Specificity of the fatty acyl moieties of diacylglycerol for the activation of calcium-activated, phospholipid-dependent protein kinase

The specificity of the fatty acyl moieties of diacylglycerol for the activation of Ca2+-activated, phospholipid-dependent protein kinase was investigated. Diacylglycerol has been previously shown to activate this enzyme by increasing the affinity for Ca2+ and phospholipid, both of which are indispensable for the enzyme activation. Diacylglycerols containing at least one unsaturated fatty acid at either position 1 or 2 are fully active in this capacity, irrespective of the chain length of the other fatty acyl moiety in the range tested, C2 to C18. Diacylglycerols containing two saturated fatty acids such as dipalmitin and distearin are far less effective. Mono- and triacylglycerols and free fatty acids are totally inactive, indicating that the diacylglycerol structure is essential.     

Differential activation of protein kinase C isozymes by short chain phosphatidylserines and phosphatidylcholines

To investigate the importance of the physical state of phospholipids for activation of protein kinase C, we have used short chain phospholipids, which, depending on their concentration, can exist as either monomers or micelles. We previously reported that short chain phosphatidylcholines (PC) can activate protein kinase C at concentrations that correlate with the critical micelle concentration of the activating lipid (Walker, J. M., and Sando, J. J. (1988) J. Biol. Chem. 263, 4537-4540). We have now expanded this work to short chain phosphatidylserine (PS) systems in order to examine the role of Ca2(+)-phospholipid interactions in the activation process. Short chain PS were synthesized from corresponding PC and purified by reverse-phase high pressure liquid chromatography. Use of the short chain system has revealed significant differences in the activation of type II and type III protein kinase C isozymes. The type II isozyme required Ca2+ in the presence of long chain PS vesicles; in the presence of the short chain phospholipid micelles (PC or PS), most of the activity was Ca2+ independent. Addition of diacylglycerol caused a small increase in type II activity in all phospholipid systems. In contrast, type III protein kinase C was Ca(+)-dependent in all of the lipid systems. The concentration of Ca2+ required to activate type III protein kinase C was independent of the phospholipid type despite large differences in the ability of these lipids to bind Ca2+. This isozyme required diacylglycerol only in the PC micelle system or with vesicles composed of long chain saturated PS. The presence of short chain PS micelles or long chain PS with unsaturated fatty acyl chains rendered this Ca2(+)-dependent protein kinase C virtually diacylglycerol independent. These results are consistent with a model in which type II protein kinase C requires Ca2+ primarily for membrane association, a requirement which is bypassed with the micelle system, whereas type III protein kinase C has an additional Ca2+ requirement for activity that does not involve Ca2(+)-phospholipid interactions.     

Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

The role of hydrophobic interactions in the phospholipid-dependent activation of protein kinase C.

The effects of hydrophobic interaction on the activation of Ca2+-stimulated phospholipid-dependent protein kinase (protein kinase C), isolated from mouse brain, by phosphatidylserine (PS) and diacylglycerol (DAG) or phorbol 12-myristate 13-acetate were studied. To maintain bilayer structure during assay conditions, phosphatidylcholine was added to the PS vesicles. The vesicular structure of all types of PS was confirmed by freeze-fracture electron microscopy. The PS-dependent activation of purified protein kinase C from mouse brain is affected by the fatty acid composition of PS: an inverse relationship between the unsaturation index of PS (isolated from bovine heart, bovine spinal cord or bovine brain) and the ability to activate protein kinase C was demonstrated. In highly saturated PS lipid dispersions, only slight additional activation of protein kinase C by DAG was found, in contrast with highly unsaturated PS lipid dispersion, where DAG increased protein kinase C activity by 2-3-fold at optimal PS concentrations. We quantified the formation of the protein kinase C-Ca2+-PS-phorbol ester complex by using [3H]phorbol 12,13-dibutyrate [( 3H]PDBu). The efficiency of complex-formation, determined as the amount of [3H]PDBu bound, is not affected by variations in the hydrophobic part of PS. These results indicate a role of the hydrophobic part of the activating phospholipid in the activation mechanism of protein kinase C and in the action of cofactors.     

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Long-chain cis-unsaturated fatty acids could substitute for phosphatidylserine and activate bovine aortic protein kinase C in assays with histone as substrate. The optimal concentration was 24-40 microM for oleic, linoleic and arachidonic acids. With arachidonic acid, the Ka for Ca2+ was 130 microM and kinase activity was maximal at 0.5 mM-Ca2+. Diolein only slightly activated the oleic acid-stimulated enzyme at low physiological Ca2+ concentrations (0.1 and 10 microM). Oleic acid also stimulated kinase C activity, determined with a Triton X-100 mixed-micellar assay. Under these conditions, the fatty acid activation was absolutely dependent on the presence of diolein, but a Ca2+ concentration of 0.5 mM was still required for maximum kinase C activity. The effect of fatty acids on protein kinase C activity was also investigated with the platelet protein P47 as a substrate, since the properties of kinase C can be influenced by the choice of substrate. In contrast with the results with histone, fatty acids did not stimulate the phosphorylation of P47 by the aortic protein kinase C. Activation of protein kinase C by fatty acids may allow the selective phosphorylation of substrates, but the physiological significance of fatty acid activation is questionable because of the requirement for high concentrations of Ca2+.     

