Rat brain protein kinase C. Kinetic analysis of substrate dependence, allosteric regulation, and autophosphorylation

Kinetic studies on the interaction of protein kinase C with cations and substrates were performed and the effects of essential activators on the interaction of protein kinase C with its substrates were studied. The catalytic fragment of protein kinase C interacted with protein substrate, MgATP, and Mg2+. The dual divalent cation requirement was shown by kinetic analysis as well as by the ability of Mn2+ to substitute for Mg2+. Analysis of kinetic data based on equilibrium assumptions suggested a random order of interaction of the catalytic fragment with its substrate and Mg2+ cofactor. Activation of intact protein kinase C required Ca2+, phosphatidylserine (PS), and diacylglycerol (DAG) as essential activators. Kinetic analysis of the interaction of activators with substrates indicated that Ca2+ and PS acted to increase the activity of the enzyme without modulating the KM for MgATP; PS and Ca2+ significantly decreased the KM for histone. DAG, on the other hand, did not affect the KM for either MgATP or histone but dramatically enhanced the kcat of the enzyme. These studies allow kinetic distinction between the effects of PS and Ca2+ on the one hand and DAG on the other. The possible interference of the kinetic analysis by histone was also examined by studying the requirements for autophosphorylation of protein kinase C; autophosphorylation showed similar dependencies on PS and DAG. There were no effects of histone on the lipid dependence of protein kinase C autophosphorylation, phorbol dibutyrate binding, and inhibition of autophosphorylation by sphingosine. These studies are discussed in relation to a kinetic model of protein kinase C activation.     

Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate.

Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1-¹⁴C]oleate and [³H]inositol, increased the production of inositol phosphates and the release of 1,2-[¹⁴C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl-4,5-bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G-proteins. Activation of protein kinase C could have been mediated by NaF-induced release of DAG.     

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 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.     

Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate.

The mechanism of protein kinase C (PKC) activation by phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI) was investigated by using Triton X-100 mixed micellar methods. The activation of PKC by PIP2, for which maximal activity was 60% of that elicited by sn-1,2-diacyglycerol (DAG), was similar to activation by DAG in several respects: (1) activation by PIP2 and DAG required phosphatidylserine (PS) as a phospholipid cofactor, (2) PIP2 and DAG reduced the concentration of Ca2+ and PS required for activation, (3) the concentration dependences of activation by PIP2 and DAG depended on the concentration of PS, and (4) PIP2 and DAG complemented one another to achieve maximal activation. On the other hand, PIP2 activation of PKC differed from activation by DAG in several respects. With increasing concentrations of PIP2, (1) the optimal concentration of PS required was constant at 12 mol%, (2) the maximal activity at 12 mol% PS increased, and (3) the cooperativity for PS decreased. PIP2 did not inhibit [3H]phorbol 12,13-dibutyrate (PDBu) binding of PKC at saturating levels of PS; however, at subsaturating levels of PS, PIP2 enhanced [3H]PDBu binding by acting as a phospholipid cofactor. PIP did not function as an activator but served as a phospholipid cofactor in the presence of PS. While PIP2, PIP, and PI did not support DAG-dependent PKC activation as phospholipid cofactors, their presence reduced the amount of PS required for maximal activation to as low as 2 mol% from 8 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Supplementation of the phosphatidyl-L-serine requirement of protein kinase C with nonactivating phospholipids.

The mechanism of protein kinase C (PKC) activation by phosphatidyl-L-serine (PS) is highly specific and occurs with high cooperativity [Lee, M.-H., & Bell, R. M. (1989) J. Biol. Chem. 264, 14797-14805]. To further investigate the multiplicity and specificity of PS cofactor requirement, some of the PS molecules present in Triton X-100 mixed micelles were substituted with nonactivating phospholipids devoid of required amino or carboxyl functional groups. The ability of these phospholipids to spare or reduce the mole percent of PS required was determined. Addition of phosphatidyl-(3-hydroxypropionate) (PP) or phosphatidate (PA) reduced the mole percent of PS required for maximal activity from 10 to 4 mol %, and also reduced the cooperativity of activation with PS. In contrast, phosphatidylethanolamine did not alter the dependence on PS. Phosphatidylethanol (P-Et) reduced the PS requirement to 2-4 mol % and cooperatively less efficiently than PP or PA. Phosphatidylglycerol and phosphatidylinositol resemble P-Et in their ability to reduce PS requirements and cooperativity. Therefore, it appears that the ability of phospholipids to substitute for PS in PKC activation depends on the negative charge in the phospholipid head group and the efficiency of substitution appears to be directly related to the negative charge density. The presence of two acyl groups within the phospholipid cofactor proved important since lyso-PS and lyso-PA replaced a portion of PS molecules required less efficiently than P-Et. Sodium oleate and sodium dodecyl sulfate behaved like lyso-PS. When other anionic lipids are present, approximately four molecules of PS per micelle are required for maximal PKC activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oleic acid promotes changes in the subcellular distribution of protein kinase C in isolated hepatocytes.

