BINDING OF PURIFIED 14-3-3-ZETA SIGNALING PROTEIN TO DISCRETE AMINO-ACID-SEQUENCES WITHIN THE CYTOPLASMIC DOMAIN OF THE PLATELET MEMBRANE GLYCOPROTEIN IB-IX-V COMPLEX

The glycoprotein (GP) Ib-PS-V complex constitutively expressed on the platelet plasma membrane mediates initial adhesion of circulating plat elets to vessel wall matrix at high shear, and shear induced platelet aggregation. In both cases. this involves binding of GP Ib-LX-V to the adhesive glycoprotein, von Willebrand Factor(vWF). vWF binding to GP Ib-IX-V rapidly induces platelet activation, leading to cytoskeletal r earrangement, shape change, and secretion that enables alpha IIB beta 3 integrin (GP IIb-IIIa)-dependent platelet aggregation. All these eve nts are critical in (patho)physiological thrombus formation. The recen t discovery that the signaling protein, 14-3-3 zeta, copurifies with t he GP lb-IX complex (minus GP V) [Du,X., Harris, S. J., Tetaz, T. J., Ginsberg, M. H., & Berndt, M. C.(1994) J. Biol. Chem. 269, 18287-18290 ] indicated a potential mechanism for vWF-dependent signaling. The aim of the present study was to identify discrete amino acid sequences th at bind 14-3-3 zeta within the cytoplasmic domain of the receptor. As an initial screening assay, overlapping synthetic peptides based on th e cytoplasmic domains of GP Ib alpha (100 residues), GP Ib beta (34 re sidues), GP IX (5 residues), and GF V (16 residues) were immobilized a nd assessed for the ability to bind purified 14-3-3 zeta. The C-termin al sequence GHSL of GP Ib alpha was identified as one 14-3-3 zeta inte ractive sequence, consistent with previous results [Du, X., Fox, J. E. , & Pei, S. (1996) J. Biol; Chem. 271, 7362-7367]. Binding of I-125-la beled 14-3-3 zeta to GHSL-containing peptides was inhibitable by unlab eled 14-3-3 zeta and by anti-14-3-3 zeta IgG. Ala-walking through the GHSL sequence suggested all residues were necessary for optimal bindin g. In addition, 14-3-3 zeta bound with lower affinity to a peptide bas ed on the central region of the GP Ib alpha cytoplasmic domain (Arg-55 7-Gly-575), whereas peptide sequences within the cytoplasmic domains o f GP Ib beta (Arg-160-Arg-175) and GP V (Lys-529-Gly-544) bound 14-3-3 zeta with comparable affinity to the GHSL-containing peptide. Soluble GHSL-containing peptides, GP Ib beta- and GP V-based peptides semidis sociated 14-3-3 zeta from GP Ib-IX-V or GP Ib-IX in platelet extracts as analyzed by immunoprecipitation, suggesting these sequences, at lea st partially, mediate the GP Ib-IX-V-14-3-3 beta interaction in cells. Further, phosphorylation of the GP Ib beta peptide at a site correspo nding to a protein kinase A phosphorylation site (Ser-166) enhanced th e affinity of 14-3-3 zeta binding by approximately 8-fold, suggesting phosphorylation as a potential mechanism for regulating 14-3-3 zeta as sociation with the GP Ib-IX-V complex.