Redistribution Of Protein Kinase C Research Articles

Role of PKCb in Signal Transduction for the 1,25D3-MARRS Receptor (ERp57/PDIA3) in Steroid Hormone-Stimulated Calcium Extrusion

Protein kinase C (PKC) is involved in the rapid 1,25(OH) 2 D 3 -mediated extrusion of calcium in chick intestinal epithelial cells. A previous report demonstrated that PKC a and PKC b redistribution corresponded to the dose- response curve for calcium extrusion. We investigated the role of both PKC a and PKC b in hormone-stimulated calcium extrusion by using siRNA in primary cultures of intestinal cells. The results indicated that there was no change in calcium extrusion in cells transfected with siRNA to PKC a , whereas the siRNA to PKC b significantly decreased calcium extrusion in 1,25(OH) 2 D 3 -treated cells when compared with the corresponding hormone-treated cells in transfected and nontransfected cells ( P < 0.01). Similar results were also found using chemical inhibitors of PKC a (safingol) and PKC b ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione]) in intestinal cell suspension. The results demonstrated that PKC b inhibited the extrusion of calcium (resulting in increased calcium uptake) from intestinal cells treated with 1,25(OH) 2 D 3 when compared with vehicle controls ( P < 0.001), the PKC a blocker did not show any difference in calcium extrusion among the treatment groups. Using confocal microscopy, we reduced the hormone exposure to 30 sec in order to view redistribution of PKC a and PKC b . Rapid redistribution of PKC a was found to significantly increase in fluorescence in the apical membrane region after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH) 2 D 3 ( P < 0.01 and 0.001, respectively). By comparison, PKC b immunofluorescence was found to increase significantly in the basal lateral region of the cells, relative to controls after the exposure of cells to 300- ( P < 0.01) or 650 pM ( P < 0.05) seco-steroid. The results suggest that PKC a is the PKC isotype involved in 1,25(OH) 2 D 3 -mediated calcium efflux in intestinal epithelial cells.

Dietary fish oil attenuates cardiac hypertrophy in lipotoxic cardiomyopathy due to systemic carnitine deficiency

1,2-Diacylglycerol (DAG), a lipid second messenger that activates protein kinase C (PKC), is increased with a distinct fatty acid composition in the heart of the juvenile visceral steatosis (JVS) mouse, which develops pathological cardiac hypertrophy with lipid accumulation induced by the perturbation of fatty acid beta-oxidation due to systemic carnitine deficiency. Fish oil (FO) may exert its beneficial effects on the cardiomyopathy in JVS mice by modifying the molecular species composition of myocardial DAG. To test this hypothesis, we investigated the effects of dietary FO on the hypertrophied hearts in JVS mice. Both control and JVS mice were fed a FO diet (containing 10% FO) or a standard diet from 4 weeks of age. At 8 weeks of age, the ventricular-to-body weight ratio in JVS mice was 2.7-fold higher than that in controls (9.9 +/- 0.1 vs. 3.7 +/- 0.1 mg/g, P < 0.01) and was reduced by dietary FO (7.7 +/- 0.1 mg/g, P < 0.01 vs. JVS mice). In JVS mice, myocardial DAG levels were elevated by 2.3-fold with a distinct fatty acid composition with increases in C18:1n-7,9 and C18:2n-6 fatty acids compared with controls; dietary FO had no effects on the total DAG levels but significantly altered the fatty acid composition of DAG with reduction of both fatty acid species. Immunoblot analysis showed that dietary FO prevented the membrane translocation of cardiac PKCs alpha, beta2, and epsilon in JVS mice. Dietary FO did not affect the plasma and myocardial total carnitine levels in JVS mice. Furthermore, dietary FO significantly improved the progressive left ventricular dysfunction and survival rate in JVS mice. Dietary FO may attenuate cardiac hypertrophy with improvements in cardiac function and survival in JVS mice via modification of the molecular species composition of myocardial DAG and the consequent inhibition of PKC redistribution. These results suggest the implication of the molecular species composition of DAG in the pathogenesis of lipotoxic cardiomyopathy due to perturbations of fatty acid beta-oxidation.