The effect of oleate on the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes and in perfused rat liver in the presence of physiological concentrations of serum albumin. A time- and dose-dependent translocation of PKC from the cytosol towards the membranes was observed at oleate concentrations that fell within the range of concentrations reached under several physiological conditions. Analysis of the membrane-bound isoenzymes of PKC by hydroxylapatite chromatography revealed that the beta isoenzyme was preferentially translocated to this compartment in hepatocytes incubated with oleate. Activation of PKC after incubation of hepatocytes with oleate involved at least three different effectors of the enzyme: the fatty acid itself, the diacylglycerol synthesized from oleate, and the rise in the cytosolic calcium concentration elicited by oleate. As a result of PKC activation, protein phosphorylation of intact hepatocytes in response to oleate exhibited an enhancement in the phosphate content of a protein of 82 kDa, similar to that phosphorylated in the presence of phorbol dibutyrate.     

Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis

The diacylglycerol (DAG) signal generated from membrane phospholipids by hormone-activated phospholipases is attenuated by mechanisms that include lipolysis or phospholipid resynthesis. To determine whether the DAG signal might also be terminated by incorporation of DAG into triacylglycerol (TAG), we studied the direct formation of TAG from endogenous DAG generated by bacterial phospholipase C (PLC). When Chinese hamster ovary (CHO) cells prelabeled with [(14)C]oleate were treated with PLC from Clostridium perfringens for 6 h, [(14)C]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of (14)C label was even greater when the cells were simultaneously exposed to PLC and 100 microM oleic acid. PLC as well as oleate treatment concomitantly increased the TAG mass within the cell. Moreover, when phospholipids were prelabeled with [(3)H]glycerol, a subsequent increase in [(3)H]TAG indicated that an intact DAG moiety was channeled into the TAG structure. Incubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhanced the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in the presence of 100 microM oleate, but not by incubation with oleate alone, indicating that the DAG released from plasma membrane phospholipids does not require the formation of a phosphatidic acid precursor for TAG synthesis. Similarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG synthesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG.These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a transport mechanism exists to move plasma membrane-derived DAG to the endoplasmic reticulum.-Igal, R. A., J. M. Caviglia, I. N. T. de Gómez Dumm, and R. A. Coleman. Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis. J. Lipid Res. 2001. 42: 88;-95.     

Heterogeneity of protein kinase C in cultured rat mesangial cells.

Two forms of protein kinase C (PKC) activity in cytosol of cultured rat mesangial cells have been characterized in vitro by using histone H1 or endogenous proteins as substrates. Histones H1-phosphorylation was significantly increased only when calcium, phosphatidylserine (PS) and 1,2-diacylglycerol (DAG) or phorbol myristate acetate (PMA) were present together in the incubation medium. EGTA, a calcium chelator, completely inhibited this activity. Upon hydroxyapatite chromatography (HPLC), the PKC activity was eluted as a main peak at 150 mM potassium phosphate with a shoulder at 180 mM. Both peaks corresponded to the type III PKC from rat brain and were identified as PKC alpha isoform by immunoblot analysis. In contrast with what was observed using histone H1, the increased phosphorylation of endogenous proteins in the presence of a mixture of Ca2+/PS, plus either DAG or PMA, was only partly reduced by EGTA. Moreover, the level of the PKC activity detected in the presence of EGTA was comparable to the level of kinase activity, measured in the presence of PS alone or associated with DAG or PMA. This suggests that mesangial cells contain PKC activity which does not absolutely require calcium. Polyacrylamide gel electrophoresis revealed that patterns of phosphorylated mesangial cell proteins are different depending on whether calcium was added or not. In the presence of calcium, PKC strongly phosphorylated the proteins of 53,000 molecular weight, a doublet of 37,000-39,000, the 24,000 and the triplet of 17,000-20,000-22,000 molecular weight. The addition of EGTA to the assays suppressed completely the labelling of most proteins; only the 20,000 molecular weight protein remained strongly labelled, while the 39,000 molecular weight band was only faintly visible. The same patterns of phosphorylations were obtained after omission of calcium in the assays containing only PS and DAG (or PMA). So, the main substrates of calcium-dependent PKC are proteins of 53,000, 39,000, 37,000, 22,000, 24,000 and 17,000 molecular weight while the protein of 20,000 molecular weight appears to be the main substrate of calcium-independent PKC. The existence in mesangial cells of at least two forms of PKC, which phosphorylate specific endogenous proteins, emphasizes the complexity of the phospholipid-dependent regulatory cascade and raises the possibility that actions of different regulators may be transduced through distinct PKC isozymes.     

