Identification of Shc as the Primary Protein Binding to the Tyrosine-phosphorylated β3 Subunit of αIIbβ3 during Outside-in Integrin Platelet Signaling*

By K. Cowan, D. Law, D. Phillips. Journal of Biological Chemistry, 2000

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Outside-in signaling mediated by the integrin αIIbβ3 (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of β3. To identify proteins that bind directly to phosphorylated β3, we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of β3. Tandem mass spectrometric sequencing and immunoblotting demonstrated that Shc was the primary protein binding to phosphorylated β3. To determine the involvement of Shc in outside-in αIIbβ3 signaling, the phosphorylation of Shc during platelet aggregation was examined; transient Shc phosphorylation was observed when thrombin-stimulated platelets were allowed to aggregate or when aggregation was induced by an LIBS (ligand-induced binding site) antibody, D3. Moreover, Shc was co-immunoprecipitated with tyrosine-phosphorylated β3 in detergent lysates of aggregated platelets. Using purified, recombinant protein, it was found that the binding of Shc to monophosphorylated (C-terminal tyrosine) and diphosphorylated β3 peptides was direct, demonstrating Shc recognition motifs on phospho-β3. Aggregation-induced Shc phosphorylation was also observed to be robust in platelets from wild-type mice, but not in those from mice expressing (Y747F,Y759F) β3, which are defective in outside-in αIIbβ3 signaling. Thus, Shc is the primary downstream signaling partner of β3 in its tyrosine phosphorylation outside-in signaling pathway.

Subfields: Hematology, Immunology and Allergy, Surgery

View Paper: https://www.semanticscholar.org/paper/57d3d9b8228654e3c5034cca56cf39884e8d0ec1

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