Translocation of the Classic Protein Kinase C Isoforms in Porcine Oocytes: Implications of Protein Kinase C Involvement in the Regulation of Nuclear Activity and Cortical Granule Exocytosis

Protein kinase C (PKC) is a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The subcellular localization of classical PKCα, -βI, and -γ in the process of porcine oocyte maturation, fertilization, and parthenogenetic activation and their involvement in cortical granule (CG) exocytosis were investigated. The results of Western blot showed that PKCα, -βI, and -γ were expressed in the oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Confocal microscopy revealed that the three PKC isoforms were concentrated in the GV but evenly distributed in the cytoplasm of MII eggs. PKCα and -γ were translocated to the plasma membrane soon after sperm penetration. cPKCs migrated into the pronucleus in fertilized eggs. Following treatment with a PKC activator, phorbol 12-myristate 13-acetate (PMA), CGs were released and PKCα and -γ were translocated to the membrane. The CG exocytosis and PKC redistribution induced by PMA could be blocked by the PKC inhibitor staurosporine. Parthenogenetic stimulation with ionophore A23187 or electrical pulse also induced cPKC translocation and CG exocytosis. Eggs injected with PKCα isoform-specific antibody failed to undergo CG exocytosis after PMA treatment or fertilization. The results suggest that cPKCs, especially the α-isotype, regulate nuclear function and CG exocytosis in porcine eggs.

Translocation of protein kinase C by halothane in cholinergic cells

Protein kinase C (PKC) is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for volatile anaesthetic actions. However, the effects of these agents on PKC activity are not yet fully understood. Volatile anaesthetics increase intracellular calcium concentration ([Ca 2+] i) in a variety of cells, thus their effects on PKC activity may be indirect due to [Ca 2+] i increase. Alternatively, the anaesthetics could directly stimulate PKC activity. In order to distinguish these two possibilities in intact cells, we used a fully functional green fluorescent protein conjugated PKC βII (GFP-PKC βII) and confocal microscopy to evaluate the dynamic redistribution of PKC in living SN56 cells, a cholinergic cell line, in response to halothane. Halothane induced PKC translocation in SN56 cells transfected with GFP-PKC βII. This effect was not suppressed by dantrolene, a drug that blocks halothane-induced Ca 2+ release from intracellular stores in these cells. These findings indicate that halothane induces PKC translocation in SN56 cells independently of its ability to release calcium from internal stores.

Age-related decreases in lymphocyte protein kinase C activity and translocation are reduced by aerobic fitness.

This study investigated the effects of advancing age and long-term aerobic fitness on lymphocyte protein kinase C (PKC) activity and translocation. Lymphocytes were obtained from young (20-36 years old) and older (61-78 years old) healthy men who were either aerobically conditioned or deconditioned. Both baseline PKC activity and the response of this enzyme to the direct PKC stimulating agent, phorbol 12-myristate, 13-acetate (PMA) or to the mitogen, phytohaemagglutinin (PHA), were measured in partially purified extracts of cytosolic and membranous fractions of lymphocytes. Basal PKC activity, PMA-induced redistribution of PKC, and PHA-induced enhancement of PKC activity were reduced among older subjects in both lymphocyte cytosolic and membranous fractions. However, the magnitudes of these reductions were smaller among the older subjects who were aerobically fit. Lymphocyte PKC activity and translocation may be biological markers of aging, and the maintenance of aerobic fitness into later life may serve to slow the rate at which activation of this enzyme declines during senescence.

Acidosis enhances translocation of protein kinase C but not Ca 2+/calmodulin-dependent protein kinase II to cell membranes during complete cerebral ischemia

Systemic hyperglycemia and hypercapnia severely aggravate ischemic brain damage when instituted prior to cerebral ischemia. An aberrant cell signaling following ischemia has been proposed to be involved in ischemic cell death, affecting protein kinase C (PKC) and the calcium calmodulin kinase II (CaMKII). Using a cardiac arrest model of global brain ischemia of 10 min duration, we investigated the effect of hyperglycemia (20 mM) and hypercapnia (pCO 2 300 mmHg) on the subcellular redistribution of PKC (α, β, γ) and CaMKII to synaptic membranes and to the microsomes, as well as the effect on PKC activity. We confirmed the marked translocation of PKC and CaMKII to cell membranes induced by ischemia, concomitantly with a decrease in the PKC activity in both the membrane fraction and cytosol. Hyperglycemia and hypercapnia markedly enhanced the translocation of PKC-γ to cell membranes while other PKC isoforms were less affected. There was no effect of acidosis on PKC activity, or on translocation of CaMKII to cell membranes. Our data strongly suggest that the enhanced translocation of PKC to cell membranes induced by hyperglycemia and hypercapnia may contribute to the detrimental effect of tissue acidosis on the outcome following ischemia.

