title: Proteomic analysis of integrin alphaIIbbeta3 outside-in signaling reveals Src-kinase-independent phosphorylation of Dok-1 and Dok-3 leading to SHIP-1 interactions.
abstract: OBJECTIVE
  Outside-in integrin alphaIIbbeta3 signaling involves a series of tyrosine kinase reactions that culminate in platelet spreading on fibrinogen. The aim of this study was to identify novel tyrosine phosphorylated signaling proteins downstream of alphaIIbbeta3, and explore their role in platelet signaling.
  
  
  RESULTS
  Utilizing proteomics to search for novel platelet proteins that contribute to outside-in signaling by the integrin alphaIIbbeta3, we identified 27 proteins, 17 of which were not previously shown to be part of a tyrosine phosphorylation-based signaling complex downstream of alphaIIbbeta3. The proteins identified include the novel immunoreceptors G6f and G6b-B, and two members of the Dok family of adapters, Dok-1 and Dok-3, which underwent increased tyrosine phosphorylation following platelet spreading on fibrinogen. Dok-3 was also inducibly phosphorylated in response to the GPVI-specific agonist collagen-related peptide (CRP) and the PAR-1 and -4 agonist thrombin, independently of the integrin alphaIIbbeta3. Tyrosine phosphorylation of Dok-1 and Dok-3 was primarily Src kinase-independent downstream of the integrin, whereas it was Src kinase-dependent downstream of GPVI. Moreover, both proteins inducibly interacted with Grb-2 and SHIP-1 in fibrinogen-spread platelets.
  
  
  CONCLUSIONS
  This study provides new insights into the molecular mechanism regulating alphaIIbbeta3-mediated platelet spreading on fibrinogen. The novel platelet adapter Dok-3 and the structurally related Dok-1 are tyrosine phosphorylated in an Src kinase-independent manner downstream of alphaIIbbeta3 in human platelets, leading to an interaction with Grb2 and SHIP-1.
authors.affiliation: Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
authors.forename: Y A
authors.initials: YA
authors.lastname: Senis
authors.forename: R
authors.initials: R
authors.lastname: Antrobus
authors.forename: S
authors.initials: S
authors.lastname: Severin
authors.forename: A F
authors.initials: AF
authors.lastname: Parguiña
authors.forename: I
authors.initials: I
authors.lastname: Rosa
authors.forename: N
authors.initials: N
authors.lastname: Zitzmann
authors.forename: S P
authors.initials: SP
authors.lastname: Watson
authors.forename: A
authors.initials: A
authors.lastname: García
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D048430:Cell Shape; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005340:Fibrinogen; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D000072181:Inositol Polyphosphate 5-Phosphatases; D013058:Mass Spectrometry; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000032:analysis; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C518789:DOK3 protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010750:Phosphoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D016601:RNA-Binding Proteins; D044463:Receptor, PAR-1; C098341:platelet membrane glycoprotein VI; D019000:Phosphotyrosine; D005340:Fibrinogen; D019061:src-Family Kinases; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; C000606430:INPP5D protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases