Structural basis of protein kinase C activation by diacylglycerols and tumor promoters.

Protein kinase C is a ubiquitous and important regulatory enzyme. The enzyme is physiologically activated in a temporary manner by (S)-diacylglycerols (DAGs), which are themselves generated by the phospholipase C mediated hydrolysis of polyphosphoinositides. The (S)-DAGs specifically bind to the regulatory domain of PKC and cause the activation of the PKC toward substrate. Minor modifications in the DAG result in inactive molecules. On the other hand, the structurally diverse, polycyclic tumor promoters also specifically activate PKC by binding to the same effector site as do the DAGs. The object of this paper is to present a discrete structural model that accounts for the activation of PKC by both the tumor promoters and the DAGs. The unique model presented is based on experimentation rather than on computer-driven hypotheses which, experience has shown, generally produce incorrect structural models when applied to PKC. The model described here begins with a structural analysis of the tumor-promoting debromoaplysiatoxins (DATs). DAT is an ideal starting molecule, because it is conformationally rigid with a known relative and absolute configuration, and it is synthetically manipulable. The pharmacophore of DAT was experimentally determined, and this pharmacophore serves as a template for further analyses. This template is used to predict the active conformer of the acylic DAGs; this conformer is then used to reveal the pharmacophore of various families of tumor promoters. The overall model presented is consistent with published structure-activity studies on the tumor promoters and makes testable predictions that have proven to be correct thus far.     

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.     

Activation of protein kinase C by naturally occurring ether-linked diglycerides

Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.     

Differential activation and inhibition of lymphocyte proliferation by modulators of protein kinase C: diacylglycerols, "rationally designed" activators and inhibitors of protein kinase C

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) can enhance or inhibit lymphocyte proliferation. Enhancement correlated with increased interleukin 2 (IL-2) production and activation of protein kinase C while inhibition correlated with decreased IL-2 and downregulation of protein kinase C activity (D.S. Grove and A.M. Mastro, Cancer Res. 51, 82-88). In this study, various activators and inhibitors of protein kinase C were used in order to try to separate the effects of TPA on this enzyme from its effects on IL-2 production and determine if protein kinase C activity was directly or indirectly related to IL-2 production. 1,2-Dioctanoylglycerol, 1-oleoyl-2-acetyl-glycerol, phospholipase C, and two "rationally designed" activators, 6-(N-decylamino)-4-hydroxy-methylindole and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, were tested. Some activators enhanced proliferation in the presence of a Ca2+ ionophore, ionomycin, but not concanavalin A. Some activators suppressed proliferation and downregulated protein kinase C. Others neither downregulated protein kinase C nor inhibited IL-2 production and proliferation. However, inhibition or downregulation of protein kinase C activity always correlated with decreased IL-2 and depressed proliferation. Thus, the evidence in this and the previous study suggests that activation of protein kinase C is directly related to IL-2 production in activated T cells.     

Activation of protein kinase C alpha by lipid mixtures containing different proportions of diacylglycerols.

Lipid activation of protein kinase C alpha (PKC alpha) was studied using a model mixture containing POPC/POPS (molar ratio 4:1) and different proportions of either DPG or POG. The lipid mixtures containing DPG were physically characterized by using different physical techniques, and a phase diagram was constructed by keeping a constant POPC/POPS molar ratio of 4:1 and changing the concentration of 1,2-DPG. The phase diagram displayed three regions delimited by two compounds: compound 1 (CO(1)) with 35 mol % of 1,2-DPG and compound 2 (CO(2)) with 65 mol % of 1,2-DPG. PKC alpha activity was assayed at increasing concentrations of 1,2-DPG, maximum activity being reached at 30 mol % 1,2-DPG, which decreased at higher concentrations. Maximum activity occurred, then, at concentrations of 1,2-DPG which corresponded to the transition from region 1 to region 2 of the phase diagram. It was interesting that this protein was maximally bound to the membrane at all DPG concentrations. Similar results were observed when the enzyme was activated by POG, when a maximum was reached at about 10 mol %. This remained practically constant up to 50 mol %, about which it decreased, the binding level remaining maximal and constant at all POG concentrations. The fact that in the assay conditions used maximal binding was already reached even in the absence of diacylglycerol was attributed to the interaction of the C2 domain with the POPS present in the membrane through the Ca(2+) ions also present. To confirm this, the isolated C2 domain was used, and it was also found to be maximally bound at all DPG concentrations and even in its absence. Since the intriguing interaction patterns observed seemed to be due then to the C1 domain, the PKC alpha mutant D246/248N was used. This mutant has a decreased Ca(2+)-binding capacity through the C2 domain and was not activated nor bound to membranes by increasing concentrations of DPG. However, POG was able to activate the mutant, which showed a similar dependence on POG concentration with respect to activity and binding to membranes. These data underline the importance of unsaturation in one of the fatty acyl chains of the diacylglycerol.     