An Essential Role for Autophosphorylation in the Dissociation of Activated Protein Kinase C from the Plasma Membrane

The cellular localization of protein kinase C (PKC) is intimately associated with the regulation of its biological activity. Previously we have demonstrated that the redistribution of PKC to the plasma membrane in response to physiological stimuli is followed by a rapid returning of PKC back to the cytoplasm (Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 10755-10762). Although the process of PKC membrane targeting has been extensively studied, the molecular mechanism underlying the dissociation of membrane-bound PKC remains unclear. In the present study, by examining the dynamic distribution of wild-type PKC betaII and its kinase-deficient mutant (K371R), we demonstrate that kinase activity is required for PKC membrane dissociation. Moreover, the inability of PKC betaII(K371R) to dissociate from the plasma membrane in cells overexpressing wild-type PKC betaII suggests that autophosphorylation activity of the kinase might be essential for its membrane dissociation. This was further supported by mutational analysis of two in vivo autophosphorylation sites on PKC betaII. The replacement of Ser660 or Thr641 by alanine (S660A or T641A) was found to synergistically reduce the reversal of PKC betaII membrane translocation, whereas the replacement of the same amino acids by glutamic acid (S660E or T641E), an amino acid commonly used to mimic phosphate, results in mutants behaving similar to wild-type PKC betaII. These findings point to an essential role for autophosphorylation in the dissociation of activated PKC from the plasma membrane and suggest that, like PKC membrane translocation, the returning of PKC to the cytoplasm after its activation is also delicately regulated.

Visualization of Dynamic Trafficking of a Protein Kinase C βII/Green Fluorescent Protein Conjugate Reveals Differences in G Protein-coupled Receptor Activation and Desensitization

Protein kinase C (PKC) links various extracellular signals to intracellular responses and is activated by diverse intracellular factors including diacylglycerol, Ca2+, and arachidonic acid. In this study, using a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII), we demonstrate a novel approach to study the dynamic redistribution of PKC in live cells in response to G protein-coupled receptor activation. Agonist-induced PKC translocation was rapid, transient, and selectively mediated by the activation of Gqalpha- but not Gsalpha- or Gialpha-coupled receptors. Interestingly, although the stimuli were continuously present, only one brief peak of PKC membrane translocation was observed, consistent with rapid desensitization of the signaling pathway. Moreover, when GFP-PKCbetaII was used to examine cross-talk between two Gqalpha-coupled receptors, angiotensin II type 1A receptor (AT1AR) and endothelin A receptor (ETAR), activation of ETARs resulted in a subsequent loss of AT1AR responsiveness, whereas stimulation of AT1ARs did not cause desensitization of the ETAR signaling. The development of GFP-PKCbetaII has allowed not only the real time visualization of the dynamic PKC trafficking in live cells in response to physiological stimuli but has also provided a direct and sensitive means in the assessment of activation and desensitization of receptors implicated in the phospholipase C signaling pathway.

Effects of withdrawal of dopamine on translocation of protein kinase C isozymes and prolactin secretion in rat lactotroph-enriched pituitary cells. Modulation of substance P-mediated responses.

