Ca2+- and phospholipid-independent activation of protein kinase C by selective oxidative modification of the regulatory domain.
The susceptibility of purified protein kinase C to oxidative inactivation by H2O2 was found to be increased by Ca2+ either alone at a high (5 mM) concentration or at a low (approximately 50 microM) concentration along with phosphatidylserine and diacylglycerol and by tumor-promoting phorbol esters even in the absence of Ca2+. This suggested that the membrane-bound and/or catalytically active form of protein kinase C is relatively more susceptible to oxidative inactivation. Although both the regulatory and catalytic domains of protein kinase C were susceptible to oxidative inactivation, a selective modification of the regulatory domain was obtained under mild oxidative conditions by protecting the catalytic site with ATP/Mg2+. Under these conditions there was a loss of both phorbol ester binding and Ca2+/phospholipid-stimulated kinase activity. However, this modified form of enzyme exhibited an increase in Ca2+/phospholipid-independent kinase activity. This suggests that selective oxidative modification of the regulatory domain may negate the requirement for Ca2+ and lipids for activation. Treatment of intact C6 glioma or B16 melanoma cells with H2O2 resulted in a time- and temperature-dependent decrease in Ca2+/phospholipid-dependent protein kinase C activity along with a concomitant transient increase in an oxidatively modified isoform of protein kinase C that exhibited activity in the absence of Ca2+ and phospholipids. Since protein kinase C can initially be activated by mild oxidative modification and subsequently inactivated by further oxidation, this dual activation-inactivation of protein kinase C in response to H2O2 suggests an effective on/off signal mechanism to influence cellular events.
Gopalakrishna
R
R
Department of Pathology, School of Medicine, University of California, Los Angeles 90024-1732.
Anderson
W B
WB
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005910:Glioma; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D010718:Phosphatidylserines; D015240:Phorbol 12,13-Dibutyrate; D004533:Egtazic Acid; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium