title: Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling.
abstract: Outside-in signaling mediated by the integrin alpha(IIb)beta(3) (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of beta(3). To identify proteins that bind directly to phosphorylated beta(3), we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of beta(3). Tandem mass spectrometric sequencing and immunoblotting demonstrated that Shc was the primary protein binding to phosphorylated beta(3). To determine the involvement of Shc in outside-in alpha(IIb)beta(3) 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 beta(3) 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 beta(3) peptides was direct, demonstrating Shc recognition motifs on phospho-beta(3). 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) beta(3), which are defective in outside-in alpha(IIb)beta(3) signaling. Thus, Shc is the primary downstream signaling partner of beta(3) in its tyrosine phosphorylation outside-in signaling pathway.
authors.affiliation: COR Therapeutics, Inc., South San Francisco, California 94080, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Cowan
authors.forename: D A
authors.initials: DA
authors.lastname: Law
authors.forename: D R
authors.initials: DR
authors.lastname: Phillips
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites / Q000276:immunology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013917:Thrombin / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D010446:Peptide Fragments; D010748:Phosphopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D021122:Protein Subunits; D011506:Proteins; D011994:Recombinant Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019000:Phosphotyrosine; D013917:Thrombin