Phospholipid metabolism and T cell activation: receptor triggering is associated with the inhibition of phosphatidylserine synthesis

Activation of Jurkat T lymphocytes to produce IL2 is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. This inhibition was obtained either with the mitogenic lectin PHA, anti-CD3 monoclonal antibodies (mAb), anti-CD2 mAb or anti-Ti mAb. Bypassing membrane receptor signalling, by using a Ca2+ ionophore or a protein phosphatase inhibitor, sodium ortho-vanadate, also results in a marked inhibition of PS synthesis. Activators of phospholipid -Ca2+ dependent protein kinase C (PKC) did not significantly modify PS synthesis, suggesting that the observed changes only involve the transduction of the first activation signal. PS being a necessary cofactor for PKC, our results strongly suggest that the inhibition of PS synthesis induced by receptor triggering exerts a feed back control on PKC therefore leading to a transient activation of the enzyme upon full lymphocyte activation.     

An update on the role of phospholipid metabolism in the action of steroidogenic agents

Most steroidogenic agents which bind to cell surface receptors activate adenylate cyclase and/or phospholipase C. Activation of either signaling system may also be associated with rapid increases in de novo phospholipid synthesis, but it is at present uncertain whether this is a secondary or parallel event. Activation of phospholipase C leads to hydrolysis of phosphatidylinositol-4',5'-PO4 (PIP2) and generation of two second messengers, inositol-triphosphate and diacylglycerol (DAG), which mobilize Ca2+ and activate protein kinase C, respectively. Increases in de novo phospholipid synthesis lead to rapid increases in phosphatidic acid, DAG and C-kinase activity. The PIP2-phospholipase C system appears to initiate the steroidogenic response to certain agents, such as angiotensin-II, and this may be amplified by concomitant increases in phospholipid synthesis. With other agonists, the role of phospholipase C activation and de novo phospholipid (and DAG) synthesis is less certain. In some tissues, activation of protein kinase C by exogeneously added DAG analogues provokes an increase in steroidogenesis. However, this is not observed in other tissues, and it is uncertain whether this rules out involvement of the C-kinase system for steroidogenesis in these tissues, or whether endogenously produced DAG is a more effective activator of the relevant C-kinase system then exogenously added DAG analogues. The role of other potential intracellular signaling substances that may be derived from phospholipase C activation and de novo phospholipid synthesis is also at present uncertain, as are the interrelationships between these two phospholipid responses, cyclic nucleotides, and other steroidogenic factors.     

Role of estrogenic compounds (diethylstibestrol, 17β‐estradiol,and bisphenol A) in the phosphorylation of substrate by protein kinase Cα

Estrogenic compounds can activate protein kinase C (PKC), which is a calcium and phospholipid‐dependent serine/threonine kinase. In the present study, we investigated the role of 17β‐estradiol (E2), diethylstibestrol (DES), and bisphenol A (BPA) in the phosphorylation of substrate by PKCα using the matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The level of phosphorylated peptide was low in the absence of phosphatidylserine (PS). Moreover, reduction of phosphorylation ratios was identified in the presence of diacylglycerol (DAG) and Ca²⁺ or PS and Ca²⁺ after adding E2, DES, and BPA. However, no change in phosphorylation ratios was found in the presence of DAG and PS. Addition of E2, DES, and BPA also had no influence on the phosphorylation reaction of substrate by cell or tissue lysate samples. Our study suggests that E2, DES, and BPA can bind to the C2 domain of PKCα but have no effects on the phosphorylation reaction of substrates in the presence of DAG and PS. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:318–323, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20294     

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.     

