Abstract
The Src homology (SH)2 domain-containing protein-tyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosine-phosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50-55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP- but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosine-phosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.
Highlights
In blood platelets, agonist-induced increases in protein-tyrosine phosphorylation is mediated primarily by cytosolic protein-tyrosine kinases, including members of the Src, Syk, Tec, focal adhesion kinase (FAK), and Jak tyrosine kinases (1, 2)
The present study was undertaken to investigate the role of the SH2 domain-containing protein-tyrosine phosphatase, SHP-1, in glycoprotein VI (GPVI) signaling in platelets
The major site of interaction of Syk, SLP-76, and Lyn with SHP-1 is through the tandem SH2 domains of the phosphatase, both Syk and Lyn kinases bind to the catalytic domain
Summary
Agonist-induced increases in protein-tyrosine phosphorylation is mediated primarily by cytosolic protein-tyrosine kinases, including members of the Src, Syk, Tec, focal adhesion kinase (FAK), and Jak tyrosine kinases (1, 2). The Src homology (SH)2 domain-containing proteintyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)selective agonist collagen-related peptide (CRP), collagen, and thrombin.
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