The present study deals with the effects of withdrawal of dopamine (DA) on the translocation of protein kinase C (PKC) isozymes and release of prolactin (Prl) in resting- and substance P (SP)-stimulated cultures of enriched rat pituitary lactotrophs. Following a brief tonic input (10 min), DA withdrawal induced a redistribution of PKC alpha- and beta-immunoreactivity (IR) to the particulate fraction with maximal levels, attained after 5 min, remaining translocated for 20 min. DA withdrawal prolonged the effect of SP-induced translocation of PKC alpha- and beta-IR. Similar effects were detected when the catalytic activity of PKC in response to DA withdrawal was evaluated. Thus, DA washout redistributed PKC catalytic activity and prolonged the effect of SP on catalytical PKC translocation to the particulate fraction. Pretreatment of cells with the protein kinase A inhibitor, rp-adenosine-3',5'-cyclic monophosphothionate (rp-cAMP), reduced the amount of PKC alpha- and beta-IR redistributed after DA withdrawal. Furthermore, this treatment also reduced the DA withdrawal effect on SP-mediated translocation of PKC alpha- and beta-IR. Methoxyverapamil, a blocker of voltage-gated Ca2+ channels, completely inhibited the redistribution of PKC isozymes after DA withdrawal, but also reduced the potentiating effect of DA withdrawal on SP-induced redistribution of PKC isozyme-IR. In perifused enriched lactotrophs, DA withdrawal induced a release of Prl that lasted 45-55 min and prolonged the effect of SP on Prl secretion. rp-cAMP did not significantly affect Prl release due to DA removal, but the prolonging effect of DA withdrawal on SP-induced Prl secretion was abolished. Methoxyverapamil completely abolished the rebound release of Prl after DA withdrawal, and the potentiating effect of DA removal on SP-mediated Prl release was also diminished. Readdition of DA after DA withdrawal was able to suppress the translocation of PKC isozyme-IR and catalytic activity and to reduce the release of Prl to baseline levels. Moreover, readdition of DA reduced the potentiating effects of DA withdrawal on the same parameters after SP-stimulation of cells. On the basis of these results it is concluded that in resting cells following DA withdrawal prolactin is released and specific PKC isozymes and concomitant catalytic activity are translocated to the particulate fraction in enriched lactotrophs. While cAMP/PKA and influx of Ca2+ seem to work in concert in translocating PKC, influx of Ca2+ is the primary mechanism responsible for the rebound release of Prl after DA withdrawal. DA withdrawal exerts a potentiating effect on SP-induced PKC translocation and Prl release. It is suggested that the biochemical events involved in these processes are cAMP/PKA and Ca2+ influx.

THE EFFECT OF PROLONGED IMIPRAMINE TREATMENT ON THE α 1-ADRENOCEPTOR-INDUCED TRANSLOCATION OF PROTEIN KINASE C IN THE CENTRAL NERVOUS SYSTEM IN RATS

The aim of the study was to investigate the effects of prolonged treatment with imipramine (10 mg kg −1/day i.p. for 21 days) on the translocation of protein kinase C (PKC) after stimulation of the α 1-adrenoceptor. Methoxamine (5–50 mg kg −1) and phenylephrine (0.1–1 mg kg −1) induced a rapid and long-term redistribution of PKC from the cytosolic to the membrane fraction in the rat frontal cortex and hippocampus. The effects of methoxamine and phenylephrine were completely blocked by pretreatment with prazosin. Prolonged pretreatment with imipramine changed the response of PKC to methoxamine and phenylephrine. Much lower doses of α 1-adrenoceptor agonists were able to induce the redistribution of PKC. Moreover, prolonged treatment with imipramine markedly increased the basal activity of PKC in the membrane fractions of the frontal cortex and hippocampus.

Addition of phosphatidylcholine-phospholipase C induces cellular redistribution and phosphorylation of protein kinase C ξ in C 6 glial cells

Phosphatidylcholine breakdown has been shown to play a critical role in signal transduction involving generation of a number of second messengers [Exton, J.H., Biochim. Biophys. Acta, 1212 (1994) 26–42]. In the present report we demonstrate by immunofluorescence that short-treatment of C 6 glial cells with phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC), changes the intracellular localization of protein kinase C (PKC) ξ from the cytoplasm to a perinuclear region. Western blot analysis also showed a redistribution of PKC ξ after incubation of cells with PC-PLC. To test whether these changes were accompanied by an activation of the enzyme, we measured the extent of phosphorylation of PKC ξ by immunoprecipitation from 32P-labelled cells. Short-treatment with PC-PLC resulted in enhanced phosphorylation of the higher Mr PKC ξ in C 6 glial cells.

Differential involvement of calcium channels and protein kinase-C activity in GnRH-induced phospholipase-C, -A 2 and -D activation in a gonadotrope cell line (αT3-1)

