Association of the Multisubstrate Docking Protein Gab1 with the Hepatocyte Growth Factor Receptor Requires a Functional Grb2 Binding Site Involving Tyrosine 1356

Abstract

Hepatocyte growth factor/scatter factor is a multifunctional factor that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Mettyrosine kinase. Upon stimulation with hepatocyte growth factor, the Met ␤ subunit becomes highly phosphorylated on tyrosine residues, one of which, tyrosine 1356 within the carboxyl terminus, is crucial for dissociation, motility, and branching tubule formation in Madin-Darby canine kidney epithelial cells. Tyrosine 1356 forms a multisubstrate binding site for the Grb2 and Shc adaptor proteins, the p85 subunit of phosphatidylinositol 3-kinase, phospholipase C␥, and a phosphatase, SHP2. To investigate additional signaling molecules that are activated by the Met receptor, we have identified hepatocyte growth factor-induced phosphoproteins in tubular epithelial cells. We have established that proteins of 100 -130 kDa are highly phosphorylated following stimulation of epithelial cells and that one of these is the Grb2-associated binding protein Gab1, a possible insulin receptor substrate-1-like signal transducer. We show that Gab1 is the major substrate for the Met kinase in vitro and in vivo. Association of Gab1 with Met requires a functional Grb2 binding site involving tyrosine 1356 and to a lesser extent tyrosine 1349. Met receptor mutants that fail to induce branching tubulogenesis are impaired in their ability to interact with Gab1, suggesting that Gab1 may play a role in these processes.

An alternatively spliced form of Met receptor is tumorigenic

“…Moreover, PI3K activity is reported to be critical for Met-mediated cell migration, in vitro tubulogenesis (Derman et al, 1995), and resistance to apoptotic agents in MDA-MB-453 cells and in glioma cell lines (Bowers et al, 2000;Fan et al, 2000). The majority of activated PI3K downstream from the Met receptor is associated with the multisubstrate adaptor protein Gab-1 (Furge et al, 2000), whose recruitment and phosphorylation requires Y15 and, to a lesser extent, Y14 (Nguyen et al, 1997). Consistent with this, the Y14, 15FF mutation abolished the transforming activity of Met-SM in both the absence and presence of HGF/SF.…”

From Tpr-Met to Met, tumorigenesis and tubes

“…In epithelial cells, Gab1 is the major substrate for the Met RTK (Nguyen et al, 1997), and genetic and cell biological studies (Maroun et al, 1999;Maroun et al, 2000Maroun et al, , 2003 have demonstrated that Gab1 is crucial for the biological responses downstream from Met. Embryos nullizygous for Gab1 display all of the defects observed in Met or HGF/SF null embryos (Itoh et al, 2000;Sachs et al, 2000).…”

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

“…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).…”