Progesterone primes zona pellucida-induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.     

Arachidonic acid,protein kinase C activators and bud formation in Hydra vulgaris

1. The effect of the protein kinase C (PKC) activators verrucosin B (VB), 1,2-sn-dioctanoylglycerol (diC₈) and phorbol-12-myristate-13-acetate (PMA), of arachidonic acid (AA) and of substances interfering with its release, re-uptake and metabolism was studied in Hydra vulgaris.2. All PKC activators potently inhibited bud formation, VB and PMA being 10,000 × more potent than diC⁸. VB effect was maximal already after 10 min incubation with hydra and persisted at 24 hr incubations.3. AA and substances inhibiting its re-uptake from cell membrane or its metabolism also inhibited bud formation, whereas oleyl-oxyethyl-phosphorylcholine (OOPC), an inhibitor of phospholipase A₂, potently induced bud formation.4. The findings described herein suggest a role for both PKC activation and AA in the inhibition of bud formation in H. vulgaris.     

Protein kinase C subspecies in adult rat hippocampal synaptosomes. Activation by diacylglycerol and arachidonic acid

Synaptosomes isolated from the adult rat hippocampus contain the alpha- and beta-subspecies of protein kinase C (PKC), but not the gamma-subspecies which is abundantly expressed in the pyramidal cells in this brain region. Although the gamma-subspecies is known to respond significantly to free arachidonic acid, it is found that both the alpha- and beta-subspecies are also activated dramatically by arachidonic acid in synergistic action with diacylglycerol. Oleic, linoleic, and linolenic acids are all active. It is possible that unsaturated fatty acids may take part in the activation of alpha- and beta-subspecies of PKC which are present in the presynaptic nerve endings terminating at the hippocampal pyramidal cells.     

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.     

Effect of arachidonic acid on the physical properties of bilayer and annular lipids of synaptic membranes

It has been found by fluorescence analysis of pyrene excimerization that arachidonic acid while decreasing microviscosity of lipid bilayer increases it in the annular lipid zone. After incorporation of fatty acid into the membrane the annular lipid acquires the properties of a cooperative system manifested in the appearance of thermal transition near 25 degrees C.     

Activation of protein kinase C in lipid monolayers

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.     

Multiple effects of protein kinase C activators on Na+ currents in mouse neuroblastoma cells

The effects of externally applied different protein kinase C (PKC) activators on Na⁺ currents in mouse neuroblastoma cells were studied using the perforated-patch (nystatin-based) whole cell voltage clamp technique. Two diacylglycerol-like compounds, OAG (1-oleoyl-2-acetyl-sn-glycerol), and DOG (1-2-dioctanoyl-rac-glycerol) attenuated Na⁺ currents without affecting the time course of activation or inactivation. The reduction in Na⁺ current amplitude caused by OAG or DOG was dependent on membrane potential, being more intense at positive voltages. The steady-state activation curve was also unaffected by these substances. However, both OAG and DOG shifted the steady-state inactivation curve of Na⁺ currents to more hyperpolarized voltages. Surprisingly, phorbol esters did not affect Na⁺ currents. Cis-unsaturated fatty acids (linoleic, linolenic, and arachidonic) attenuated Na⁺ currents without modifying the steady-state activation. As with DOG and OAG, cis-unsaturated fatty acids also shifted the steady-state inactivation curve to more negative voltages. Interestingly, inward currents were more effectively attenuated by cis-fatty acids than outward currents. Oleic acid, also a cis-unsaturated fatty acid, enhanced Na⁺ currents. This enhancement was not accompanied by changes in kinetic or steady-state properties of currents. Enhancement of Na⁺ currents caused by oleate was voltage dependent, being stronger at negative voltages. The inhibitory or stimulatory effects caused by all PKC activators on Na⁺ currents were completely prevented by pretreating cells with PKC inhibitors (calphostin C, H7, staurosporine or polymyxin B). By themselves, PKC inhibitors did not affect membrane currents. Trans-unsaturated or saturated fatty acids, which do not activate PKC's, did not modify Na⁺ currents. Taken together, the experimental results suggest that PKC activation modulates the behavior of Na⁺ channels by at least three distinct mechanisms. Because qualitatively different results were obtained with different PKC activators, it is not clear how Na⁺ currents would respond to activation of PKC under physiological conditions.This work was supported in part by a grant-in-aid from the American Heart Association (National Center), and by Loyola University Medical Center. Dr. Godoy is a recipient of a fellowship from Conselho Nacional de Pesquisas e Desenvolvimento (Brazil).     

