Protein kinase C mediates translocation of type II phosphatidylinositol 5-phosphate 4-kinase required for platelet alpha-granule secretion.
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 alpha-granule secretion. SFLLRN-stimulated alpha-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 IIbeta PtdIns 5-phosphate 4-kinase. In contrast, recombinant type IIbeta PtdIns 5-phosphate 4-kinase augmented SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets. SFLLRN-stimulated alpha-granule secretion was inhibited by a protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated alpha-granule secretion was inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase and augmented by recombinant type IIbeta 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 bisindolylmaleimide I. In addition to stimulating alpha-granule secretion, both SFLLRN and PMA enhanced the association of a fluorescein isothiocyanate-labeled peptide derived from the PtdIns (4,5)P(2)-binding domain of gelsolin to permeabilized platelets. Agonist-induced recruitment of the PtdIns (4,5)P(2)-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)P(2)-binding proteins.
Rozenvayn
Nataliya
N
Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Flaumenhaft
Robert
R
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D011493:Protein Kinase C