Protein kinase C mediates translocation of type II phosphatidylinositol 5-phosphate 4-kinase required for platelet α-granule secretion

Abstract

To better understand the molecular mechanisms of platelet granule secretion, we have evaluated the role of type II phosphatidylinositol (PtdIns) 5-phosphate 4-kinase in agonist-induced platelet α-granule secretion. SFLLRN-stimulated a-granule secretion from SL-O-permeabilized platelets was inhibited by either antibodies directed at type II PtdIns 5-phosphate 4-kinase or by a kinase-impaired point mutant of type IIβ PtdIns 5-phosphate 4-kinase. In contrast, recombinant type IIβ PtdIns 5-phosphate 4-kinase augmented SFLLRN-stimulated α-granule secretion from SL-O-permeabilized platelets. SFLLRN-stimulated α-granule secretion was inhibited by a protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated a-granule secretion was inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the kinase-impaired point mutant of type IIβ PtdIns 5-phosphate 4-kinase and augmented by recombinant type IIβ PtdIns 5-phosphate 4-kinase. Immunoblot analysis demonstrated that type II PtdIns 5-phosphate 4-kinase remained associated with SL-O-permeabilized platelets when incubated in the presence, but not the absence, of SFLLRN. This SFLLRN-induced translocation of type II PtdIns 5-phosphate 4-kinase was blocked by either the protein kinase C-specific inhibitor peptide or bisindolyl-maleimide I. In addition to stimulating α-granule secretion, both SFLLRN and PMA enhanced the association of a fluorescein isothiocyanate-labeled peptide derived from the PtdIns (4,5)P2-binding domain of gelsolin to permeabilized platelets. Agonist-induced recruitment of the PtdIns (4,5)P2-binding domain was inhibited by neomycin, bisindolylmaleimide I, and anti-type II PtdIns 5-phosphate 4-kinase antibody. These results suggest a mechanism whereby protein kinase C-mediated translocation of type II PtdIns 5-phosphate 4-kinase leads to the recruitment of PtdIns (4,5)P2-binding proteins.