A Novel Role for Gab1 and SHP2 in Epidermal Growth Factor-induced Ras Activation

Abstract

SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr 317 were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.The GTPase Ras is a crucial signaling relay of receptor tyrosine kinases, and the mechanisms controlling its activation by growth factors appear well understood. For example, the activated epidermal growth factor (EGF) 1 receptor (EGFR) autophosphorylates on tyrosine residues, which creates docking sites for phosphotyrosine-binding domains (e.g. SH2) of adaptor proteins, including Grb2 and Shc. Because Grb2 is constitutively associated with Sos, a guanine nucleotide exchange factor of Ras, the binding of Grb2 to phosphorylated EGFR results in the recruitment of Sos to the plasma membrane and has been proposed as a model for activation of membrane-bound Ras. Once activated, Ras can stimulate several effectors, notably the protein Ser/Thr kinase Raf1 and the downstream activated mitogen-activated protein kinases Erk1/2, major regulators of cell proliferation, differentiation, and survival (1). Although this pathway has reached a canonical status, the docking protein Gab1 and one of its binding partners, SHP2, were recently found to participate in Ras/Erk activation in response to EGF and other growth factors (2-5). SHP2 is a protein-tyrosine phosphatase with two SH2 domains and phosphorylation sites with affinity for Grb2 SH2 domains. Its catalytic activity is stimulated by engagement of its SH2 domains with specific phosp...

Differential phosphorylation of the docking protein Gab1 by c-Src and the hepatocyte growth factor receptor regulates different aspects of cell functions

“…When a mutation was introduced to this site, the extent of Src-induced Gab1 phosphorylation was enhanced by almost twofolds (Figure 5a), which correlated with increased interaction between Gab1 and Crk II and PLCg ( Figure 5b). Among Y242, Y259, Y317 and Y373 of Gab1, Y317 and Y373 have also been reported to bind to the Ras GTPase-activating protein and PLCg, respectively (Gual et al, 2000;Montagner et al, 2005). We found that Src enhanced the interaction of Gab1 with PLCg, but not RasGTPase-activating protein (data not shown).…”

Reduced phosphatase activity of SHP‐2 in LEOPARD syndrome: Consequences for PI3K binding on Gab1

“…After transfection of these constructs in mammalian cells, we measured in vitro the catalytic activity of each mutant isolated by immunoprecipitation. Vero cells were chosen for their low basal level of SHP-2-dependent signalling pathways once incubated overnight in serum-free medium [5,13]. Following immunoprecipitation, the PTP activity of the different constructs was assayed using as substrate a synthetic phosphopeptide encompassing Y529 of Src, a standard in vitro substrate of PTP [3,9].…”

MET signalling: principles and functions in development, organ regeneration and cancer

“…The promiscuous docking motif in the C-terminal tail of MET binds numerous Src-homology-2 domain (SH2 domain)-containing effectors, such as PI3K 37 , the non-receptor tyrosine kinase Src 37 , the growth factor receptor-bound protein 2 (GRB2) and SH2 domain-containing transforming protein (SHC) adaptors 37,45,46 , SHP2 (also known as PTPN11; an upstream activator of Src and Ras) 46 , phospholipase Cγ1 (PLCγ1) 37 and the transcription factor STAT3 (Refs 47,48). MET also associates with GRB2-associated-binding protein 1 (GAB1), a multi-adaptor protein that, upon phosphorylation by the MET receptor, provides extra binding sites for SHC, PI3K, SHP2, CRK, PLCγ1 and p120 Ras-GTPase-activating protein (p120-Ras-GAP) 43,44,49,50,51,52,53 . The association between MET and GAB1 occurs directly, through a unique 13-amino-acid MET binding site (MBS) on GAB1, and indirectly, through MET-bound GRB2 (Refs 51,54).…”

“…MET activates Ras through the GRB2-SOS complex, which can interact directly with the multifunctional docking site in the C-terminal tail of MET or can be associated indirectly through the SHC adaptor protein 37,45 . Another route leading to Ras-ERK activation involves the tyrosine phosphatase SHP2, which dephosphorylates the p120-Ras-GAP binding site on GAB1; this impedes the recruitment of p120-Ras-GAP, which usually deactivates Ras, to promote Ras activation 44,53 (Fig. 2a).…”