Role of substrate in determining the phospholipid specificity of protein kinase C activation

The phospholipid selectivity of protein kinase C (PKC) activation was examined by using two substrates, histone and a random copolymer of lysine and serine [poly(lysine, serine)] (PLS), plus phospholipids provided as vesicles or as Triton-mixed micelle preparations. The results indicated that substrate-phospholipid interaction was an essential component of PKC activation and that many in vitro properties of PKC activation are attributable to this interaction. The substrate histone interacted with phospholipid-Triton mixed micelles containing phosphatidylserine (PS), but not with those containing phosphatidylinositol (PI) or phosphatidylglycerol (PG). In direct correlation, only PS-Triton mixed micelles were effective in supporting PKC activity. Also, the minimum PS composition (4 mol % in Triton) required to induce significant histone-PS interaction coincided with the minimum composition required for phosphorylation of histones. Moreover, the PS composition required for maximum activity varied with the histone concentration of the reaction. In contrast to histone, PLS interacted with phospholipid-Triton mixed micelles containing either PS, PI, or PG, and all these mixed micelles supported the phosphorylation of PLS. In fact, by selection of appropriate experimental conditions (e.g., concentration of substrate and phospholipid), any of the three mixed micelles could appear the most effective in supporting PKC activity. Phospholipid vesicles containing PS, PG, or PI were found to interact with both histone and PLS and to support the activity of PKC. Physical properties of the solution and conditions used for preparation of phospholipid vesicles had considerable influence on PKC activation. At high phospholipid concentrations, vesicles containing PS, PI, or PG supported the activity of PKC to essentially the same level, provided that the physical differences among the phospholipid vesicles were minimized.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Direct demonstration of involvement of protein kinase Calpha in the Ca2+-induced platelet aggregation

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin alphaIIbbeta3. However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca2+-induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of alphaIIbbeta3, suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKCalpha and beta from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCalpha. Furthermore, the addition of purified PKCalpha in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKCalpha is involved in the regulation of Ca2+-induced platelet aggregation.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids and diacylglycerols. Triton X-100 mixed micelles containing 8 mol % phosphatidylserine (PS) and 2.5 mol % sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1 did in the standard assay. However, protein kinase C activity was more responsive to diC18:1 in the mixed micellar assay than the standard assay. At 8 mol % PS and 100 microM Ca2+, diC18:1 stimulated maximally at 1 mol %. At 2.5 mol % diC18:1 and 100 microM Ca2+, PS did not activate until 3 mol % and then did so cooperatively with maximal stimulation occurring at 6-8 mol %. Direct evidence for a Ca2+-, PS-, and diC18:1-dependent interaction of protein kinase C with mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data permit inferences pertaining to the number of diC18:1 and PS molecules/micelle which are required for activation. For diC18:1, a single molecule may be sufficient but no more than 2 molecules are required. For PS, greater than 4 but less than 10 molecules are required. These data establish that a phospholipid bilayer is not required for protein kinase C activation and that activation of monomeric protein kinase C occurs.     

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)     

Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine

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.     

Insulin receptor aggregation and autophosphorylation in the presence of cationic polyamino acids

Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the [32P]phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptor aggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.     

Regulation of platelet protein kinase C by oleic acid. Kinetic analysis of allosteric regulation and effects on autophosphorylation, phorbol ester binding, and susceptibility to inhibition

The regulation of protein kinase C by oleic acid was studied, and parameters that characterize the activation of protein kinase C by oleic acid and distinguish its effects from those of diacylglycerol (DAG) and phosphatidylserine (PS) were delineated. Activation of protein kinase C by sodium oleate required the presence of calcium and showed mild cooperative behavior (Hill number of 1.25) suggesting that Ca(oleate)2 is the active species. Kinetic analysis of the interaction of sodium oleate with substrates indicated that sodium oleate acted to increase the activity of the enzyme without modulating the KM for either MgATP or histone substrates. In this respect, sodium oleate action resembled that of DAG but not PS. However, multiple parameters distinguished the effects of sodium oleate from those of DAG. Unlike DAG, sodium oleate was unable to inhibit phorbol dibutyrate binding to protein kinase C. Sodium oleate also failed to interact with micelle-bound protein kinase C and preferentially activated "soluble" protein kinase C. The addition of histone caused protein/lipid aggregation in the presence of DAG but not in the presence of oleate. Activation of protein kinase C by sodium oleate or by PS/DAG demonstrated differential susceptibility to the action of inhibitors. Sphingosine and NaCl were more potent in inhibiting activation of protein kinase C by PS/DAG than by sodium oleate. Sodium oleate also expressed PS-like activity in that calcium and oleate acted as cofactors in activation of protein kinase C by DAG. Similar to PS, the ability of oleate to act in synergy with DAG resulted from "competitive" activation with a decrease in KM(app) of protein kinase C for DAG. Finally, sodium oleate was unable to induce autophosphorylation of protein kinase C. These studies demonstrate that oleate activates protein kinase C by a mechanism that is distinct from PS/DAG but partially overlaps the kinetic effects of both PS and DAG. The significance of these studies is discussed in relation to mechanisms of protein kinase C activation and to the possible physiological relevance of activation of protein kinase C by fatty acids.     

