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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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The adaptor protein Grb-2-associated binding protein-1 (Gab1) associates with Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) and phosphatidylinositol 3-kinase on platelet activation. These associations are enhanced in the absence of platelet endothelial cell adhesion molecule-1 (PECAM-1). Gab1 was immunoprecipitated from washed human platelets and platelets derived from PECAM-1-deficient and wild-type (WT) mice following stimulation of PECAM-1 signaling by antibody crosslinking (XL) (A, B) or collagen (C–F) for 90 s. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotted to detect SHP-2 (A, C, E) and p85 (B, D, F). Numerical data represent the percentage change of Gab1–SHP-2 or Gab1–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05. IP, immunoprecipitation.
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fig03: The adaptor protein Grb-2-associated binding protein-1 (Gab1) associates with Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) and phosphatidylinositol 3-kinase on platelet activation. These associations are enhanced in the absence of platelet endothelial cell adhesion molecule-1 (PECAM-1). Gab1 was immunoprecipitated from washed human platelets and platelets derived from PECAM-1-deficient and wild-type (WT) mice following stimulation of PECAM-1 signaling by antibody crosslinking (XL) (A, B) or collagen (C–F) for 90 s. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotted to detect SHP-2 (A, C, E) and p85 (B, D, F). Numerical data represent the percentage change of Gab1–SHP-2 or Gab1–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05. IP, immunoprecipitation.

Mentions: In different cell types, Gab1 has been shown to contain a number of different docking sites that mediate independent interactions with SH2 domain-containing proteins such as SHP-2 and the p85 subunit of PI3K. The formation of these complexes is involved in signaling events mediated by cytokine and tyrosine kinase receptors [36,37]. Given our finding that, in human platelets, SHP-2 interacts directly with p85 in a manner that depends on the presence of PECAM-1, we hypothesized that PECAM-1 may bind to SHP-2–p85 complexes and interfere with the ability of either of these molecules to bind to Gab1. To test this hypothesis, we investigated the effect of PECAM-1 crosslinking or PECAM-1 deficiency on the ability of SHP-2 and p85 to interact with Gab1 in GPVI-activated platelets. PECAM-1 crosslinking had no effect on the levels of association of either SHP-2 (Fig. 3A) or p85 (Fig. 3B) with Gab1 in unstimulated platelets. We found that the levels of association of SHP-2 and p85 with Gab1 in Gab1 immunoprecipitates, which are low in resting human platelets, increased upon stimulation of platelets with collagen (Fig. 3C,D). Gab1–SHP-2 interactions were also found to be increased in SHP-2 immunoprecipitates (Fig. S3F). The effect of PECAM-1 on levels of association of p85 or SHP-2 with Gab1 was investigated with mouse platelets deficient in PECAM-1. Significantly higher levels of association of SHP-2 (Fig. 3E) or p85 (Fig. 3F) with Gab1 were observed in collagen-stimulated platelets derived from PECAM-1-deficient mice than in those from wild-type mice. On the basis of these findings, we conclude that PECAM-1 competes with Gab1 for association with SHP-2 in GPVI-stimulated platelets. Furthermore, the ability of PECAM-1-associated SHP-2 to complex with the p85 subunit of PI3K limits the amount of p85 available to bind to Gab1 downstream of GPVI stimulation.


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)

The adaptor protein Grb-2-associated binding protein-1 (Gab1) associates with Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) and phosphatidylinositol 3-kinase on platelet activation. These associations are enhanced in the absence of platelet endothelial cell adhesion molecule-1 (PECAM-1). Gab1 was immunoprecipitated from washed human platelets and platelets derived from PECAM-1-deficient and wild-type (WT) mice following stimulation of PECAM-1 signaling by antibody crosslinking (XL) (A, B) or collagen (C–F) for 90 s. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotted to detect SHP-2 (A, C, E) and p85 (B, D, F). Numerical data represent the percentage change of Gab1–SHP-2 or Gab1–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05. IP, immunoprecipitation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: The adaptor protein Grb-2-associated binding protein-1 (Gab1) associates with Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) and phosphatidylinositol 3-kinase on platelet activation. These associations are enhanced in the absence of platelet endothelial cell adhesion molecule-1 (PECAM-1). Gab1 was immunoprecipitated from washed human platelets and platelets derived from PECAM-1-deficient and wild-type (WT) mice following stimulation of PECAM-1 signaling by antibody crosslinking (XL) (A, B) or collagen (C–F) for 90 s. Proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotted to detect SHP-2 (A, C, E) and p85 (B, D, F). Numerical data represent the percentage change of Gab1–SHP-2 or Gab1–p85 association in stimulated samples as compared with the control (mean ± standard error of the mean; n = 4). t-test: *P ≤ 0.05. IP, immunoprecipitation.
Mentions: In different cell types, Gab1 has been shown to contain a number of different docking sites that mediate independent interactions with SH2 domain-containing proteins such as SHP-2 and the p85 subunit of PI3K. The formation of these complexes is involved in signaling events mediated by cytokine and tyrosine kinase receptors [36,37]. Given our finding that, in human platelets, SHP-2 interacts directly with p85 in a manner that depends on the presence of PECAM-1, we hypothesized that PECAM-1 may bind to SHP-2–p85 complexes and interfere with the ability of either of these molecules to bind to Gab1. To test this hypothesis, we investigated the effect of PECAM-1 crosslinking or PECAM-1 deficiency on the ability of SHP-2 and p85 to interact with Gab1 in GPVI-activated platelets. PECAM-1 crosslinking had no effect on the levels of association of either SHP-2 (Fig. 3A) or p85 (Fig. 3B) with Gab1 in unstimulated platelets. We found that the levels of association of SHP-2 and p85 with Gab1 in Gab1 immunoprecipitates, which are low in resting human platelets, increased upon stimulation of platelets with collagen (Fig. 3C,D). Gab1–SHP-2 interactions were also found to be increased in SHP-2 immunoprecipitates (Fig. S3F). The effect of PECAM-1 on levels of association of p85 or SHP-2 with Gab1 was investigated with mouse platelets deficient in PECAM-1. Significantly higher levels of association of SHP-2 (Fig. 3E) or p85 (Fig. 3F) with Gab1 were observed in collagen-stimulated platelets derived from PECAM-1-deficient mice than in those from wild-type mice. On the basis of these findings, we conclude that PECAM-1 competes with Gab1 for association with SHP-2 in GPVI-stimulated platelets. Furthermore, the ability of PECAM-1-associated SHP-2 to complex with the p85 subunit of PI3K limits the amount of p85 available to bind to Gab1 downstream of GPVI stimulation.

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