The mode of action of GnRH on pituitary gonadotropes involves metabolism of phospholipids, protein kinase-C (PKC) and voltage sensitive Ca 2+ channels (VSCC) activation. We have studied the differential role of PKC and VSCC on the coupling of the GnRH receptor with phospholipases-C (PLC), -A 2 (PLA 2) and -D (PLD) activities in a gonadotrope cell line (αT3-1), by measuring the production of inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that in these cells GnRH stimulated through a specific receptor, IPs formation, a rapid and sustained diacylglycerol generation, consequently AA release and a delayed PEt production in a dose-dependent manner. In contrast to GnRH-induced PLC activity, the PLA 2 and PLD stimulation by the neuropeptide involved Ca 2+ mobilization via VSCC activation. BAY-K8644 a VSCC agonist significantly potentiated, while the VSCC antagonist nitrendipine markedly inhibited GnRH-induced AA release and PEt production. TPA, a phorbol ester which induced a rapid and important redistribution of PKC, although unable to elicit PLC or PLA 2 stimulation, specifically provoked PLD activation in a PKC-dependent but Ca 2+-independent manner. The PKC stimulation by TPA significantly inhibited the GnRH-stimulated IPs and AA formation, while it potentiated the GnRH-evoked PEt production. This negative feed-back of PKC on GnRH-induced PLC and PLA 2 activities was reversed when PKC was either down regulated after long TPA treatments or inhibited by the PKC inhibitors, staurosporine or GF109203X. The GnRH-induced PEt formation was markedly diminished in PKC depleted cells or after PKC inhibition. Under such conditions, both agonist and antagonist of VSCC became less effective in modulating the remaining GnRH-evoked PEt formation. These results suggest that PKC, in coordination with Ca 2+, plays a key role in regulating the cross-talk between the multiple phospholipases implicated in the GnRH signal transduction.

Pronounced activation of protein kinase C, ornithine decarboxylase and c- jun proto-oncogene by paraquat-generated active oxygen species in WI-38 human lung cells

Paraquat (methyl viologen, PQ) is a widely used herbicide that produces oxygen-derived free radicals and severely injures human lungs. In this study we examined the effects of PQ on the protein kinase C (PKC), ornithine decarboxylase (ODC) and c- jun oncogene expression in WI-38 human lung cells. Exposure of cells to 25–200 μM PQ resulted in an increase of [3H]phorbol dibutyrate (PDBu) binding and PKC redistribution in a dose-dependent manner. Interestingly, a superoxide dismutase mimic, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol, 2.5 mM) and catalase (400 μg/ml) could significantly reduce the PQ-stimulated increase of phorbol ester binding and particular PKC phosphorylatiog activity, but dimethylsulfoxide (DMSO, 1.5%), an effective ·OH trapping agent, failed to prevent this stimulation. In addition, an endogenous substrate of PKC, 80 kDa protein, was found to be highly phosphorylated in intact WI-38 cells treated with 50 AM PQ. The increase of phosphorylated proteins could be completely or partly abolished by Tempol or catalase, but only the phosphorylation of 80 kDa protein was diminished by protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7). A maximal peak of ODC activity was observed at 6 h of treatment with 50 μM PQ. PQ induced activity was reduced at the following rates, Tempol 85%, DMSO 80% and catalase 45%, but H-7 failed to do so. Furthermore, we found that the level of c- jun mRNA was transiently increased by PQ and the peak appeared at 1 h of treatment. When correlated with the PKC result, Tempol, catalase and H-7 all effectively blocked PQ-elicited c- jun transcript expression, but DMSO only exhibited a weakly inhibitory effect. We therefore propose that superoxide anion (O 2 − and H 2O 2 generated by PQ could activate PKC and lead to induction of c- jun gene expression; on the other hand, O 2 − and ·OH might trigger other kinase pathways to elevate ODC activity. Finally, the sequential expression of c- jun oncogene, and ODC may cooperate to relieve the oxidative damages elicited by PQ.

Arachidonic Acid, but Not Sodium Nitroprusside, Stimulates Presynaptic Protein Kinase C and Phosphorylation of GAP‐43 in Rat Hippocampal Slices and Synaptosomes