Regulation of protein kinase C activity by cooperative interaction of Zn2+ and Ca2+

cis-Fatty acids such as oleic acid or linoleic acid have been previously shown to induce full activation of protein kinase C in the absence of Ca2+ and phospholipids (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193; Murakami, K., Chan, S.Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429). In this study, we have investigated the effects of various metal ions on protein kinase C activity without the interference of Ca2+ since cis-fatty acid requires no Ca2+ for protein kinase C activation. Here we report a specific interaction of Zn2+ with protein kinase C in either a positive or negative cooperative fashion in concert with Ca2+. At low concentrations (approximately 5 microM) of Ca2+, Zn2+ enhances protein kinase C activity induced by both oleic acid and phosphatidylserine/diolein. In contrast, Zn2+ inhibits the activity at higher concentrations (over 50 microM) of Ca2+. In the absence of Ca2+, Zn2+ shows no effect on protein kinase C activity. Our results suggest that Zn2+ does not recognize or interact with protein kinase C in the absence of Ca2+, that protein kinase C possesses high and low affinity Ca2+-binding sites, and that at least one Zn2+-binding site exists which is distinct from Ca2+-binding sites.     

Activation of protein kinase C by short chain phosphatidylcholines

The acidic phospholipid requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme) activation has been well established, although the molecular nature of this lipid-protein interaction is unclear. The additional requirement for Ca2+ has provided the basis for several models involving charge interactions. We now report that short chain neutral phosphatidylcholines also activate the kinase. Examination of a large series of phosphatidylcholines of varying acyl chain length revealed a close correlation between the ability to form micelles and the ability to support kinase activity. Peak activation occurred in the concentration range just before the critical micelle concentration of each phospholipid. Activation was absolutely dependent on the presence of Ca2+ and diacylglycerol. The possible roles of Ca2+ and phospholipid in the activation process are reexamined in light of these unexpected results.     

Activation of protein kinase C by cis- and trans-octadecadienoic acids in intact human platelets and its potentiation by diacylglycerol

Octadecadienoic acids (linoleic acid and linolelaidic acid) and the diacylglycerol, 1-oleoyl-2-acetyl-rac-glycerol (OAG) concentration-dependently induced activation of gel-filtered human platelets, i.e. aggregation and phosphorylation of 20 kDa and 47 kDa peptides. In contrast, octadecenoic acids (oleic and elaidic acid) and octadecanoic (stearic) acid were inactive. Octadecadienoic acid-induced platelet activation was suppressed by the protein kinase C inhibitor, polymyxin B, but not by the cyclooxygenase inhibitor, indomethacin. OAG-induced activation was potentiated by octadecadienoic acids present at non-stimulatory concentrations. Our data suggest that octadecadienoic acids and diacylglycerol synergistically induce platelet activation via protein kinase C. Furthermore, linolelaidic acid may provide a useful experimental tool to study fatty acid regulation of protein kinase C in intact cells.     

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.     

Cytoplasmic lipid bodies of neutrophils: formation induced by cis- unsaturated fatty acids and mediated by protein kinase C

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.     

Purification and characterization of the zeta isoform of protein kinase C from bovine kidney.

The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.     

ATP-sensitive K+ channels in insulinoma cells are activated by nonesterified fatty acids.

Both 86Rb+ efflux experiments and electrophysiological studies have shown that arachidonic acid and other nonesterified fatty acids activate ATP-sensitive K+ channels in insulinoma cells (HIT-T15). Activation was observed with arachidonic, oleic, linoleic, and docosahexaenoic acid but not with myristic, stearic, and elaidic acids. Fatty acid activation of ATP-sensitive K+ channels was blocked by antidiabetic sulfonylureas such as glibenclamide. The activating effect of arachidonic acid was unaltered by indomethacin and by nordihydroguaiaretic acid, indicating that it is not due to metabolites of arachidonic acid via cyclooxygenase or lipoxygenase pathways. Moreover, the nonmetabolizable analogue of arachidonic acid, eicosatetraynoic acid, was an equally potent activator. Activation of ATP-sensitive K+ channels by fatty acids was potentiated by diacylglycerol and was inhibited by calphostin C, an inhibitor of protein kinase C. These findings indicate that fatty acid activation of ATP-sensitive K+ channels is most likely due to the participation of arachidonic acid (and other fatty acid)-activated protein kinase C isoenzymes. Activation of ATP-sensitive K+ channels by nonesterified fatty acids is not involved in the control of insulin secretion since arachidonic acid stimulates insulin secretion from insulinoma cells instead of inhibiting it.     

