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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.

Moraes LA, Barrett NE, Jones CI, Holbrook LM, Spyridon M, Sage T, Newman DK, Gibbins JM - J. Thromb. Haemost. (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. l.a.moraes@reading.ac.uk

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Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates the association of p85 with SHP-2. Washed human platelets (A, B) and platelets derived from PECAM-1-deficient and wild-type (WT) mice (D) were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to PECAM-1 stimulation by antibody crosslinking (XL) (A) or stimulation with collagen for 90 s (B, D). The levels of p85 associated with SHP-2 were detected before equivalent protein loading was verified by reprobing for Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2). (C) Far-western blotting for SHP-2–p85 interaction was performed on lysates of cells stimulated with collagen (25 μg mL−1) for 90 s, resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to poly(vinylidine difluoride) membranes. The membranes were incubated with glutathione-S-transferase (GST) fusion protein containing the N-terminal SH2 domain of p85 (GST–p85-N-SH2) or GST control, followed by anti-GST and secondary antibodies. Blots were washed and incubated for 2 h with horseradish peroxidase-conjugated anti-goat IgG antibody (1 : 8000), and signals were detected by chemifluorescence. Numerical data represent the percentage change of SHP-2–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: **P ≤ 0.01 and ***P ≤ 0.001. IP, immunoprecipitation.
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fig02: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates the association of p85 with SHP-2. Washed human platelets (A, B) and platelets derived from PECAM-1-deficient and wild-type (WT) mice (D) were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to PECAM-1 stimulation by antibody crosslinking (XL) (A) or stimulation with collagen for 90 s (B, D). The levels of p85 associated with SHP-2 were detected before equivalent protein loading was verified by reprobing for Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2). (C) Far-western blotting for SHP-2–p85 interaction was performed on lysates of cells stimulated with collagen (25 μg mL−1) for 90 s, resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to poly(vinylidine difluoride) membranes. The membranes were incubated with glutathione-S-transferase (GST) fusion protein containing the N-terminal SH2 domain of p85 (GST–p85-N-SH2) or GST control, followed by anti-GST and secondary antibodies. Blots were washed and incubated for 2 h with horseradish peroxidase-conjugated anti-goat IgG antibody (1 : 8000), and signals were detected by chemifluorescence. Numerical data represent the percentage change of SHP-2–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: **P ≤ 0.01 and ***P ≤ 0.001. IP, immunoprecipitation.

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 [38]. 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.


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.

Moraes LA, Barrett NE, Jones CI, Holbrook LM, Spyridon M, Sage T, Newman DK, Gibbins JM - J. Thromb. Haemost. (2010)

Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates the association of p85 with SHP-2. Washed human platelets (A, B) and platelets derived from PECAM-1-deficient and wild-type (WT) mice (D) were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to PECAM-1 stimulation by antibody crosslinking (XL) (A) or stimulation with collagen for 90 s (B, D). The levels of p85 associated with SHP-2 were detected before equivalent protein loading was verified by reprobing for Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2). (C) Far-western blotting for SHP-2–p85 interaction was performed on lysates of cells stimulated with collagen (25 μg mL−1) for 90 s, resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to poly(vinylidine difluoride) membranes. The membranes were incubated with glutathione-S-transferase (GST) fusion protein containing the N-terminal SH2 domain of p85 (GST–p85-N-SH2) or GST control, followed by anti-GST and secondary antibodies. Blots were washed and incubated for 2 h with horseradish peroxidase-conjugated anti-goat IgG antibody (1 : 8000), and signals were detected by chemifluorescence. Numerical data represent the percentage change of SHP-2–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: **P ≤ 0.01 and ***P ≤ 0.001. IP, immunoprecipitation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3298659&req=5

fig02: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates the association of p85 with SHP-2. Washed human platelets (A, B) and platelets derived from PECAM-1-deficient and wild-type (WT) mice (D) were treated with EGTA (1 mm), apyrase (2 U mL−1) and indomethacin (10 μm) prior to PECAM-1 stimulation by antibody crosslinking (XL) (A) or stimulation with collagen for 90 s (B, D). The levels of p85 associated with SHP-2 were detected before equivalent protein loading was verified by reprobing for Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2). (C) Far-western blotting for SHP-2–p85 interaction was performed on lysates of cells stimulated with collagen (25 μg mL−1) for 90 s, resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to poly(vinylidine difluoride) membranes. The membranes were incubated with glutathione-S-transferase (GST) fusion protein containing the N-terminal SH2 domain of p85 (GST–p85-N-SH2) or GST control, followed by anti-GST and secondary antibodies. Blots were washed and incubated for 2 h with horseradish peroxidase-conjugated anti-goat IgG antibody (1 : 8000), and signals were detected by chemifluorescence. Numerical data represent the percentage change of SHP-2–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: **P ≤ 0.01 and ***P ≤ 0.001. IP, immunoprecipitation.
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 [38]. 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.

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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular & Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK. l.a.moraes@reading.ac.uk

Show MeSH
Related in: MedlinePlus