Disruption of 14-3-3ζ binding to GPIb-IX by mutagenesis and a novel inhibitor. (A) Schematic depicting GPIb-IX and the reported 14-3-3ζ binding sites (black boxes). Also shown is the GPIbα 14-3-3ζ binding sequence from which a novel membrane-permeable inhibitor of 14-3-3ζ-GPIbα interaction, MPαC, is derived. (B) CHO cells expressing wild-type, GPIb-IX (1b9), or GPIb-IX mutants bearing 14-3-3ζ binding disrupting mutations, S609A (GPIbα Ser609 to alanine mutation), S166A (GPIbβ Ser166 to alanine mutation), and S609A/S166A double mutations, were solubilized in Triton X-100 containing lysis buffer and immunoprecipitated with the anti-GPIbα antibody LJ-P3 (P3), or control IgG. The immunoprecipitates were immunoblotted with anti-GPIbα and anti-14-3-3ζ antibodies. (C) Platelets were preincubated with MPαC, control peptides (MαC, MPαCsc, or MαCsc), or DMSO. Platelets were then solubilized and immunoprecipitated with an anti-GPIbα antibody (P3). Immunoprecipitates were immunoblotted with anti-GPIbα and anti-14-3-3ζ antibodies.
