Further studies on the specificity of diacylglycerol for protein kinase C activation

Specificity of 1,2-diacylglycerol for the activation of protein kinase C was investigated with various synthetic products. 1-Stearoyl-2-arachidonylglycerol, a major species of diacylglycerol derived from the receptor-mediated hydrolysis of inositol phospholipids, was most active, but many other diacylglycerols having naturally occurring fatty acids were almost equally active in this role. Hormone-sensitive lipase could produce potentially active diacylglycerols during lipolysis. The lack of the specificity may be reconciled with the possibility that the stearoyl-arachidonyl species is the diacylglycerol with which protein kinase C indeed comes in contact in the membrane when the receptor is stimulated, and that diacylglycerols from other sources are produced in distinct compartments and are not intercalated into the phospholipid bilayer.     

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.     

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.     

Structural requirements of lyngbyatoxin A for activation and downregulation of protein kinase C.

Structure-activity studies of novel synthetic analogues of lyngbyatoxin A reveal that the lactam ring but not the 7-linalyl moiety of lyngbyatoxin A is essential for the in vitro stimulation of protein kinase C (PKC). (-)-Indolactam V (ILV), which contains no hydrophobic substituent at C-7, or analogues containing either a linalyl or n-hexyl group at C-7 were equally efficacious in stimulating HeLa cell PKC in vitro and in competing with phorbol 12,13-dibutyrate for binding to PKC in intact cells. The hydrophobicity of alkyl groups at C-7, however, influenced the potency of these compounds to bind to and activate PKC. In addition, these compounds exhibited differences in their ability to translocate PKC. Lyngbyatoxin A (0.1 microM) like TPA induced a rapid translocation of PKC from the cytosol to the membrane and subsequently led to a sustained decrease in both cytosolic and membrane PKC activity. In contrast, (-)-n-hexylILV (0.1 microM) and (-)-ILV (1 microM) produced a transient and attenuated decrease in cytosolic PKC activity. At concentrations that produced half-maximal PKC stimulation, (-)-ILV did not cause any downregulation of PKC whereas lyngbyatoxin A and (-)-n-hexylILV led to 60% and 40% PKC downregulation, respectively. Western blot analyses with monoclonal antibodies to PKC isoforms indicated that reduction in PKC activity by chronic exposure to TPA or lyngbyatoxin A analogues could be explained by downregulation of PKC alpha. Constitutive expression of PKC beta and PKC gamma isoforms was low in HeLa cells and was not affected significantly by TPA or lyngbyatoxin A analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Structural analogies between protein kinase C activators

Phorbol esters and diacylglycerols activate protein kinase C but specific structural parameters appear to be required for the enzyme activation. We have analyzed the conformation of potent and not potent diacylglycerols and phorbol esters. The orientation of the CH20H group at C3 of 1,2 diolein is remarkably similar to that of the same group at C-20 of 4 beta phorbol didecanoate and crucial for potency in activating the enzyme. Our data suggest that the new conformational approach here described could be used to rationally design specific inhibitors preventing the effects of tumor promoters and to predict the structure of potential tumor promoters.     

Effect of phospholipid unsaturation on protein kinase C activation.

To examine the hypothesis that physical features of the membrane contribute to protein kinase C activation, phosphatidylcholine/phosphatidylserine/diolein (70:25:5) vesicles of defined acyl chain composition were tested for their ability to activate the enzyme. Maximal activation was found to correlate with the mole percent unsaturation in the system. Unsaturation could be provided by either the phosphatidylserine or the phosphatidylcholine component. Vesicles containing 5 mol% diolein but lacking any unsaturation in the phospholipid did not support activity, indicating that acidic head groups alone are not sufficient for activity. The saturated lipid vesicles could be rendered effective but only at very high (25 mol%) concentrations of diolein. The degree of acyl chain unsaturation and the positioning of the double bond had little effect on the activity, suggesting that the effect of the unsaturation is due to some physical property of the lipid rather than to a specific lipid-protein interaction. Addition of cholesterol to both saturated and unsaturated systems indicated that fluidity, as assessed by fluorescence anisotropy, did not correlate with activity. These results suggest that a physical property of the membrane other than fluidity is important for the activation of protein kinase C. A model for protein kinase C activation involving phase separation and/or head group spacing is discussed.     

Effect of protein kinase C inhibitors on porcine oocyte activation

The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.     

Role of the kinase activation loop on protein kinase C theta activity and intracellular localisation

Multiple protein kinase C (PKC) theta species, identified in an erythroleukaemia cell line, have been characterised in terms of their molecular properties and intracellular distribution. PKCthetas localised in the detergent-soluble cell fraction have an Mr of 76 kDa (theta-76) and contain Thr538 or pThr538 in the kinase activation loop. In contrast, PKCthetas localised in the Golgi complex have an Mr of 85 kDa (theta-85) and, although unphosphorylated at Thr538, are catalytically active. Strikingly, only theta-76 species which are unphosphorylated at Thr538 can undergo autocatalytic conversion to theta-85. Moreover, a Thr538-->Ala PKCtheta mutant is constitutively localised in the Golgi complex, confirming that changes in the phosphorylation state of this residue play a pivotal role in the overall control of catalytic properties and localisation of this kinase.     

