HUMAN SIGNALING PROTEIN 14-3-3-ZETA INTERACTS WITH PLATELET GLYCOPROTEIN IB SUBUNITS IB-ALPHA AND IB-BETA

The initiation of primary hemostasis is mediated by interaction of the platelet glycoprotein Ib (GPIb) surface receptor and its arterial sub endothelial von Willebrand factor (vWF) ligand, The intracellular sign aling immediately following GPIb receptor occupancy connecting the adh esive event to platelet activation and aggregation has not been well c haracterized. The 14-3-3 proteins are a 27- to 30-kD ubiquitous protei n family with diverse biologic roles, including functional modulation of several prominent signaling proteins. We used the yeast two-hybrid system and confocal microscopy to characterize the recently described interaction between GPIb and platelet 14-3-3 zeta, and provide evidenc e for the potential signaling role of this protein, Two-hybrid interac tions suggest that platelet 14-3-3 zeta associates with the cytoplasmi c domain of GPIb subunits Ib alpha and Ib beta in transformed yeast ce lls. The 14-3-3 interaction with GPIb beta may be partly mediated thro ugh the latter's phosphorylated serine 166 residue as its mutagenesis results in 20% to 40% reduced interaction, There was 51% to 59% reduce d interaction between GPIb and three 14-3-3 zeta deletion mutants comp ared with full-length 14-3-3 zeta, suggesting that either the N-termin al dimerization or membrane-binding domains or more than one noncontig uous 14-3-3 zeta element may be required for optimal GPIb interaction, Confocal studies of platelets and a megakaryocyte cell line provided additional evidence for interaction of 14-3-3 zeta with GPIb alpha and GPIb beta, We also found that, similar to the signaling mediators pho sphatidylinositol 3-kinase and Src, platelet cytoskeletal 14-3-3 zeta content is increased following vWF and ristocetin stimulation. We sugg est that platelet 14-3-3 zeta interacts with GPIb alpha and Ib beta, t hat this interaction may be partly mediated through phosphoserine reco gnition, and that 14-3-3 zeta cytoskeletal translocation may serve as a GPIb post-receptor occupancy signaling event. (C) 1998 by The Americ an Society of Hematology.