Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells.
Bottom Line: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI-Fc receptor (FcR)γ-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT).An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses.PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling.
Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. firstname.lastname@example.orgShow MeSH
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Mentions: Previous studies in other cell models have suggested that the SH2 domains of p85 direct the interaction of the PI3K complex with activated growth factor receptors and signaling intermediate molecules such SHP-2, Gab1, Grb-2-associated binding protein-2, Grb2, and SHIP . Given the role of PECAM-1 in the negative regulation of platelet function and the recruitment of SHP-2 to this ITIM-containing receptor, we investigated whether the p85 subunit of PI3K associates with SHP-2 upon PECAM-1 crosslinking or GPVI stimulation. As shown in Fig. 2A,B, SHP-2 was immunoprecipitated from the lysates of resting platelets and following stimulation of PECAM-1 and GPVI signaling. Low levels of p85 were found to be present in SHP-2 immunoprecipitates from unstimulated platelets, and this association was increased notably following stimulation of PECAM-1 or activation of platelets with collagen. In order to explore a potential direct interaction between SHP-2 and the p85 subunit of PI3K, we used GST–p85-N-SH2 in far-western blots. Resting and collagen-stimulated samples were lysed, and SHP-2 was immunoprecipitated. Immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes. After incubation with GST–p85-N-SH2 or GST alone (control), the presence of bound fusion protein was detected with an anti-GST antibody and chemifluorescence detection. An increase in GST–p85-N-SH2 binding to immunoprecipitated SHP-2 following GPVI stimulation (Fig. 2C) suggested that the p85 subunit of PI3K is capable of binding directly to SHP-2.
Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. email@example.com