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.     

Protein kinase C activation induces phosphatidylserine exposure on red blood cells

We have shown previously that red blood cells (RBCs) can be induced to influx Ca(2+) when treated with lipid mediators, such as lysophosphatidic acid and prostaglandin E(2), that are released during clot formation. Since calcium loading of RBCs can lead to both protein kinase C (PKC) activation and phosphatidylserine (PS) exposure, we decided to investigate the possible linkage between PKC activation and membrane PS scrambling using phorbol 12-myristate-13-acetate (PMA), a commonly used activator of PKC. Treatment of RBCs with PMA in a calcium-containing buffer caused immediate PS exposure in an RBC subpopulation. The size of the subpopulation did not change upon further incubation, indicating that not all RBCs are equally susceptible to this treatment. Using a fluorescent indicator, we found a subpopulation of RBCs with elevated intracellular calcium levels. In the absence of extracellular calcium, no PS exposure was found. However, we did find cells with high levels of calcium that did not expose PS, and a variable percentage of PS-exposing cells that did not show elevated calcium concentrations. Inhibition of PKC with either calphostin C, a blocker of the PMA binding site, or chelerythrine chloride, an inhibitor of the active site, diminished the level of formation of PS-exposing cells. However, the inhibitors had different effects on calcium internalization, indicating that a high calcium concentration alone was not responsible for inducing PS exposure in the absence of PKC activity. Moreover, PKC inhibition could prevent PS exposure induced by calcium and ionophore treatment of RBCs. We conclude that PKC is implicated in the mechanism of membrane phospholipid scrambling.     

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.     

Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism.

The role of protein kinase C (PKC) in the regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) was investigated. In membranes from Chinese hamster lung fibroblasts that had been incubated with [14C]choline to label endogenous phosphatidylcholine, phorbol 12-myristate 13-acetate (PMA) failed to stimulate production of [14C]choline. However, stimulation was observed if fibroblast cytosolic fraction or PKC partially purified from this fraction was added. When incubated with membranes in the presence of PMA, pure PKC from rat brain stimulated [14C]choline production in a concentration-dependent manner, with a maximal 2-3-fold effect. PMA similarly stimulated [14C]phosphatidylpropanol formation from propanol using membranes from [14C]myristic acid-prelabeled cells, confirming the activation of PLD. None of the effects described required exogenous ATP. To probe the role of phosphorylation in the PKC effect, we included high concentrations of apyrase in the assay. This ATPase had no effect on the ability of PKC to activate PLD, but under exactly the same conditions, it eliminated autophosphorylation of PKC. The results provide conclusive evidence for the involvement of PKC in the activation of PLD and suggest that ATP-dependent phosphorylation is not required.     

Protein kinase C activation by platelet-activating factor is independent of enzyme translocation

