Ca2+- and phospholipid-independent activation of protein kinase C by selective oxidative modification of the regulatory domain.

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...

Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium

“…Interestingly, when a preparation of PKC from rat brain was used, a decrease in PKC activity was obtained with thimerosal. Similar results have been obtained with the oxidizing agent H # O # [55]. These results suggest that probably only a small fraction of thimerosal enters the cells, and activates PKC by an indirect mechanism.…”

Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C

“…A selective oxidative modification of the regulatory domain results in a Ca2*/lipid-independent activation of the kinase with a loss of phorbol ester binding [6]. Alternately, a selective oxidative modification of the kinase domain results in the generation of a modified form of PKC which exhibits only phorbol ester binding [8].…”

“…Alternately, a selective oxidative modification of the kinase domain results in the generation of a modified form of PKC which exhibits only phorbol ester binding [8]. Some oxidants such as H20, induce irreversible oxidative modification [6], whereas others such as m-periodate induce reversible oxidative modification [8]. Furthermore, PKC (the phorbol ester receptor) was shown to be regulated by lipid peroxidation [9], redoxy cycling quinones [10], and oxidant-generating systems [11].…”

“…Terpenoid tumor promoters such as phorbol esters induce not only the activation of PKC by direct binding to the enzyme but also the downregulation of PKC by an indirect mechanism [1][2][3][4][5]. Oxidant tumor promoters either activate or inactivate PKC depending on the extent of oxidative modification [6][7][8].…”

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

“…There are several excellent reviews for a more complete description of PKC structure and function [120,143,153]. Oxidation of the cysteine residues in the regulatory domain have been shown to induce the conformational changes necessary to relieve autoinhibition of PKC and render the kinase constitutively active [51] while oxidation of the cysteine residues in the catalytic domain have been shown to induce [160] as well as inhibit [50] kinase activity. There is still much we do not understand regarding the effects of the direct oxidation of the cysteine residues on PKC.…”