A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility

Abstract

Background Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A 2 (cPLA 2 ) metabolite glycerophosphoinositol 4-phosphate (GroPIns4 P ), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4 P activates Src remains unknown. Methods Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4 P- interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4 P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. Results We find that Shp1 is the direct cellular target of GroPIns4 P . GroPIns4 P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4 P show significantly enhanced wound healing capability, indicating that GroPIns4 P has a stimulatory role to activate fibroblast migration. GroPIns4 P is produced by cPLA 2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4 P was shown to mediate the EGF-induced cell motility. Conclusions This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA 2 metabolite GroPIns4 P . We show that GroPIns4 P is required for EGF-induced fibroblast migration and that it is part of a cPLA 2 /GroPIns4 P/ Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer. El...

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“…Our group has recently shown that Shp1 regulates actin remodeling and cell motility through yet another mechanism involving Src kinase (45). We identified Shp1 as the cellular receptor of the bioactive metabolite glycerophosphoinositol-4 phosphate (GroPIns4P) and as a novel component in the signaling pathway activated by EGF to control cell motility (45). In NIH3T3 cells, activation of the EGF receptor leads to activation of the cytosolic phospholipase A 2 and results in the hydrolysis of phosphatidylinositol-4 phosphate and formation of GroPIns4P that binds to Shp1 and promotes its association with Src.…”

“…In NIH3T3 cells, activation of the EGF receptor leads to activation of the cytosolic phospholipase A 2 and results in the hydrolysis of phosphatidylinositol-4 phosphate and formation of GroPIns4P that binds to Shp1 and promotes its association with Src. This in turn activates Src by direct dephosphorylation of the inhibitory phosphotyrosine in position 530 and triggers a signaling cascade which ends with the formation of the Tiam/Rac complex at the cell membrane and the concomitant induction of plasma membrane ruffles leading to cell motility (45,46).…”