A mechanism underlying stimulation and inhibition of protein kinase C by lyso-PC: A role of membrane physical state

ＰｒｏｔｅｉｎｋｉｎａｓｅＣｉｓａｃｒｕｃｉａｌｅｎｚｙｍｅｆａｍｉｌｙｉｎｔｈｅｃｏｎｔｒｏｌｏｆｓｉｇｎａｌｔｒａｎｓｄｕｃｔｉｏｎａｎｄｉｓｉｎｖｏｌｖｅｄｉｎｄｉｖｅｒｓｅｃｅｌｌａｃｔｉｏｎｓ[1].ＴｈｅａｃｔｉｖｉｔｙｏｆｃｌａｓｓｉｃａｌＰＫＣ(ｃＰＫＣ),ｗｈｉｃｈｉｓｏｎｅｏｆｔｈｅｔｈｒｅｅｓｕｂｇｒｏｕｐｓ,ｎｏｔｏｎｌｙｄｅｐｅｎｄｓｏｎｃａｌｃｉｕｍａｎｄｐｈｏｓｐｈａｔｉｄｙｌｓｅｒｉｎｅ,ｂｕｔａｌｓｏｉｓｒｅｇｕｌａｔｅｄｂｙｄｉａｃｙｌｇｌｙｃｅｒｏｌｓ,ｕｎｓａｔｕｒ…     

Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes

The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.     

Calcium alters the acyl chain composition and lipid fluidity of rat hepatocyte plasma membranes in vitro

Calcium ion decreases the lipid fluidity of isolated rat hepatocyte plasma membranes by modulating the activity of membrane enzymes which alter the lipid composition. To explore the mechanism of the effect of the cation, eight fluorophores were used to assess lipid fluidity via estimations of either steady-state fluorescence polarization or excimer fluorescence intensity. The results demonstrate that the reduction in fluidity occurs in the hydrophobic interior of the bilayer and that both the dynamic and static (lipid order) components of fluidity are affected by treatment with calcium. Analysis of the membrane lipids demonstrates that calcium treatment decreases the arachidonic acid content of the polar lipid fraction and, thereby, reduces the double-bond index of the fatty acids. This change in composition, which is expected to reduce the lipid fluidity, may result from activation by calcium of the endogenous hepatocyte plasma membrane phospholipase A2.     

Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of β1 Integrin

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl phorbol 13-acetate (TPA) further increases the relative content of F-actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKCϵ, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plama membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of β1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading. J. Cell. Physiol. 173:361–370, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Ceramides modulate protein kinase C activity and perturb the structure of Phosphatidylcholine/Phosphatidylserine bilayers.

