Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Regulation of protein kinase C by lysophospholipids. Potential role in signal transduction. 336 Aug 11

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.
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PMID:Fatty acid activation of protein kinase C: dependence on diacylglycerol. 336 53

The activity of rat brain protein kinase C, measured in the presence of diacylglycerol, phosphatidylserine and Ca+2, was found to be greatly increased by micromolar amounts of long chain acyl-CoAs, using two different assay systems (lipids added as sonicated dispersion or as mixed micelles with Triton X-100). The potentiation phenomenon required the presence of both diacylglycerol and phosphatidylserine; it was observed at low and saturating concentrations of these effectors, and it was inhibited at high, non physiological Ca+2 concentrations. Under similar conditions, fatty acids alone or coenzyme A were ineffective. The data strongly suggest that acyl-CoAs at the intracellular concentration levels, are important in the modulation of protein kinase C, after activation of the enzyme by the phospholipase C/phosphatidylinositol pathway.
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PMID:Diacylglycerol activation of protein kinase C is modulated by long-chain acyl-CoA. 337 82

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.
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PMID:Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates. 340 77

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been purified to electrophoretic homogeneity from mouse brain cytosol. [20-3H]phorbol 12,13-dibutyrate binding activity was found to coelute quantitatively with the protein kinase activity throughout the purification procedure. The crude extract was first run over a DE52 column. Fractions containing peak activities were then chromatographed using a fast protein liquid chromatography system with a Mono Q column followed by chromatography on the same column run in the presence of 1 mM adenosine triphosphate. The adenosine triphosphate specifically shifted the elution position of the Ca2+- and phospholipid-dependent protein kinase, providing a powerful step in the purification procedure. The remaining minor contaminants were removed by hydrophobic chromatography on a TSK-phenyl-5-PW column. This purification procedure required less than 2 days after the initial large batch DE52 column chromatography. The molecular weight of the purified receptor was estimated to be 81,000 by its mobility on sodium dodecyl sulfate/polyacrylamide gels, in agreement with the published values. Optimal conditions for the storage of the purified receptor were sought. Both protein kinase and phorbol ester binding activities were stable for 2 mo when stored in the presence of 0.01% Triton X-100 at -70 degrees C. Polyclonal antibodies to the purified receptor have been prepared from rabbits. These antibodies recognized the purified receptor in electroblotting assays and were able to immunoprecipitate the purified receptor.
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PMID:Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography. 345 69

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C.
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PMID:Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols. 345 78

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.
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PMID:Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine. 345 28

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.
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PMID:Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. 346 88

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.
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PMID:Phorbol ester-induced contraction in chemically skinned vascular smooth muscle. 346 15

Trypsinization of rat brain protein kinase C (80 kDa) into 50- and 32-kDa fragments occurred without inhibition of [3H]phorbol dibutyrate ([3H]PDBu) binding activity. The 50-kDa fragment, the catalytic domain (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616), was further degraded by trypsin, whereas the 32-kDa fragment was resistant. Protein kinase activity and the [3H]PDBu binding activity were completely separated upon gel filtration of a solution containing Triton X-100/phosphatidylserine mixed micelles and trypsinized protein kinase C. Pooled fractions of the [3H]PDBu binding activity contained a 32-kDa fragment exclusively. The binding of [3H]PDBu to this fragment was dependent on calcium and phosphatidylserine and was of high affinity (Kd = 2.8 nM) and of essentially identical specificity to that of native protein kinase C. It is concluded that the 32-kDa fragment represents a lipid binding, regulatory domain of protein kinase C.
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PMID:The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity. 346 1


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