem tyrosines inhibit enzymatic activity by blocking the active site. Upon Y-->F mutation of Y1252-1253, neither lig and binding nor Tpr-mediated dimerization ran release this block. Here we s how 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 mu tants strictly depend on Y1252-1253 for activity. Surprisingly, however, th ey constitutively activate the isolated cytoplasmic TK domain of Met (Cyto- Met), These data indicate that the two mutations operate via distinct mecha nisms.