Regulation of platelet protein kinase C by oleic acid. Kinetic analysis of allosteric regulation and effects on autophosphorylation, phorbol ester binding, and susceptibility to inhibition.
The regulation of protein kinase C by oleic acid was studied, and parameters that characterize the activation of protein kinase C by oleic acid and distinguish its effects from those of diacylglycerol (DAG) and phosphatidylserine (PS) were delineated. Activation of protein kinase C by sodium oleate required the presence of calcium and showed mild cooperative behavior (Hill number of 1.25) suggesting that Ca(oleate)2 is the active species. Kinetic analysis of the interaction of sodium oleate with substrates indicated that sodium oleate acted to increase the activity of the enzyme without modulating the KM for either MgATP or histone substrates. In this respect, sodium oleate action resembled that of DAG but not PS. However, multiple parameters distinguished the effects of sodium oleate from those of DAG. Unlike DAG, sodium oleate was unable to inhibit phorbol dibutyrate binding to protein kinase C. Sodium oleate also failed to interact with micelle-bound protein kinase C and preferentially activated "soluble" protein kinase C. The addition of histone caused protein/lipid aggregation in the presence of DAG but not in the presence of oleate. Activation of protein kinase C by sodium oleate or by PS/DAG demonstrated differential susceptibility to the action of inhibitors. Sphingosine and NaCl were more potent in inhibiting activation of protein kinase C by PS/DAG than by sodium oleate. Sodium oleate also expressed PS-like activity in that calcium and oleate acted as cofactors in activation of protein kinase C by DAG. Similar to PS, the ability of oleate to act in synergy with DAG resulted from "competitive" activation with a decrease in KM(app) of protein kinase C for DAG. Finally, sodium oleate was unable to induce autophosphorylation of protein kinase C. These studies demonstrate that oleate activates protein kinase C by a mechanism that is distinct from PS/DAG but partially overlaps the kinetic effects of both PS and DAG. The significance of these studies is discussed in relation to mechanisms of protein kinase C activation and to the possible physiological relevance of activation of protein kinase C by fatty acids.
el Touny
S
S
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Khan
W
W
Hannun
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006657:Histones / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008823:Micelles; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood*
D004075:Diglycerides; D006657:Histones; D008823:Micelles; D009829:Oleic Acids; D019301:Oleic Acid; D015240:Phorbol 12,13-Dibutyrate; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D002118:Calcium; C013965:diolein