Forskolin and prostacyclin inhibit fluoride induced platelet activation and protein kinase C dependent responses

Platelet activation (cytosolic [Ca2+] increase, aggregation and ATP secretion) was induced with A1F-4. This agent presumably interacts with a G protein which appears to mediate the coupling of the receptors for Ca mobilizing hormones and phospholipase C. All the A1F-4 evoked responses were inhibited by treatment with forskolin or prostacyclin, agents known to increase cellular cAMP. Thus the G protein-phospholipase C system appears to be the site of cAMP inhibition. Unexpectedly forskolin and prostacyclin also inhibited secretion and aggregation induced by the activators of protein kinase C, diglyceride and phorbol ester, suggesting that cAMP can also inhibit directly the protein kinase C dependent responses.     

A rapid effect of thyroxine on accumulation of diacylglycerols and on activation of protein kinase C in liver cells

L-Thyroxine rapidly stimulated the accumulation of diacylglycerols in isolated hepatocytes and in liver when lipids were prelabeled with [14C]oleic acid or with [14C]CH3COONa. Perfusion of the liver of hypothyroid animals with L-thyroxine-containing solution or incubation of liver fragments with the hormone increased the content of diacylglycerols in the liver cells. The increase in [14C]diacylglycerol level in the liver cells was accompanied by a decrease in the level of [14C]phosphatidylcholine, whereas contents of other 14C-labeled phospholipids, such as phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns4P), and phosphatidylinositol-4,5-bis-phosphate (PtdIns(4,5)P2), and of 14C-labeled fatty acids were the same as in the control. The L-thyroxine-induced accumulation of diacylglycerols in hepatocytes was not affected by neomycin but was inhibited by propranolol. Incubation of hepatocytes prelabeled with [14C]oleic acid with L-thyroxine and ethanol (300 mM) was accompanied by generation and accumulation of [14C]phosphatidylethanol that was partially suppressed by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). L-Thyroxine was responsible for the translocation of protein kinase C from the cytosol into the membrane fraction and for a many-fold activation of the membrane-bound enzyme. D-Thyroxine failed to affect the generation of diacylglycerols in hepatocytes and the activity of protein kinase C.     

Learning-induced activation of protein kinase C

PKC activation has been shown to mimic the biophysical consequences of classical conditioning in both rabbit hippocampus and Hermissenda type B cells. Furthermore, conditioning in rabbits results in the 24 h translocation of PKC from cytosol to membrane, which is probably responsible for mediating the biophysical consequences of conditioning. A model has been presented that suggests that long-term translocation of PKC occurs via the synergistic activation of a DG dependent pathway that activates PKC and a calcium dependent pathway that activates CaM kinase. Translocation of PKC to the plasma membrane, by altering ion channel properties, could subserve memory lasting for days, whereas translocation to the nuclear membrane could induce cellular change, by genomic regulation, lasting beyond days. We are, therefore, suggesting that protein kinase C may play a critical role in the formation of short, intermediate, and long-term associative memory.     

Differences in the effects of phorbol esters and diacylglycerols on protein kinase C

The binding of protein kinase C (PKC) to membranes and appearance of kinase activity are separable events. Binding is a two-step process consisting of a reversible calcium-dependent interaction followed by an irreversible interaction that can only be dissociated by detergents. The irreversibly bound PKC is constitutively active, and the second step of binding may be a major mechanism of PKC activation [Bazzi & Nelsestuen (1988) Biochemistry 27, 7589]. This study examined the activity of other forms of membrane-bound PKC and compared the effects of phorbol esters and diacylglycerols. Like the membrane-binding event, activation of PKC was a two-stage process. Diacylglycerols (DAG) participated in forming an active PKC which was reversibly bound to the membrane. In this case, both activity and membrane binding were terminated by addition of calcium chelators. DAG functioned poorly in generating the constitutively active, irreversible PKC-membrane complex. These properties differed markedly from phorbol esters which activated PKC in a reversible complex but also promoted constitutive PKC activation by forming the irreversible PKC-membrane complex. The concentration of phorbol esters needed to generate the irreversible PKC-membrane complex was slightly higher than the concentration needed to activate PKC. In addition, high concentrations of phorbol esters (greater than or equal to 50 nM) activated PKC and induced irreversible PKC-membrane binding in the absence of calcium. Despite these striking differences, DAG prevented binding of phorbol esters to high-affinity sites on the PKC-membrane complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts

Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.     

The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation

The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation.     

