Roles of Gab1 and SHP2 in Paxillin Tyrosine Dephosphorylation and Src Activation in Response to Epidermal Growth Factor

Abstract

Epidermal growth factor (EGF) induces paxillin tyrosine dephosphorylation and Src activation, but the signaling pathways that mediate these responses were largely undefined. We found that Gab1, a docking protein for the SHP2 protein-tyrosine phosphatase in EGFstimulated cells, was associated with paxillin. SHP2 dephosphorylated paxillin and caused dissociation of Csk, a negative regulator of Src, from paxillin but had no effect on paxillin-Src association. A lower level of Src Tyr-530 phosphorylation was detected in paxillin-associated Src in EGF-stimulated cells. Expression of an SHP2 binding defective mutant of Gab1 (Gab1FF) or a catalytically inactive mutant of SHP2 (SHP2DN) prevented paxillin tyrosine dephosphorylation and Src activation induced by EGF. Importantly, Gab1FF blocked paxillin-SHP2 complex formation, Src Tyr-530 dephosphorylation, Erk activation, and cell migration induced by EGF. Inhibition of Src tyrosine kinase activity abrogated EGF-stimulated Erk activation and cell migration. Together, these results reveal that Gab1 recruits SHP2 to dephosphorylate paxillin, leading to dissociation of Csk from the paxillin-Src complex and Src activation and that Src is an SHP2 effector involved in EGF-stimulated Erk activation and cell migration.

Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking

“…Together, these findings indicate that PHPS1 mainly acts through interference with the sustained Shp2-dependent Ras/MAP kinase pathway. The focal adhesion component paxillin has recently been shown in EGFstimulated mammary gland carcinoma cells and in a reconstituted system using purified components to be a direct target of Shp2 phosphatase activity (17). We found that HGF/SF induces dephosphorylation of paxillin in MDCK cells, and that PHPS1 significantly inhibited this dephosphorylation, in particular at later time points (Fig.…”

“…Genetic experiments in Drosophila (11) and Caenorhabditis elegans (12) and biochemical experiments in vertebrates (10) have shown that Shp2 acts upstream of the Ras/MAP kinase pathway to promote its activation. Several direct targets of Shp2 have been identified, including the platelet-derived growth factor receptors [PDGFR (13)/Torso (14)], the multiadaptor protein Gab1 (15), Csk-binding protein [Cbp/PAG (16)], and paxillin (17). Downstream of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met, Shp2 is activated by association with Gab1 and is both essential and sufficient for Met function (18,19).…”

The interplay between Src and integrins in normal and tumor biology

“…Hence, shp2À/À cells exhibit hyperphosphorylated Src carboxy-terminal tyrosine residues (Zhang et al, 2004). A separate study has indicated that EGF stimulation induces Shp2-dependent dephosphorylation of paxillin and dissociation of Csk from the paxillin-Src complex, leading to Src activation (Ren et al, 2004). Given that paxillin, a focal adhesioncontaining protein, is involved in the EGF-mediated regulation of Src kinase activity may indicate a mechanism of Src activation via crosstalk between integrin-and growth factor-mediated signaling pathways.…”

TIMP-2 upregulates RECK expression via dephosphorylation of paxillin tyrosine residues 31 and 118

“…Paxillin also interacts with protein tyrosine phosphatases (PTPs), such as SHP-2 and PTP-PEST, which have been reported to affect the phosphorylation levels of paxillin and thereby the accessibility of Csk to Src (Turner, 2000;Ren et al, 2004). To test the possibility that these PTPs are involved in the TIMP-2-mediated inhibition of Src kinase activity, we examined their molecular interaction with paxillin by immunoprecipitation, but found no significant changes (Figure 1d).…”