We studied the effects of natural ceramide and a series of ceramide analogs with different acyl chain lengths on the activity of rat brain protein kinase C (PKC) and on the structure of bovine liver phosphatidylcholine (BLPC)/dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylserine (DPPS) (3:1:1 molar ratio) bilayers using (2)H-NMR and specific enzymatic assays in the absence or presence of 7.5 mol % diolein (DO). Only a slight activation of PKC was observed upon addition of the short-chain ceramide analogs (C(2)-, C(6)-, or C(8)-ceramide); natural ceramide or C(16)-ceramide had no effect. In the presence of 7.5 mol % DO, natural ceramide and C(16)-ceramide analog slightly attenuated DO-enhanced PKC activity. (2)H-NMR results demonstrated that natural ceramide and C(16)-ceramide induced lateral phase separation of gel-like and liquid crystalline domains in the bilayers; however, this type of membrane perturbation has no direct effect on PKC activity. The addition of both short-chain ceramide analogs and DO had a synergistic effect in activating PKC, with maximum activity observed with 20 mol % C(6)-ceramide and 15 mol % DO. Further increases in C(6)-ceramide and/or DO concentrations led to decreased PKC activity. A detailed (2)H-NMR investigation of the combined effects of C(6)-ceramide and DO on lipid bilayer structure showed a synergistic effect of these two reagents to increase membrane tendency to adopt nonbilayer structures, resulting in the actual presence of such structures in samples exceeding 20 mol % ceramide and 15 mol % DO. Thus, the increased tendency to form nonbilayer lipid phases correlates with increased PKC activity, whereas the actual presence of such phases reduced the activity of the enzyme. Moreover, the results show that short-chain ceramide analogs, widely used to study cellular effects of ceramide, have biological effects that are not exhibited by natural ceramide.     

In vitro linoleic acid activation of protein kinase C

The importance of membrane fluidity in the activation of protein kinase C (PKC) was examined using the membrane fluidizer, linoleic acid, in a well-defined model membrane system. Biochemical and biophysical properties of the system were monitored. Linoleic acid activated PKC to a level of 50% of that observed for diacylglycerol. In contrast, linoleic acid did not directly interact with the phorbol ester binding site as did diacylglycerol. This was determined by the lack of involvement of the ionizable group of the fatty acid with activity and the enhancement of phorbol ester binding by linoleic acid and its ester analogs. The membrane fluidity of this model membrane system in the presence of linoleic acid was increased as determined by fluorescence polarization. This increased the availability of phospholipids, thus, explaining the linoleic acid-induced enhancement of phorbol ester binding. The PKC conformation as determined from intrinsic tryptophan fluorescence spectra was different for lipid mixtures containing linoleic acid or diacylglycerol correlating with the difference in biochemical activation properties. This study provides evidence that membrane fluidization is not the predominant function of the lipid activator in PKC activation, but may play a role in obtaining the preferred membrane state for maximal activation.     

The C1 domain of protein kinase C as a lipid bilayer surface sensing module

The activity of membrane-associated protein kinase C (PKC) is tightly controlled by the physical properties of the membrane lipid bilayer, in particular, curvature stress, which is induced by bilayer-destabilizing lipid components. An important example of this is the weakened lipid headgroup interactions induced by phosphatidylethanolamine (PE) and cholesterol. In this work our previous observation with a mixed isoform PKC showing a biphasic dependence of activity as a function of membrane curvature stress [Slater et al. (1994) J. Biol. Chem. 269, 4866-4871] was here extended to individual isoforms. The Ca(2+)-dependent PKCalpha, PKCbeta, and PKCgamma, along with Ca(2+)-independent PKCdelta, but not PKCepsilon or PKCzeta, displayed a biphasic activity as a function of membrane PE content. The fluorescence anisotropy of N-(5-dimethylaminonaphthalene-1-sulfonyl)dioleoylphosphatidylserine (dansyl-PS), which probes the lipid environment of PKC, also followed a biphasic profile as a function of PE content for full-length PKCalpha, PKCbetaIotaIota, and PKCgamma as did the isolated C1 domain of PKCalpha. In addition, the rotational correlation time of both PKCalpha and PKCdelta C1-domain-associated sapintoxin D, a fluorescent phorbol ester, was also a biphasic function of membrane lipid PE content. These results indicate that the C1 domain acts as a sensor of the bilayer surface properties and that its conformational response to these effects may directly underlie the resultant effects on enzyme activity.     

