Mutations in the met oncogene unveil a "dual switch" mechanism controlling tyrosine kinase activity.
The met oncogene, encoding the high affinity hepatocyte growth factor receptor, is the only known gene inherited in human cancer that is invariably associated with somatic duplication of the mutant locus. Intriguingly, mutated Met requires ligand stimulation in order to unleash its transforming potential. Furthermore, individuals bearing a germ line met mutation develop cancer only late in life and with incomplete penetrance. To date, there is no molecular explanation for this unique behavior, which is unusual for a dominant oncogene. Here we investigate the molecular mechanisms underlying met oncogenic conversion by generating antibodies specific for the differently phosphorylated forms of the Met protein. Using these antibodies, we show that activation of wild-type Met is achieved through sequential phosphorylation of Tyr1235 and Tyr1234 in the activation loop and that mutagenesis of either tyrosine dramatically impairs kinase function. Surprisingly, oncogenic Met mutants never become phosphorylated on Tyr1234 despite their high enzymatic activity, and mutagenesis of Tyr1234 does not affect their biochemical or biological function. By analyzing the enzymatic properties of the mutant proteins in different conditions, we demonstrate that oncogenic mutations do not elicit constitutive kinase activation but simply overcome the requirement for the second phosphorylation step, thus reducing the threshold for activation. In the presence of activating signals, these mutations result therefore in a dynamic imbalance toward the active conformation of the kinase. This explains why mutant met provides an oncogenic predisposition but needs a second activating "hit," provided by sustained ligand stimulation or receptor overexpression, to achieve a fully transformed phenotype.
Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Torino Medical School, I-10060 Candiolo, Torino, Italy.
Federica
F
Chiara
Paolo
P
Michieli
Luisa
L
Pugliese
Paolo M
PM
Comoglio
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D004789:Enzyme Activation; D017353:Gene Deletion; D015870:Gene Expression; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011817:Rabbits; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D000906:Antibodies; D010446:Peptide Fragments; D014443:Tyrosine; D017228:Hepatocyte Growth Factor; D011505:Protein-Tyrosine Kinases; D019859:Proto-Oncogene Proteins c-met