Amplification of diacylglycerol activation of protein kinase C by cholesterol

The combined effects of cholesterol, a major cell membrane component, and the lipid second messenger diacylglycerol on the activity of protein kinase C (PK-C) and the structure of phosphatidylcholine/phosphatidylserine bilayers were investigated using specific PK-C assays and ²H NMR. Whereas the classical activation of PK-C was observed as an effect of diacylglycerol, in the absence of this second messenger, cholesterol did not affect PK-C activity. A novel effect of amplified PK-C activation was observed in the presence of both cholesterol and diacylglycerol concentrations within the physiological range of each of these components. ²H NMR results suggest that this phenomenon is due to cholesterol- and diacylglycerol-induced increased propensity of the lipids to adopt nonbilayer phases, effectively destabilizing the bilayer structure. The magnitude of the effect was a function of cholesterol concentration, implying that laterally separated cell membrane domains with distinct cholesterol concentrations have the capacity to differ in their sensitivity to extracellular stimuli.     

Control of platelet protein kinase C activation by cyclic AMP

Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP. These changes were also observed when platelet cAMP was increased by prostacyclin (PGI2), forskolin, or theophylline. The ADP scavenger creatine phosphate/creatine phosphokinase (CP/CPK) and the cyclooxygenase inhibitor indomethacin also diminished the aggregation and p47 phosphorylation responses to PMA or OAG. Pre-incubation of platelets with dibutyryl cAMP significantly potentiated the inhibition of aggregation and p47 phosphorylation effected by CP/CPK and indomethacin. These results are consistent with the model that PMA- or OAG-induced activation of platelets is amplified by secreted ADP and that the response to secreted ADP is inhibited by cAMP. Furthermore, the findings that increased intracellular cAMP inhibits PMA- or OAG-induced p47 phosphorylation in excess of that due solely to CP/CPK, and that cAMP significantly potentiates the effects of ADP removal and inhibition of cyclooxygenase in blocking p47 phosphorylation suggest that cAMP also exerts non-ADP-mediated inhibitory effects on PKC in intact platelets.     

The stereospecific activation of protein kinase C

Protein kinase C is synergistically activated by the presence of calcium, certain phospholipids and a diacylglycerol. The physiological activation of the enzyme appears to be determined by the availability of the diacylglycerol which is itself a product of (poly) phosphoinositol turnover. It is shown here that the diacylglycerol activation effect is stereospecific, with only the 1,2-sn-diglycerides being active. This demonstrates for the first time a stereospecific effector role for a membrane-bound lipid. Furthermore, this work strengthens the link forged between the highly potent and specific tumor promoters (such as the phorbol esters) and the diglycerides as activators of protein kinase C.     

Flavonoid modulation of protein kinase C activation

The flavonoid quercetin exhibited a biphasic effect on calcium and phospholipid-dependent protein kinase (protein kinase C) activity from rat brain and pig thyroid. At a low concentration (10(-7) M) quercetin stimulated the enzyme activity whereas at higher concentrations quercetin was inhibitory. By contrast the synthetic penta-0-ethylquercetin stimulated protein kinase C activity in a dose-dependent manner. When fresly dispersed pig thyroid cells were treated with penta-0-ethylquercetin or 12-0-tetradecanoylphorbol 13-acetate (TPA), a 50% decrease of the cytosolic protein kinase C activity was observed. These results suggest that the lipophilicity as well as other structural determinants may be crucial for the ability of flavonoids to regulate (inhibit or activate) the enzyme activity.     

Mitogen activated protein kinase and protein kinase C activation mediate promotion of sAPPalpha secretion by deprenyl

The beta amyloid cascade plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Therefore, drugs that regulate amyloid precursor protein (APP) processing toward the nonamyloidgenic pathway may have therapeutic potential. Many anti-dementia drugs can regulate APP processing in addition to their pharmacological properties. Deprenyl is a neuroprotective agent used to treat some neurodegenerative diseases, including AD. In the present study, the effects of deprenyl on APP processing were investigated. Using SK-N-SH and PC12 cells, it was demonstrated that deprenyl stimulated the release of the nonamyloidogenic alpha-secretase form of soluble APP (sAPPalpha) in a dose-dependent manner without affecting cellular APP expression. The increase of sAPPalpha secretion by deprenyl was blocked by the mitogen activated protein (MAP) kinase inhibitor U0126 and PD98059, and by the protein kinase C (PKC) inhibitor GF109203X and staurosporine, suggesting the involvement of these signal transduction pathways. Deprenyl induced phosphorylation of p42/44 MAP kinase, which was abolished by specific inhibitors of MAP kinase and PKC. Deprenyl also phosphorylated PKC and its major substrate, and myristoylated alanine-rich C kinase (MARCKS) at specific amino acid residues. The data also indicated that 10microM deprenyl successfully induced two PKC isoforms involved in the pathogenesis of AD, PKCalpha and PKCepsilon, to translocate from the cytosolic to the membrane fraction. This phenomenon was substantiated by immunocytochemistry staining. These data suggest a novel pharmacological mechanism in which deprenyl regulates the processing of APP via activation of the MAP kinase and PKC pathways, and that this mechanism may underlie the clinical efficacy of the drug in some AD patients.