title: A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling.
abstract: The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block ((396)PAIPPKKPRP(405)) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395-407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity.
authors.affiliation: From the Departments of Medicine.
authors.forename: Tanvir
authors.initials: T
authors.lastname: Khatlani
authors.affiliation: From the Departments of Medicine.
authors.forename: Subhashree
authors.initials: S
authors.lastname: Pradhan
authors.affiliation: From the Departments of Medicine.
authors.forename: Qi
authors.initials: Q
authors.lastname: Da
authors.affiliation: From the Departments of Medicine.
authors.forename: Tanner
authors.initials: T
authors.lastname: Shaw
authors.affiliation: the School of Biosciences, Cardiff University, Wales CF10 3AX, United Kingdom.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Buchman
authors.affiliation: From the Departments of Medicine, Pediatrics, and Molecular Physiology and Biophysics, Baylor College of Medicine and Center for Translational Research on Inflammatory Diseases (CTRID), Michael E. DeBakey Veterans Affairs Medical Center (MEDVAMC), Houston, Texas 77030 and.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Cruz
authors.affiliation: From the Departments of Medicine, Pediatrics, and Molecular Physiology and Biophysics, Baylor College of Medicine and Center for Translational Research on Inflammatory Diseases (CTRID), Michael E. DeBakey Veterans Affairs Medical Center (MEDVAMC), Houston, Texas 77030 and vvijayan@bcm.edu.
authors.forename: K Vinod
authors.initials: KV
authors.lastname: Vijayan
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D002352:Carrier Proteins / Q000494:pharmacology; D000071679:Glycogen Synthase Kinase 3 beta / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism*; D000071636:Protein Phosphatase 2C / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D036341:Intercellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C433810:SH3KBP1 protein, human; C485059:peptide P3; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000607456:PPM1A protein, human; D000071636:Protein Phosphatase 2C
keywords: adaptor protein; adhesion; fibrinogen; integrin; platelet; protein phosphatase 2A