Intracellular Ca2+ and protein kinase C interact to regulate alpha 1-adrenergic- and bradykinin receptor-stimulated phospholipase A2 activation in Madin-Darby canine kidney cells.

Alpha 1-Adrenergic receptors and bradykinin receptors are two distinct membrane receptors that stimulate phospholipid breakdown and arachidonic acid and arachidonic acid metabolite release. In the current studies, we have examined several mechanisms to assess their possible contribution to arachidonic acid release in the Madin-Darby canine kidney cell line by agonist stimulation of these receptors: 1) activation of phospholipase A2 (PLA2); 2) sequential activation of phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase; and 3) inhibition of the sequential action of fatty acyl-CoA synthetase and lysophosphatide acyltransferase. Experiments were conducted to measure the stimulation of lysophospholipid production by epinephrine and bradykinin, the rate of incorporation of [3H]arachidonic acid into stimulated and unstimulated cells, and the effect on [3H]arachidonic acid release of treating cells with exogenous phospholipase C. The data indicate that stimulation of PLA2 activity is regulated by alpha 1-adrenergic and bradykinin receptors and that this stimulation is mediated, at least in part, by the activation of protein kinase C. We find that the role of diacylglycerol in arachidonic acid release is as an activator of protein kinase C and not as a substrate for a lipase. Moreover, the hormonal agonists do not appear to inhibit fatty acid reacylation. Experiments using the Ca2(+)-sensitive dye fura-2 and the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid suggest that bradykinin activates PLA2 by a transient elevation of intracellular Ca2+. This action appears to be less important for activation of PLA2 by epinephrine. Taken together, these data are consistent with the following conclusions. 1) Hormone-stimulated arachidonic acid release in Madin-Darby canine kidney-D1 cells occurs as a consequence of PLA2 activation. 2) The ability of an agonist both to mobilize Ca2+ and to activate protein kinase C contributes to its efficacy as a stimulator of PLA2-mediated arachidonic acid release.     

Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS), and sn-1,2-dioleoylglycerol (diC18:1) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC18:1. Activity of protein kinase C was dependent on PS, diC18:1, and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC18:1 mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC18:1 rapidly equilibrate among the mixed micelles. At saturating calcium, the diC18:1 dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC18:1 gave maximal activity whereas 6 mol % PS and 6 mol % diC18:1 did not give maximal activity. diC18:1 dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC18:1/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC18:1 increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all the phospholipids tested in the mixed micelle assay. diC18:1 was found to modulate the amount of calcium required for maximal activity. As the level of Ca2+ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.     

Regulation of protein kinase C by lysophospholipids. Potential role in signal transduction

Certain lysophospholipids, lysophosphatidylcholine (lyso-PC) in particular, stimulated protein kinase C at low concentrations (less than 20 microM) but, conversely, inhibited it at high concentrations (greater than 30 microM). Protein kinase C stimulation by lyso-PC required the presence of phosphatidylserine (PS) and Ca2+ and was associated with a decreased Ka for PS and increased Ka for Ca2+ of the enzyme. Cardiolipin and phosphatidic acid could partially substitute for PS in supporting the stimulatory effect of lyso-PC. Lyso-PC also biphasically regulated protein kinase C activated by diolein. Of several synthetic lyso-PC preparations tested, the oleoyl, myristoyl and palmitoyl derivatives were most active. Data from the Triton X-100 mixed micellar assay indicated that 1.4 and 14.0 mol of lyso-PC/micelle produced a maximal stimulation and a complete abolishment of the stimulation of protein kinase C, respectively. Protein kinase C stimulation by lyso-PC, with a pH optimum of about 7.5, was observed for phosphorylation of histone H1, myelin basic protein, and the 35- and 47-kDa proteins from the rat brain, but not for that of other histone subfractions and protamine. Lyso-PC acted synergistically with diacylglycerol in stimulating protein kinase C, whereas the stimulation by lyso-PC was additive to that by oleic acid. Protein kinase C inhibitors (alkyllysophospholipid, sphingosine, tamoxifen, and polymyxin B) inhibited more potently the protein kinase C activity stimulated by PS/Ca2+/lyso-PC than that stimulated by PS/Ca2+. The stimulatory and inhibitory effects of lyso-PC were not observed for myosin light chain kinase and cAMP-dependent protein kinase, indicating a specificity of its actions. The present findings suggested that lyso-PC, likely derived from membrane PC by the action of phospholipase A2, might play a role in signal transduction via a dual regulation of protein kinase C, and that it could further modulate the enzyme and hence the cellular activity by interplaying with diacylglycerol and unsaturated fatty acid, the two other classes of cellular mediators also shown to be activators of protein kinase C.     

