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

β4 integrin activates a Shp2–Src signaling pathway that sustains HGF-induced anchorage-independent growth

Despite being a cell–matrix adhesion molecule, β4 integrin can prompt the multiplication of neoplastic cells dislodged from their substrates (anchorage-independent growth). However, the molecular events underlying this atypical behavior remain partly unexplored. We found that activation of the Met receptor for hepatocyte growth factor results in the tyrosine phosphorylation of β4, which is instrumental for integrin-mediated recruitment of the tyrosine phosphatase Shp2. Shp2 binding to β4 enhances the activation of Src, which, in turn, phosphorylates the multiadaptor Gab1 predominantly on consensus sites for Grb2 association, leading to privileged stimulation of the Ras–extracellular signal-regulated kinase (ERK) cascade. This signaling axis can be inhibited by small interfering RNA–mediated β4 depletion, by a β4 mutant unable to bind Shp2, and by pharmacological and genetic inhibition of Shp2 or Src. Preservation of the β4 docking sites for Shp2 as well as the integrity of Shp2, Src, or ERK activity are required for the β4-mediated induction of anchorage-independent growth. These results unravel a novel pathway whereby β4 directs tyrosine kinase–based signals toward adhesion-unrelated outcomes

Hepatocyte growth factor/scatter factor stimulates the Ras-guanine nucleotide exchanger

Hepatocyte growth factor/scatter factor (HGF/SF) induces mitogenesis and cell dissociation upon binding to the protein-tyrosine kinase receptor encoded by the MET proto-oncogene (p190MET). The signal transduction pathways downstream from the receptor activation are largely unknown. We show that HGF/SF activates Ras protein. HGF/SF stimulation of metabolically labeled A549 cells raised the amount of Ras-bound radiolabeled guanine nucleotides by over 5-fold. Furthermore, following HGF/SF stimulation of these cells, 50% of Ras was in the GTP-bound active state. The uptake by Ras of radiolabeled GTP was also increased by 5-fold following HGF/SF stimulation in digitonin-permeabilized A549 cells. Moreover, HGF/SF treatment of A549 cells leads to stimulation of the cytosolic Ras-guanine nucleotide exchange activity, measured as accelerated release of [3H]GDP from purified recombinant Ras protein in vitro, in a dose- and time-dependent manner. Likewise, treatment with the protein-tyrosine kinase inhibitor 3-(1',4'-dihydroxytetralyl)methylene-2-oxindole of GTL-16 cells (featuring a p190MET receptor constitutively active) significantly decreased the cytosolic Ras-guanine nucleotide exchange activity. These data demonstrate that HGF/SF activates Ras protein by shifting the equilibrium toward the GTP-bound state and increases the uptake of guanine nucleotides by Ras, through mechanism(s) including the activation of a Ras-guanine nucleotide exchanger

The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase.

The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids

Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235

Generation of a truncated hepatocyte growth factor receptor in the endoplasmic reticulum.

The hepatocyte growth factor (HGF) receptor (p190MET) is a tyrosine kinase composed of two disulfide-linked chains, alpha of 50 kDa and beta of 145 kDa. We have previously described an isoform (p140MET) containing a beta chain of 85 kDa, lacking the cytoplasmic kinase domain. The two receptor variants originate by post-translational processing of a common single-chain precursor of 170 kDa (Pr170). In the endoplasmic reticulum a fraction of Pr170 is cleaved at the cytosolic side generating an intermediate product of 120 kDa (Pr120). This molecule 1) is already detectable after 15 min of pulse labeling, 2) contains high mannose-branched oligosaccharides, and 3) accumulates upon treatments inhibiting the export from the endoplasmic reticulum. A second cleavage, occurring after 30 min of chase in the trans-Golgi network, converts the single-chain precursors Pr170 and Pr120 into the mature heterodimers p190MET and p140MET. This process is inhibited by brefeldin A treatment. Conditions leading to Pr170 accumulation in the endoplasmic reticulum, such as receptor overexpression, induce kinase activation and overproduction of Pr120. Conversely, cells expressing a kinase-defective HGF receptor lack the truncated isoform. The proteolytic cleavage of the cytoplasmic domain may thus represent a safety mechanism aimed at preventing ligand-independent intracellular activation of the HGF receptor kinase

Hepatocyte growth factor (HGF) receptor expression is inducible and is part of the delayed-early response to HGF.

The c-MET proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), also known as scatter factor, a potent mitogen and motogen for epithelial cells. The level of the HGF receptor expressed by epithelial cells varies in different growth conditions, being lower in growth arrested confluent monolayers and higher in growing sparse cells. The amount of HGF receptor mRNA increases from 3- to 5-fold after stimulation of confluent monolayers by serum and up to 10-fold after stimulation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA). An increased level of the receptor mRNA was also observed after cell stimulation with nanomolar concentration of HGF itself. The effect was transient, dose, and time-dependent. Transcription of a reporter gene under control of the cloned 297 base pair c-MET promoter was also stimulated by serum, TPA, or HGF. The accumulation of specific mRNA is followed by appearance of the HGF receptor precursor protein, which is further processed to the receptor mature form. After HGF stimulation, HGF receptor expression follows c-FOS and c-JUN induction with a peak approximately 4 h. Pretreatment with the protein synthesis inhibitor puromycin strongly reduced the response to HGF, while cycloheximide alone increased the level of the receptor mRNA. These data show that c-MET behaves as a delayed early-response gene and suggest that the HGF response is autoamplified by inducing the specific receptor