Characterization of protein kinase C in rat and human prostates

The properties of protein kinase C (PKC) activity have been studied in cytosolic and membrane fractions from rat and human prostate. Ion exchange chromatography indicated the existence of different PKC isoforms, PKC from rat ventral prostate behaved as a classical Ca²⁺- and phospholipid-dependent enzyme and was activated by 1,2-diacylglycerol as well as by high concentrations of arachidonic acid. PKC activity in the cytosolic fraction was higher and presented different cofactor requirements than that in the membrane fraction. PKC from human benign hyperplastic prostate was also phospholipid dependent, activated by tumor-promotong phorbol esters, and appeared to belong to the group of PKC isozymes which lack Ca²⁺ sensitivity. Human prostatic PKC activity appeared to be of similar nature in both membrane and cytosolic fractions but the specific activity was higher in the particulate preparation which could be related to the stage of endogenous activation of the enzyme. These results extend previous observations in rat ventral prostate and present evidences on the human counterpart. Forthcoming experiments are needed to establish the exact nature of PKC isozymes and their physiological and pathophysiological role in this gland.     

Synergistic effect of arachidonic acid and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of arachidonic acid and cAMP on glucose transport was examined in 3T3-L1 adipocytes. In cells pre-treated with arachidonic acid and increasing concentrations of 8-bromo cAMP for 8 h, although either agent alone enhanced glucose uptake, the simultaneous presence of both agents dramatically increased 2-deoxyglucose uptake in a synergistic fashion. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was abolished in the presence of cycloheximide. Immunoblot analysis revealed that the contents of ubiquitous glucose transporter (GLUT1) in total cellular and plasma membranes were similarly augmented in cells pre-treated with both arachidonic acid and 8-bromo cAMP, to a greater extent than the additive effect of each agent alone. The content of GLUT4, on the other hand, was not altered under the same experimental conditions. In cells pre-treated with 4beta-phorbol 12beta-myristate 13alpha-acetate (PMA) for 24 h to down-regulate protein kinase C (PKC), the subsequent synergistic effect of arachidonic acid and 8-bromo cAMP was greatly inhibited. In addition, pre-treatment with both PMA and 8-bromo cAMP enhanced glucose transport in a similarly synergistic fashion. Thus the present study seems to indicate that arachidonic acid may act with cAMP in a synergistic way to increase glucose transport by a PKC-dependent mechanism. The increased activity may be accounted for by increased GLUT1 synthesis.     

Activation mechanisms of protein kinase C: maturation,catalytic activation,and targeting

The biological function of protein kinase C (PKC) depends on its catalytic activity and spatial localization. Its catalytic competence and localization in the resting state are regulated by serine/threonine phosphorylations, i.e., "maturation." Upon stimulation of various receptors, PKC is catalytically activated by several activators including diacylglycerol. In addition, PKC often translocates to particular subcellular compartments including the plasma membrane and Golgi complex, and such translation is here referred to as "targeting." In short, the physiological function of PKC is controlled by the three events: maturation, catalytic activation, and targeting. Catalytic activation and targeting contribute to temporal, spatial, and isotype-specific regulation of PKC. This review summarizes the evidence for the role of these three events in the isotype-specific activation of PKC, with particular emphasis on catalytic activation and targeting by lipid mediators.     

The use of fluorescent phorbol esters in studies of protein kinase C-membrane interactions

The family of protein kinase C (PKC) isozymes belongs to a growing class of proteins that become active by associating with membranes containing anionic phospholipids, such as phosphatidylserine. Depending on the particular PKC isoform, this process is mediated by Ca(2+)-binding to a C2 domain and interaction of activators such as 1,2-diacyl-sn-glycerol or phorbol esters with tandem C1 domains. This cooperation between the C1 and C2 domains in inducing the association of PKC with lipid membranes provides the energy for a conformational change that consists of the release of a pseudosubstrate sequence from the active site, culminating in activation. Thus, the properties of the interactions of the C1 and C2 domains with membranes, both as isolated domains, and as modules in the full length PKC isoforms, have been the subject of intense scrutiny. Here, we review the findings of studies in which fluorescent phorbol esters have been utilized to probe the properties of the C1 domains of PKC with respect to the interaction with activators, the subsequent interaction with membranes, and the role of the activating conformational change that leads to activation.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.