IL-1 signaling for IL-2 production in T cells involves a rise in phosphatidylserine synthesis

Activation of Jurkat T cells with anti-TCR, anti-CD3, anti-CD2, or PHA is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. The inhibition of the synthesis of this phospholipid could be partially reversed by IL-1. In Jurkat cells, IL-1 did not activate phosphodiesterases as demonstrated by the lack of change of inositol triphosphate and diacylglycerol levels as well as the lack of change in cytosolic Ca2+ concentration. Furthermore, IL-1 did not modify the intracellular level of cGMP and cAMP, suggesting that the observed rise of PS synthesis could play the role of mediator IL-1 action. As PS is a necessary cofactor for the activation of protein kinase C, our results suggest strongly that IL-1 modulate protein kinase C activity in the activated lymphocyte through its action on PS synthesis.     

Rapid microassay for protein kinase C translocation in Swiss 3T3 cells

The Ca2+/phosphatidylserine-stimulated protein kinase C (PKC) appears to exist as interconvertible inactive, soluble and active, membrane-bound forms. Changes in the bimodal distribution of PKC induced by diacylglycerol or tumor-promoting phorbol esters have been proposed to regulate the activity of this kinase [Nishizuka, Y. (1984) Nature (London) 308, 693-698]. A rapid microassay for assessment of protein kinase C translocation between cytosol and membranes was developed. This procedure, which relied on the selective digitonin-mediated release of cytoplasmic proteins, eliminated potential homogenization and fractionation artifacts. PKC activity toward histone H1 was determined after limited trypsinolysis, which abolished the Ca2+/phospholipid requirement of the enzyme and prevented interference by inhibitory proteins. Complete translocation of PKC to the membrane fraction and subsequent down-regulation of the kinase in response to 12-O-tetradecanoylphorbol-13-acetate treatment of Swiss 3T3 cells could be demonstrated by this method. Platelet-derived growth factor, insulin-like growth factor 1, vasopressin, and prostaglandin F2 alpha facilitated partial conversions of PKC to the membrane-bound form in quiescent 3T3 cells.     

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.     

Fatty acyl esters potentiate fatty acid induced activation of protein kinase C

Purified rat pancreas protein kinase C (PKC) is activated by unsaturated free fatty acids (oleic and arachidonic). The ethyl esters of these fatty acids are ineffective as enzyme activators. However, when the ethyl esters are added in combination with a free fatty acid, there is significant enhancement of enzyme activation. Nearly optimal PKC activation was obtained when non-activating ethyl oleate or ethyl arachidonate was added to sub-optimally activating concentrations of oleic or arachidonic acids. In addition to the ethyl esters, 1-monooleylglycerol also had a potentiating effect on PKC activation by oleic acid. However, the degree of activation observed in the presence of a free fatty acid and an acyl ester of the fatty acid quantitatively never surpassed that produced by sn-1,2-dioleylglycerol. Our findings indicate that significant PKC activation can be achieved by presenting the enzyme with an environment which we believe approximates the structural characteristics of the endogenous activator, sn-1,2-diacylglycerol.     

The nature of protein kinase C activation by physically defined phospholipid vesicles and diacylglycerols

Protein kinase C is activated by a 1,2-sn-diacylglycerol and phospholipid at low calcium concentrations. Of the various phospholipids studied, phosphatidylserine has been shown to be the most effective one and is usually used in assaying the enzyme (Kaibuchi, K., Takai, Y., and Nishizuka, Y. (1981) J. Biol. Chem. 256, 7146-7149). It is shown here that under the conditions of the enzymatic assay, phosphatidylserine does not form typical fluid bilayer structures as seen by electron microscopy and fluorescence polarization. On the other hand, 1:4 phosphatidylserine/phosphatidylcholine bilayer vesicles can be formed which support protein kinase C activation. They have the advantage in that they are characterizable, form physiologically relevant bilayer structures, and are readily and reproducibly formed. In addition, they do not support protein kinase C activation in the absence of added diacylglycerol, a property that makes them invaluable in studying the role of diacylglycerol structure in protein kinase C activation. It is further demonstrated that the rat brain enzyme is activated by 1,2-sn-diolein but not by 2,3-sn-diolein nor 1,3-diolein, demonstrating the high specificity of the kinase toward the glycerol backbone. 1,2-rac-Dielaidin, 1,2-rac-distearin, and 1,2-sn-dipalmitin are all active, which is consistent with the idea that the specificity of protein kinase C is not directed toward the fatty acid side chain of the diacylglycerols.