Although the biochemical mechanism of action of phorbol ester tumour promoters is not fully understood, it is known that phorbol ester binding to the cell surface causes rapid changes in calcium flux and phospholipid metabolism. A protein kinase activity has recently been described which is dependent on calcium and acidic phospholipids and is further enhanced by diacylglycerol. Previously, we have observed that phorbol ester treatment of EL4 mouse thymoma cells causes a rapid decrease in cytosolic calcium, phospholipid-dependent protein kinase (Ca, PL-PK) activity, which is mediated through the specific phorbol ester cell-surface receptors identified on EL4 cells. We now show that treatment of parietal yolk sacs (PYS-2) cells with biologically active 12-O-tetradecanoyl phorbol-13-acetate (TPA) provokes a rapid decrease in cytosolic Ca, PL-PK activity that is accompanied by a significant increase in the amount of Ca, PL-PK activity associated with the plasma membrane fraction. These results suggest that the rapid and tight association of Ca, PL-PK activity with the plasma membrane may be an early event in mediating some of the effects of phorbol esters.

No abstract

No abstract

The susceptibility of purified protein kinase C to oxidative inactivation by H202 was found to be increased by Ca2W either alone at a high (5 mM) concentration or at a low (=50 ,uM) concentration along with phosphatidylserine and diacylglycerol and by tumor-promoting phorbol esters even in the absence of Ca+. This suggested that the membrane-bound and/or catalyticaily active form of protein kinase C is relatively more susceptible to oxidative inactivation. Although both the regulatory and catalytic doin 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 ofintact C6 glioma or B16 melanoma cells with H202 resulted in a time-and temperaturedependent 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 Ca+ 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 H202 suggests an effective on/off signal mechanism to influence cellular events.Protein kinase C (PKC) plays a crucial role in cellular signal transduction and also serves as an intracellular receptor for tumor-promoting phorbol esters (1). Besides the reversible activation ofPKC by Ca2+, phospholipids (PL), diacylglycerol, unsaturated fatty acids, and phorbol esters, this enzyme is also irreversibly activated by limited proteolysis involving the intracellular protease calpain (1-5). The calpain-mediated proteolysis of PKC also has been implicated in the down-regulation of this kinase in cells treated for prolonged periods of time with phorbol esters (6-8). Recently, we suggested that the oxidative inactivation of PKC, particularly the membrane-bound form, also may play a role in the down-regulation of this enzyme (9). Since phorbol esters induce not only the membrane association of PKC (10, 11) but also the generation of reactive oxygen species (12, 13), increased oxidative inactivation of PKC may occur in phorbol ester-treated cells (9). In turn, as demonstrated for other proteins (14), this inactive, oxidatively modified form of PKC may be more susceptible to proteolysis.In this communication we identify factors that regulate the rate of H202-mediated oxidative inactivation of PKC. Further, it is shown that selective oxidative modification of the regulatory (phorbol ester/PL-binding) domain, under conditions wh...