Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.
Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.
Robinson
P J
PJ
Endocrine Unit, John Hunter Hospital, New South Wales, Australia.
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000494:pharmacology*; D034281:Dynamins; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010718:Phosphatidylserines; D010750:Phosphoproteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; D034281:Dynamins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium