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

MET Oncogene Controls Invasive Growth by Coupling with NMDA Receptor

SIMPLE SUMMARY: The MET oncogene, encoding the tyrosine kinase receptor for a hepatocyte growth factor (HGF), plays a key role in the onset and progression of aggressive forms of breast cancer. Recently, it was found that the glutamate receptor, which has a well-known role in the nervous system, is expressed in many types of tumors outside the nervous system and contributes to metastatic behavior in breast cancer cells. Here, we highlight that MET protein physically interacts with glutamate receptors in two highly metastatic breast cancer cell lines. HGF, which creates a supportive proinvasive microenvironment for the tumor cells, stabilizes this interaction. Pharmacological inhibition of glutamate receptors blunts the migration and invasion elicited by HGF, suggesting drug repurposing of glutamate receptor antagonists for anticancer therapy. ABSTRACT: The N-methyl-D-aspartate receptor (NMDAR) is a glutamate-gated ion channel involved in excitatory synaptic transmission. Outside the nervous system, the NMDAR is expressed in a variety of tissues and in cancers, notably in the highly invasive and metastatic triple-negative breast carcinoma. MET encodes the tyrosine kinase receptor for HGF and is a master regulator gene for “invasive growth”. In silico analysis shows that high expression of the NMDAR2B subunit is a negative prognostic factor in human invasive breast carcinoma. Here, we show that in triple-negative breast cancer cell lines NMDAR2B and MET proteins are coexpressed. HGF stimulation of these cells is followed by autophosphorylation of the MET kinase and phosphorylation of the NMDAR2B subunit at tyrosines 1252 and 1474. MET and phosphorylated NMDAR2B are physically associated, as demonstrated by co-immunoprecipitation, confocal immunofluorescence, and proximity ligation assays. Notably, pharmacological inhibition of NMDAR by MK801 and ifenprodil blunts the biological response to HGF. These results demonstrate the existence of a MET-NMDAR crosstalk driving the invasive program, paving the way for a new combinatorial therapy