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Grb2 promotes integrin-induced focal adhesion kinase (FAK) autophosphorylation and directs the phosphorylation of protein tyrosine phosphatase ? by the Src-FAK kinase complex.

ABSTRACT: The integrin-activated Src-focal adhesion kinase (FAK) kinase complex phosphorylates PTP? at Tyr789, initiating PTP?-mediated signaling that promotes cell migration. Recruitment of the BCAR3-Cas complex by PTP?-phospho-Tyr789 at focal adhesions is one mechanism of PTP? signaling. The adaptor protein Grb2 is also recruited by PTP?-phospho-Tyr789, although the role of the PTP?-Grb2 complex in integrin signaling is unknown. We show that silencing Grb2 expression in fibroblasts abolishes PTP?-Tyr789 phosphorylation and that this is due to two unexpected actions of Grb2. First, Grb2 promotes integrin-induced autophosphorylation of FAK-Tyr397. This is impaired in Grb2-depleted cells and prohibits FAK activation and formation of the Src-FAK complex. Grb2-depleted cells contain less paxillin, and paxillin overexpression rescues FAK-Tyr397 phosphorylation, suggesting that the FAK-activating action of Grb2 involves paxillin. A second distinct role for Grb2 in PTP?-Tyr789 phosphorylation involves Grb2-mediated coupling of Src-FAK and PTP?. This requires two phosphosites, FAK-Tyr925 and PTP?-Tyr789, for Grb2-Src homology 2 (SH2) binding. We propose that a Grb2 dimer links FAK and PTP?, and this positions active Src-FAK in proximity with other, perhaps integrin-clustered, molecules of PTP? to enable maximal PTP?-Tyr789 phosphorylation. These findings identify Grb2 as a new FAK activator and reveal its essential role in coordinating PTP? tyrosine phosphorylation to enable downstream integrin signaling and migration.

SUBMITTER: Cheng SY

PROVIDER: S-EPMC3911518 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Grb2 promotes integrin-induced focal adhesion kinase (FAK) autophosphorylation and directs the phosphorylation of protein tyrosine phosphatase α by the Src-FAK kinase complex.

Cheng Suzanne Y S SY Sun Guobin G Schlaepfer David D DD Pallen Catherine J CJ

Molecular and cellular biology 20131118 3

The integrin-activated Src-focal adhesion kinase (FAK) kinase complex phosphorylates PTPα at Tyr789, initiating PTPα-mediated signaling that promotes cell migration. Recruitment of the BCAR3-Cas complex by PTPα-phospho-Tyr789 at focal adhesions is one mechanism of PTPα signaling. The adaptor protein Grb2 is also recruited by PTPα-phospho-Tyr789, although the role of the PTPα-Grb2 complex in integrin signaling is unknown. We show that silencing Grb2 expression in fibroblasts abolishes PTPα-Tyr789 ...[more]

PMID: 24248601

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Project description:PurposeRegulation of lens development involves an intricate interplay between growth factor (e.g. FGF and TGFbeta) and extracellular matrix (ECM) signaling pathways. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays key roles in transmitting ECM signals by integrins. In this study, we delineated patterns of FAK expression and tyrosine phosphorylation (Y397) in the developing lens and investigated its regulation by FGF2. We also examined FAK expression and activation during disrupted fiber differentiation in mice expressing a dominant-negative TGFbeta receptor.MethodsFAK expression and activation (phosphorylation on Y397) was studied in embryonic and postnatal rodent lenses by in situ hybridization, immunofluorescence, and western blotting. Rat lens explants were used to investigate the effects of FGF2 on FAK expression and activation. Immunofluorescence and western blotting were used to examine FAK expression and phosphorylation in transgenic mice that express a dominant-negative TGFbeta receptor.ResultsFAK is widely expressed and phosphorylated during embryonic stages of lens morphogenesis and differentiation. However, in postnatal lenses its expression and activation becomes restricted to the posterior germinative zone and the transitional zone at the lens equator. While both NH2- and COOH-terminal antibodies revealed cytoplasmic and membrane-associated staining in lens cells, the NH2-terminal antibody also showed FAK was present in fiber cell nuclei. In vitro, FAK expression and phosphorylation on Y397 were increased by concentrations of FGF2 that initiate lens epithelial cell migration (10 ng/ml) and differentiation (50 ng/ml) but not proliferation (5 ng/ml). Moreover, reactivity for Y397 phosphorylated FAK is prominent in the nuclei of differentiating fibers both in vivo and in vitro. Disruption of TGFbeta-like signals by ectopic expression of a dominant-negative TGFbeta receptor (TbetaRII(D/N)) results in abnormal lens fiber differentiation in transgenic mice. While FAK expression is initiated normally in the posterior germinative zone of TbetaRII(D/N) transgenic lenses, as fiber differentiation proceeds, FAK becomes localized to a perinuclear compartment, decreases its association with the cytoskeleton and is poorly phosphorylated on Y(397).ConclusionsFAK is widely expressed and activated during early lens morphogenesis. During secondary lens fiber differentiation, FAK is expressed and phosphorylated on Y397 as epithelial cells exit the cell cycle, initiate migration at the equator, and undergo differentiation in the transitional zone. During terminal fiber differentiation an NH2-terminal fragment of FAK, including Y397, is translocated to the nucleus. The expression, activation, and nuclear localization of FAK are regulated, at least partly, by FGF2. FAK activity and subcellular localization are also modulated by TGFbeta-like signals. In fiber cells of TbetaRII(D/N) transgenic lenses, FAK is abnormally retained in a perinuclear compartment, loses its association with the cytoskeleton, and is poorly phosphorylated. These data suggest that integrin signaling via FAK plays important roles during lens differentiation, mediated by FGFs and TGFbeta-superfamily signals.

Dataset Information

Grb2 promotes integrin-induced focal adhesion kinase (FAK) autophosphorylation and directs the phosphorylation of protein tyrosine phosphatase ? by the Src-FAK kinase complex.

Publications

Grb2 promotes integrin-induced focal adhesion kinase (FAK) autophosphorylation and directs the phosphorylation of protein tyrosine phosphatase α by the Src-FAK kinase complex.

Similar Datasets

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