The subcellular distribution and activation state of protein kinase C (PKC) was studied after short-term exposure of rabbit platelets to platelet-activating factor (PAF). Cytosolic and nonidet P-40-solubilized particulate extracts prepared from treated platelets were subjected to analytical column chromatography on MonoQ, hydroxylapatite and Superose 6/12. PKC activity was assayed by the ability of the enzyme to phosphorylate the following substrates: (i) histone H1 in the presence of the activators calcium, diacylglycerol and phosphatidylserine; (ii) histone H1 following proteolytic activation of PKC with 0.5 micrograms trypsin/ml; and (iii) protamine in the absence of calcium and lipid. PAF treatment for 1-20 min elicited a rapid 2-4-fold activation of both cytosolic and particulate-derived PKC as assessed by all three methods. On the other hand, there were no significant PAF-induced changes in the level of [3H]phorbol-12,13-dibutyrate binding by soluble and particulate-associated PKC. Hydroxyapatite column chromatography revealed that in non-treated rabbit platelets the type II (beta) form of PKC predominated, but PAF appeared to induce a shift in the elution profile from this resin. The stability of the PAF activation of PKC to column chromatography and the altered binding affinity to hydroxylapatite indicated that the stimulation might be a consequence of covalent modification, albeit minor, since PKC still eluted as an 80 kDa protein from Superose 6/12. As the PAF-induced increases in the kinase activity of PKC were preserved even after proteolytic activation with trypsin, but were without effect on the phorbol ester binding activity, such a putative modification may have occurred within or near the catalytic domain of PKC. These findings imply that PAF may directly modulate the activity of preexisting membrane-associated PKC by a novel mechanism, rather than by eliciting its recruitment from the cytoplasm.     

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues; highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T(191) and Y(193) in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T(187) showed reduced enzymatic activity against exogenous substrates but retained autophosphorylation activity. Phosphorylation was confirmed by the use of a phospho-specific T(187) antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed.     

Combining precision spin-probe partitioning with time-resolved fluorescence quenching to study micelles. Application to micelles of pure lysomyristoylphosphatidylcholine (LMPC) and LMPC mixed with sodium dodecyl sulfate

Micelles of lysomyristoylphosphatidylcholine (LMPC) and mixed micelles of LMPC with anionic detergent sodium dodecyl sulfate (SDS) have been characterized by spin-probe-partitioning electron paramagnetic resonance (SPPEPR) and time-resolved fluorescence quenching (TRFQ) experiments. SPPEPR is a novel new method to study structure and dynamics in lipid assemblies successfully applied here for the first time to micelles. Several improvements to the computer program used to analyze SPPEPR spectra have been incorporated that increase the precision in the extracted parameters considerably from which micelle properties such as effective water concentration and microviscosity may be estimated. In addition, with this increased precision, it is shown that it is feasible to study the rate of transfer of a small spin probe between micelles and the surrounding aqueous phase by SPPEPR. The rate of transfer of the spin probe di-tert-butyl nitroxide (DTBN) and the activation energy of the transfer process in LMPC and LMPC-SDS micelles have been determined with high precision. The rate of transfer increases with temperature and SDS molar fraction in mixed micelles, while it remains constant with LMPC concentration in pure LMPC micelles. The activation energy of DTBN transfer in pure lysophospholipid micelles does not change with LMPC concentration while it decreases with the increasing molar fraction of SDS in mixed LMPC-SDS micelles. Both this decrease in activation energy and the increase in the rate of transfer are rationalized in terms of an increasing micelle surface area per molecule (decreasing compactness) as SDS molecules are added. This decreasing compactness as a function of SDS content is confirmed by TRFQ measurements showing an aggregation number that decreases from 122 molecules for pure LMPC micelles to 80 molecules for pure SDS micelles. The same increase in surface area per molecule is predicted to increase the effective water concentration in the polar shell of the micelles. This increase in hydration with SDS molar fraction is confirmed by measuring the effective water concentration in the polar shell of the micelles from the hyperfine spacing of DTBN. This work demonstrates the potential to design mixed lysophospholipid surfactant micelles with variable physicochemical properties. Well-defined micellar substrates, in terms of their physicochemical properties, may improve the studies of protein structure and enzyme kinetics.     

Regulation of receptor-mediated protein kinase C membrane trafficking by autophosphorylation

Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. In response to activation of cell-surface receptors, PKC is directed to the plasma membrane by two membrane-targeting domains, namely the C1 and C2 regions. This is followed by the return of the enzyme to the cytoplasm, a process shown recently to require PKC autophosphorylation (Feng, X., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 26870-26874). In the present study, we examined mechanisms of translocation and reverse translocation and the role of autophosphorylation in these processes. By visualizing the trafficking of wild-type as well as mutant PKCbetaII in live cells, we demonstrated that in response to cell-surface receptor activation, the function of the C1 region is required but not sufficient for recruitment of the enzyme to the plasma membrane. The C2 region is also critical in anchoring the enzyme to the plasma membrane. Furthermore, the inability of a kinase-deficient PKC to undergo reverse translocation was restored by the addition of intracellular calcium chelators, suggesting a role for the C2 region in the persistent phase of translocation. On the other hand, the inability of a C2 deletion mutant (C1 region intact) to translocate in response to agonist was reversed in mutants lacking kinase activity or by mutation of the Ser(660) autophosphorylation site to alanine, suggesting that autophosphorylation of this site is required for opposing the action of the C2 region. Therefore, the membrane-targeting function of the C1 region is facilitated by the C2 region and appears to be opposed by autophosphorylation. Taken together, these findings provide novel evidence of the functional regulation of reversible PKC membrane localization by autophosphorylation, and they show that the dynamic translocation of PKC in response to agonists is tightly regulated in a collaborative fashion by the C1 and C2 regions in balance with the effects of autophosphorylation.     

The antioxidant butylated hydroxytoluene (BHT) inhibits the dioctanoylglycerol-evoked platelet response but potentiates that elicited by ionomycin.

Preincubation of aspirin-treated human platelets with butylated hydroxytoluene (BHT) inhibits secretion, aggregation, and protein phosphorylation induced by dioctanoylglycerol or phorbol 12-myristate 13-acetate (PMA). BHT alone elicits a rapid and transient phosphorylation of a 47-kDa protein, which is indistinguishable from the well-recognized major substrate of protein kinase C (PKC). Inhibition of diacylglycerol- or PMA-induced platelet activation is also observed after decay to the basal level of the BHT-evoked phosphorylation of the 47-kDa protein. By contrast BHT potentiates platelet responses elicited by the calcium ionophore ionomycin. In the presence of the PKC inhibitor staurosporine BHT fails to increase the ionomycin-promoted platelet aggregation, indicating that its effect occurs through a PKC activation, even if no correlation with the 47-kDa protein phosphorylation is observed. BHT does not significantly modify the affinity of protein kinase C purified from calf brain for Ca2+ or dioctanoylglycerol. It is concluded that: (a) a short exposure of platelets to BHT induces an activation, whereas a long exposure an inhibition of PKC, (b) at variance with diacylglycerols BHT decreases the platelet responses promoted by subsequent challenge with PKC activators themselves, and (c) similarly to other PKC activators BHT potentiates the cellular response elicited by calcium ionophores most likely by activating the phospholipase A2.     

Effects of a fecapentaene on protein kinase C

Protein kinase C (PKC) is a Ca2(+)- and phospholipid-dependent serine and threonine protein kinase which binds and is activated by tumor promoters such as the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). PKC can be activated in vitro by phosphatidylserine (PS) plus Ca2+. We report here that the compound fecapentaene-12 can replace the requirement for PS in the activation of PKC by Ca2+. In addition, at low concentrations fecapentaene-12 can enhance the activation of PKC by Ca2+ and PS. It can also either enhance or inhibit activation of PKC by the tumor promoter teleocidin, depending on the assay conditions. These results are of interest since fecapentaene is known to be a potent mutagen that is produced by Bacteroides species present in the lumen of the human colon. The present studies raise the possibility that this compound might also play a role in colon cancer by altering the activity of PKC.     

Purification of octyl beta-D-glucopyranoside and re-estimation of its micellar size

The commercial non-ionic detergent octyl beta-D-glucopyranoside is often contaminated by significant amounts of UV absorbing and/or ionic compounds that can associate with membrane proteins. Such impurities can be monitored by several techniques (i.e., spectrophotometry, size exclusion chromatography, and pH, conductivity, and surface tension measurements) and can be removed using mixed-bed ion exchange chromatography. High performance size exclusion chromatography, dynamic light scattering, and ultracentrifugation have been used to re-estimate the size of micelles of octyl beta-D-glucopyranoside since previously published data varied over a wide range. Aggregation numbers were 27 to 100 for micellar molecular weights 8000 to 29,000. Direct physical methods that do not perturbate the sample indicated a large size for the micelles (hydrodynamic radius 23 +/- 3 A; Mr 22,000 +/- 3000; aggregation number 75 +/- 10 for a 34 mM aqueous solution). In contrast the chromatographic micellar size appeared to be smaller (hydrodynamic radius 15 +/- 1 A; Mr 8000 +/- 1000; aggregation number 27). This underestimation may be the result of adsorption and/or alteration of the micelles.