Gab1 coupling to the HGF/Met receptor multifunctional docking site requires binding of Grb2 and correlates with the transforming potential

Abstract

Activation of the HGF receptor, encoded by the c-MET protooncogene (Met receptor), triggers motility, matrixinvasion and branching morphogenesis in epithelial cells. It has recently been shown that the Met receptor interacts with Gab-1, an IRS-like adaptor protein, via the docking site (Y 1349 VHVNATY 1356 VNV) known to bind Grb2 and multiple SH2-containing signal transducers. Here we show that Gab1 is the major phosphorylation-substrate of the Met receptor and of its oncogenic variant Tpr-Met. A series of point mutations in the docking site established a direct correlation between the ability to recruit and phosphorylate Gab1 and the transforming potential. Interestingly, the mutations of either Y 1356 or N 1358 abolished the binding of both Grb2 and Gab1 in intact cells. Furthermore, peptides designed to block either the SH2 or the SH3 domains of Grb2 interfered with the receptor-Gab1 interaction. These data indicate that Gab1 coupling to the Met receptor requires binding of Grb2 and correlates with the transforming potential of Tpr-Met.

Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis

“…The two carboxy-terminal multifunctional tyrosines in the receptor are relatively low a nity binding sites for p85 (Ponzetto et al, 1993;Songyang et al, 1993). Conversely, three`optimal' p85 binding motifs are present in the multiadaptor GAB-1, a Met substrate which binds the receptor via Grb2 (Holgado-Madruga et al, 1996;Weidner et al, 1996;Bardelli et al, 1997b;Fixman et al, 1997). Thus, PI 3-kinase activation in vivo is likely to occur mainly indirectly, through Grb2-mediated GAB-1 phosphorylation.…”

“…Met signaling and Tpr ± Met mediated transformation are based on the activation of multiple pathways triggered by phosphorylation of two carboxy-terminal tyrosines (Y 1349 VHVNATY 1356 VNV, Ponzetto et al, 1993Ponzetto et al, , 1994Fixman et al, 1995). These tyrosine residues are part of a consensus sequence (YVH/NV) which mediates coupling of the receptor with several e ectors, including the Grb2/SoS complex (Ponzetto et al, 1994;Fixman et al, 1995), the p85 regulatory subunit of PI-3-kinase (Ponzetto et al, 1993), Stat-3 (Boccaccio et al, 1998), and the multiadaptor protein Gab1 (Weidner et al, 1996;Bardelli et al, 1997b;Nguyen et al, 1997). Grb2, in particular, requires an Asn in the +2 position for binding, and is thus linked to the receptor via the Y 1356 VNV motif Maina et al, 1996;Fixman et al, 1997).…”

Tyrosine residues 654 and 670 in β-catenin are crucial in regulation of Met–β-catenin interactions

“…In turn, the signal is transduced downstream, either directly or through its adaptor molecules-Gab1 or Grb2, which trigger activation of signaling such as ERK1/2/MAPK, PLC-γ, PI3K, SHP2 and others [4][5][6][7][8][9]. Some proteins such as Stat3 and Src bind directly to met [10][11][12][13].…”

Loss of the exon encoding the juxtamembrane domain is essential for the oncogenic activation of TPR-MET

“…Arrows indicate bands corresponding to Tpr-Met (60 KD) and Tpr-juxtaMet (70 KD) Figure 7 Tpr-juxtaMet interacts with signal transducers and induces Map Kinase activation less e ciently than Tpr-Met. Lysates from ®broblasts expressing Tpr-Met or Tpr-juxtaMet, prepared as described in Ponzetto et al (1994) were incubated in the presence of phosphatase inhibitors either with GST-Grb2 (a) or with Gab-1 GST-MBD domain (b) immobilized on glutathione sepharose Bardelli et al, 1997b). The amount of Tpr-Met or Tpr-juxtaMet proteins associated with Grb-2 or with the MBD domain was revealed by immunoblotting with anti-Met antibodies (C-12).…”