Abstract
BackgroundShp1, 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 A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown.MethodsAffinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P 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.ResultsWe find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P 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 GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility.ConclusionsThis study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.
Highlights
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
This procedure yielded 30 potential GroPIns4P-binding proteins. Functional annotation of these proteins allowed their classification under five major clusters: cellular motility and membrane trafficking components; stress and folding proteins; metabolic enzymes; proteins associated with DNA and RNA processing; and cell-cycle regulation proteins. We searched these clusters for proteins that could be related to the activities of GroPIns4P on actin dynamics
GroPIns4P induces actin ruffle formation in fibroblasts through a signalling cascade that begins with Src activation; no direct interaction occurs between GroPIns4P and Src [11]
Summary
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. A similar pathway controlling actin dynamics was observed in Jurkat T-cells, in which exogenously-added GroPIns4P promotes actin polymerisation by activating the Lck kinase, and inducing the phosphorylation of the GDP/ GTP exchanger Vav and subsequent activation of the GTPase Rac, resulting in increased cell motility [9, 12]. This GroPIns4P-dependent Lck activation potentiates the transactivation of the TCR by the SDF1α-stimulated CXCR4 receptor enhancing T-cell chemotaxic response [12]. These effects of GroPIns4P might play a role in the immune response by mediating the recruitment of T-cells toward the injured site [9, 12]
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