Protein kinase C activation without membrane contact in platelets stimulated by bryostatin.
The tumor promoter phorbol myristate acetate (PMA) directly activates protein kinase C (PKC) and, in human platelets, induces aggregation, release of granular contents, mobilization of intracellular Ca2+ as detected by the photoprotein aequorin, and phosphorylation of the 47-kDa substrate (p47) of PKC. Whether PKC activation by PMA or other agonists requires translocation of PKC from the cytoplasm to the lipids of the platelet surface membrane, however, is not known. Bryostatin-1, a macrocyclic lactone that binds to PKC at or near the PMA-binding site and thus blocks PMA's proliferative effects, induced concentration-dependent p47 phosphorylation, platelet aggregation, release of dense granule contents, and a rise in cytoplasmic Ca2+ (ED50 = 2.0 nM) in intact aequorin-loaded platelets. Neither alpha-, beta-, delta-, or zeta-PKC isozymes translocated to the plasma membrane following bryostatin-1, although translocation of both alpha- and beta-PKC isozymes was seen in PMA-stimulated platelets. In a cell-free assay, bryostatin-1 activated PKC purified from platelets if the membrane-derived lipid phosphatidyl serine was present; however, if 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid or arachidonic acid, two lipid products formed in the cytoplasm of activated platelets, were present, no membrane-derived lipids were required for bryostatin-induced PKC activation. Thus, phosphorylation of PKC substrates and associated functional changes do not require isozyme translocation to the surface membrane, even when these events are induced by a direct activator of PKC.
Grabarek
J
J
Harvard-Thorndike Laboratory, Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215.
Ware
J A
JA
D001692:Biological Transport; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D054713:Bryostatins; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology*; D018942:Macrolides; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D012701:Serotonin; C046785:bryostatin 1; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium