A Conserved Inositol Phospholipid Binding Site within the Pleckstrin Homology Domain of the Gab1 Docking Protein Is Required for Epithelial Morphogenesis
ABSTRACT Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4, 5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.
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- "The mechanisms of Gab1 function in EGFR and Met signalling are only partly understood. The EGFR does not bind Gab1 directly, but can engage Gab1 via Grb2   , and there is evidence that activation of Biochimica et Biophysica Acta 1833 (2013) 3286–3294 Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; Gab1, Grb2-associated binding protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Grb2, growth factor receptorbound protein 2; HGF, hepatocyte growth factor; PH, pleckstrin homology; PI3K, phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; RTK, receptor tyrosine kinase ⁎ Corresponding author. "
ABSTRACT: Grb2-assosiated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.Biochimica et Biophysica Acta 10/2013; 1833(12). DOI:10.1016/j.bbamcr.2013.10.004 · 4.66 Impact Factor
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- "Vol. 14, April 2003 1703 ificity for PIP3 (Isakoff et al., 1998; Maroun et al., 1999b; Rodrigues et al., 2000). However, discrimination between a requirement for the Gab1 PH domain for biological activity and membrane localization had not been established. "
ABSTRACT: The hepatocyte growth factor receptor tyrosine kinase Met promotes cell dissociation and the inherent morphogenic program of epithelial cells. In a search for substrates downstream from Met, we have previously identified the Grb2-associated binder-1 (Gab1) as critical for the morphogenic program. Gab1 is a scaffold protein that acts to diversify the signal downstream from the Met receptor through its ability to couple with multiple signal transduction pathways. Gab1 contains a pleckstrin homology (PH) domain with specificity for phosphatidylinositol 3,4,5-trisphosphate. The phospholipid binding capacity of the Gab1 PH domain is required for the localization of Gab1 at sites of cell-cell contact in colonies of epithelial cells and for epithelial morphogenesis, suggesting that PH domain-dependent subcellular localization of Gab1 is a prerequisite for function. We have investigated the requirement for membrane localization of Gab1 for biological activity. We show that substitution of the Gab1 PH domain with the myristoylation signal from the c-Src protein is sufficient to replace the Gab1 PH domain for epithelial morphogenesis. The membrane targeting of Gab1 enhances Rac activity in the absence of stimulation and switches a nonmorphogenic noninvasive response to epidermal growth factor to a morphogenic invasive program. These results suggest that the subcellular localization of Gab1 is a critical determinant for epithelial morphogenesis and invasiveness.Molecular Biology of the Cell 05/2003; 14(4):1691-708. DOI:10.1091/mbc.E02-06-0352 · 4.47 Impact Factor
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- "Gab1 contains an amino-terminal pleckstrin homology (PH) domain that binds PIP3 in a PI3-kinase– dependent manner (Isakoff et al., 1998; Maroun et al., 1999b; Rodrigues et al., 2000). This association is required for localization of Gab1 at cell-cell junctions in epithelial cells and for Gab1-dependent morphogenic responses (Maroun et al., 1999b). Gab1 also contains two binding sites for the C-terminal Src homology 3 (SH3) domain of Grb2 (Lock et al., 2000; Schaeper et al., 2000; Lewitzky et al., 2001). "
ABSTRACT: The Gab family of docking proteins (Gab1 and Gab2) are phosphorylated in response to various cytokines and growth factors. Gab1 acts to diversify the signal downstream from the Met receptor tyrosine kinase through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. To determine whether Gab1 and Gab2 are functionally redundant, we have examined the role of Gab2 in epithelial cells. Both Gab1 and Gab2 are expressed in epithelial cells and localize to cell-cell junctions. However, whereas overexpression of Gab1 promotes a morphogenic response, the overexpression of Gab2 fails to induce this response. We show that Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. In contrast, Gab1 recruitment to Met is both Grb2 dependent and Grb2 independent. The latter requires a novel amino acid sequence present in the Met-binding domain of Gab1 but not Gab2. Mutation of these residues in Gab1 impairs both association with the Met receptor and the ability of Gab1 to promote a morphogenic response, whereas their insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. We propose that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis.Molecular Biology of the Cell 07/2002; 13(6):2132-46. DOI:10.1091/mbc.02-02-0031. · 4.47 Impact Factor