Further characterization of tumor-promoter-mediated activation of protein kinase C

Tumor promoting phorbol esters are able to activate Ca2+-sensitive, phospholipid-dependent protein kinase (protein kinase C) in a reconstituted system. Indol alkaloid teleocidin, a tumor promoter, has been found to be as potent as tumor promoters from the series of phorbol esters and mezerein in activating the mouse brain enzyme. Chemically unrelated tumor promoters such as tetrachlorodibenzo-p-dioxin, anthralin and phenobarbital are devoid of effect. Diacylglycerol 1,2 diolein strongly activated the enzyme whereas 1,3 diolein like 1,2 distearin were poor activators and 1,3 distearin was inactive. Although tumor-promoter-or diacylglycerol-mediated activation of protein kinase C was observed in the presence of 0.5mM EGTA, the reaction requires traces of Ca2+. Tumor promoters did not prevent inhibitory action of antipsychotic phenothiazines and local anesthetics but appear to increase IC50 of these drugs.     

Effect of protein kinase C activation on mast cell histamine release

The role of protein Kinase C activators in the process of histamine secretion has been studied in rat peritoneal mast cells purified by a density gradient. TPA (12-O-tetradecanoyl-phorbol-13-acetate), a tumor promoter which activates protein kinase C, induced histamine release in the presence and in the absence of external free Ca2+. TPA and the calcium ionophore A23187 have an additive effect on secretion. Histamine release induced by TPA is energy-dependent. In the presence of 100 microM KCN secretion was moderately inhibited, however when glucose was removed from the incubation medium TPA-induced histamine release in the presence of KCN was strongly depressed.     

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.     

Insulin activation of protein kinase C: a reassessment

Although insulin is known to activate several protein serine/threonine protein kinases, its ability to activate protein kinase C remains controversial. We reinvestigated this question, taking advantage of several technical advances such as the development of fibroblast cell lines that overexpress normal human insulin receptors, and the development of antibodies to and expression vectors for the myristoylated, alanine-rich C kinase substrate (MARCKS) protein, a major cellular substrate for protein kinase C. In HIR 3.5 cells, a mouse 3T3 cell derivative that expresses about 6 x 10(6) human insulin receptors/cell, insulin (70 nM for 10 min) stimulated phosphorylation of the MARCKS protein by approximately 2-fold (p less than 0.005). This phosphorylation was not further increased by different times of insulin exposure, different insulin concentrations, or longer periods of serum deprivation. The insulin stimulation represented about 14% of the response to phorbol 12-myristate 13-acetate and about 17% of the response to 10% fetal calf serum. No significant stimulation of MARCKS protein phosphorylation was seen in four other insulin-sensitive cell lines, in which insulin is known to activate other protein serine/threonine kinases: HIRC-B, BC3H-1, 3T3-L1 adipocytes, and H35 rat hepatoma cells made to stably express the MARCKS protein. In these four cell lines, serum and/or phorbol 12-myristate 13-acetate exerted a large stimulatory effect on MARCKS protein phosphorylation. We conclude that insulin may activate protein kinase C to a minor extent in certain cell types that vastly overexpress insulin receptors; however, we believe that this effect of insulin is unlikely to be of physiological importance.     

Fatty acid activation of protein kinase C: dependence on diacylglycerol

The kinetics of activation of protein kinase C by oleic acid have been reinvestigated, using highly purified preparations of the rat brain and bovine spleen enzymes. Activation of both enzymes by oleic acid is enhanced dramatically by diolein, contrary to previous reports. In the presence of 9.7 microM diolein, the concentrations of oleic acid required for half-maximal activation are 5 microM and 9 microM for the rat brain and bovine spleen enzymes respectively, indicating that the system is much more sensitive to activation by fatty acids than previously recognized. Both enzymes also exhibit a pronounced lag in the activation at low concentrations of oleic acid. The kinetics of activation are very similar to those reported by Hannun et al. (J. Biol. Chem 260, 10039-10043), who characterized the activation of the rat brain enzyme by mixed micelles containing Triton X-100, phosphatidylserine and diolein.     

Thrombin rapidly induces protein kinase D phosphorylation,and protein kinase C delta mediates the activation

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKC delta phosphorylation and that the PKC delta-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKC delta mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKC delta inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKC epsilon nor PKC zeta affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKC delta in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKC delta in mediating thrombin-induced PKD activation in vascular smooth muscle cells.     

Alkyl-linked diglycerides inhibit protein kinase C activation by diacylglycerols

Alkylacylglycerols are synthesized when choline-phospholipids are degraded by a phospholipase C. This class of compounds has been shown to have biological activities; however, the mechanism of action is unknown. A series of alkyl-linked diglycerides were synthesized and tested for activity in an in vitro assay for protein kinase C. When protein kinase C activity was stimulated with the synthetic diacylglyceride analog 1-oleoyl-2-acetyl-sn-glycerol, the addition of alkyl glycerides caused a concentration-dependent inhibition of protein kinase C activity. Comparison of the protein kinase C inhibition by this series of 1-O-alkyl-2-acyl analogs revealed that both saturated and unsaturated long-chain groups in position 1 were effective and that dietherglycerols with short-chain moieties in position 2 were also effective. It is concluded from these studies that the biological activity of alkyl-linked glycerides may be expressed through protein kinase C inhibition.