Binding of purified 14-3-3 ζ 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 platelets 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 rearrangement, shape change, and secretion that enables alpha IIB beta 3 integrin (GP IIb-IIIa)-dependent platelet aggregation. All these events are critical in (patho)physiological thrombus formation. The recent discovery that the signaling protein, 14-3-3 zeta, copurifies with the 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 that bind 14-3-3 zeta within the cytoplasmic domain of the receptor. As an initial screening assay, overlapping synthetic peptides based on the cytoplasmic domains of GP Ib alpha (100 residues), GP Ib beta (34 residues), GP IX (5 residues), and GF V (16 residues) were immobilized and assessed for the ability to bind purified 14-3-3 zeta. The C-terminal sequence GHSL of GP Ib alpha was identified as one 14-3-3 zeta interactive sequence, consistent with previous results [Du, X., Fox, J. E., & Pei, S. (1996) J. Biol; Chem. 271, 7362-7367]. Binding of I-125-labeled 14-3-3 zeta to GHSL-containing peptides was inhibitable by unlabeled 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 binding. In addition, 14-3-3 zeta bound with lower affinity to a peptide based on the central region of the GP Ib alpha cytoplasmic domain (Arg-557-Gly-575), whereas peptide sequences within the cytoplasmic domains of 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 semidissociated 14-3-3 zeta from GP Ib-IX-V or GP Ib-IX in platelet extracts as analyzed by immunoprecipitation, suggesting these sequences, at least 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 corresponding to a protein kinase A phosphorylation site (Ser-166) enhanced the affinity of 14-3-3 zeta binding by approximately 8-fold, suggesting phosphorylation as a potential mechanism for regulating 14-3-3 zeta association with the GP Ib-IX-V complex.