Oleate and eicosapentaenoic acid attenuate palmitate-induced inflammation and apoptosis in renal proximal tubular cell

Free fatty acid (FFA)-bound albumin, which is filtrated through the glomeruli and reabsorbed into proximal tubular cells, is one of the crucial mediators of tubular damage in proteinuric kidney disease. In this study, we examined the role of each kind of FFA on renal tubular damage in vitro and tried to identify its molecular mechanism. In cultured proximal tubular cells, a saturated fatty acid, palmiate, increased the expression of monocyte chemoattractant protein-1 (MCP-1), but this effect was abrogated by co-incubation of monounsaturated fatty acid, oleate, or ω-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA). Palmitate led to intracellular accumulation of diacylglycerol (DAG) and subsequent activation of protein kinase C protein family. Among the several PKC inhibitors, rottlerin, a PKCθ inhibitor, prevented palmitate-induced MCP-1 expression via inactivation of NFB pathway. Overexpression of dominant-negative PKCθ also inhibited palmitate-induced activation of MCP-1 promoter. Furthermore, palmitate enhanced PKCθ-dependent mitochondrial apoptosis, which was also prevented by co-incubation with oleate or EPA through restoration of pro-survival Akt pathway. Moreover, oleate and EPA inhibited palmitate-induced PKCθ activation through the conversion of intracellular DAG to triglyceride with the restoration of diacylglycerol acyltransferase 2 expression. These results suggest that oleate and EPA have protective effects against the palmitate-induced renal tubular cell damage by inhibiting PKCθ activation.     

Aminoacridines, potent inhibitors of protein kinase C

Acridine orange, acridine yellow G, and related compounds potently inhibited protein kinase C (Ca2+/phospholipid-dependent enzyme) activity and phorbol dibutyrate binding. Inhibition was investigated in vitro using Triton X-100 mixed micellar assays (Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043 and Hannun, Y. A., and Bell, R. M. (1986) J. Biol. Chem. 261, 9341-9347). Inhibition by the acridine derivatives was subject to surface dilution; therefore, the relevant concentration unit is mol % rather than the bulk molar concentration. Fifty percent inhibition of protein kinase C activity occurred at concentrations of these compounds comparable to concentrations of sn-1,2-diacylglycerol (DAG) and phosphatidylserine (PS) required for enzyme activation (i.e. 1-6 mol %). The mechanism of inhibition appeared to be complex: both the catalytic and regulatory sites of protein kinase C were affected. Acridine orange was a competitive inhibitor with respect to MgATP when the catalytic fragment of protein kinase C was employed. Inhibition at the active site was overcome by the addition of Triton X-100 micelles or phospholipid vesicles. When the activity of intact protein kinase C was measured, inhibition was noncompetitive with respect to MgATP. Further kinetic analysis suggested a competitive type of inhibition with respect to PS and DAG implying an interaction of acridine compounds with the regulatory lipid cofactors or with the regulatory domain of protein kinase C. This was further supported by demonstrating inhibition of phorbol dibutyrate binding to both protein kinase C and the lipid-binding domain generated by trypsin hydrolysis. Acridine orange and acridine yellow G also inhibited thrombin-induced 40-kDa phosphorylation in human platelets and phorbol dibutyrate binding to platelets. These effects were also subject to surface dilution. These results suggest that acridine derivatives have multiple interactions with protein kinase C with the predominant effect being inhibition of activation within the regulatory domain of the enzyme. Some of the biologic effects of acridine derivatives including anti-tumor action may occur as a consequence of protein kinase C inhibition.     

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.     

Interleukin-1 stimulated activation of the COT catalytic subunit through the phosphorylation of Thr290 and Ser62

The protein kinase COT/Tpl2 is activated by interleukin-1 (IL-1), TNFalpha and lipopolysaccharide, and its activation by these agonists involves the IkappaB kinase beta (IKKbeta) catalysed phosphorylation of the p105 regulatory subunit. Here, we show that COT activation also requires catalytic subunit phosphorylation, since IL-1beta induced a 5-10-fold activation of a COT mutant unable to bind p105. Activation was paralleled by the phosphorylation of Thr290 and Ser62 and unaffected by the IKKbeta inhibitor PS1145 at concentrations which prevented the degradation of IkappaBalpha. Mutagenesis experiments indicated that COT activation is initiated by Thr290 phosphorylation catalysed by an IL-1-stimulated protein kinase distinct from IKKbeta, while Ser62 phosphorylation is an autophosphorylation event required for maximal activation.