Activation of protein kinase C (PKC) and phosphorylation of its presynaptic substrate, the 43-kDa growth-associated protein GAP-43, may contribute to the maintenance of hippocampal long-term potentiation (LTP) by enhancing the probability of neurotransmitter release and/or modifying synaptic morphology. Induction of LTP in rat hippocampal slices by high-frequency stimulation of Schaffer collateral-CA1 synapses significantly increased the PKC-dependent phosphorylation of GAP-43, as assessed by quantitative immunoblotting with a monoclonal antibody that recognizes an epitope that is specifically phosphorylated by PKC. The stimulatory effect of high-frequency stimulation on levels of immunoreactive phosphorylated GAP-43 was not observed when 4-amino-5-phosphonovalerate (50 microM), an N-methyl-D-aspartate (NMDA) receptor antagonist, was bath-applied during the high-frequency stimulus. This observation supports the hypothesis that a retrograde messenger is produced postsynaptically following NMDA receptor activation and diffuses to the presynaptic terminal to activate PKC. Two retrograde messenger candidates--arachidonic acid and nitric oxide (sodium nitroprusside was used to generate nitric oxide)--were examined for their effects in hippocampal slices on PKC redistribution from cytosol to membrane as an indirect measure of enzyme activation and PKC-specific GAP-43 phosphorylation. Bath application of arachidonic acid, but not sodium nitroprusside, at concentrations that produce synaptic potentiation (100 microM and 1 mM, respectively) significantly increased translocation of PKC immunoreactivity from cytosol to membrane as well as levels of immunoreactive, phosphorylated GAP-43. The stimulatory effect of arachidonic acid on GAP-43 phosphorylation was also observed in hippocampal synaptosomes. These results indicate that arachidonic acid may contribute to LTP maintenance by activation of presynaptic PKC and phosphorylation of GAP-43 substrate. The data also suggest that nitric oxide does not activate this signal transduction system and, by inference, activates a distinct biochemical pathway.

Insulin action on cardiac glucose transport: studies on the role of protein kinase C

Isolated ventricular cardiomyocytes from adult rat have been used to elucidate a possible relationship between protein kinase C (PKC) and the stimulatory action of insulin on cardiac glucose transport. Cells were incubated in the presence of either insulin or phospholipase C from Clostridium perfringens (PLC-Cp) and intracellular sn-1,2-diacylglycerol (DAG) levels and initial rates of 3- O-methylglucose transport were determined. Insulin had no effect on the DAG mass level, whereas it was elevated by PLC-Cp to 200% of control. Under these conditions the hormone produced a 2.7-fold stimulation of glucose transport with no significant effect of PLC-Cp. Insulin was unable to produce a redistribution of PKC, whereas phorbol 12-myristate 13-acetate (PMA) increased membrane associated PKC twofold. The PKC inhibitors tamoxifen and staurosporine did not interfere with glucose transport stimulation by insulin. Furthermore, cells treated with PMA exhibited unaltered basal and maximally insulin stimulated rates of glucose transport. In contrast, at physiological concentrations of insulin the stimulatory action of the hormone was significantly reduced. We conclude from our data that PKC is not involved in insulin action on cardiac glucose transport. However, activation of this enzyme may lead to a modified insulin sensitivity of the cardiac cell.

Human endothelial cells are targets for platelet-activating factor (PAF). Activation of alpha and beta protein kinase C isozymes in endothelial cells stimulated by PAF.

We evaluated the role of the protein kinase C (PKC) and its isozymes in the activation of human endothelial cells (EC) stimulated by platelet-activating factor (PAF). Exposure of confluent EC to PAF resulted in a rapid and concentration-dependent redistribution of PKC from cytosol to plasma-membrane, rearrangement of cytoskeleton (i.e. decrease in F-actin content and redistribution of vinculin), and finally increase in the transendothelial flux of 125I-albumin. Stimulation of EC with oleylacetylglycerol or phorbol 12-myristate 13-acetate induced the modification of the cytoskeletal structures and the increase of 125I-albumin clearance. Inhibitors of PKC prevented the effects induced by PAF on the cytoskeleton and on the barrier function of the EC monolayer. Confluent EC expressed only alpha, beta, and epsilon PKC isoforms. Biochemical and immunochemical analysis showed that the time course of the PKC isozymes translocation from cytosol to the membrane fraction of EC stimulated by PAF was different: beta isoform was redistributed more quickly than alpha isoform. PAF did not induce translocation of PKC epsilon. These results suggest that activation of PKC alpha and beta is an important signal transduction pathway by which PAF activates endothelial monolayer and modify its function of barrier to macromolecules.

Time Course of the Translocation and Inhibition of Protein kinase C During Complete Cerebral Ischemia in the Rat

The time course for the ischemia-induced changes in the subcellular distribution of protein kinase C (PKC) (alpha), (beta II), and (gamma) and the activity of PKC were studied in the neocortex of rats subjected to 1, 2, 3, 5, 10, and 15 min of global cerebral ischemia. In the particulate fraction, a 14-fold increase in PKC (gamma) levels was seen at 3 min of ischemia, which further increased at 5-15 min of ischemia. At 15 min of ischemia, PKC (alpha) and (beta II) levels had increased two- and six-fold, respectively. In the cytosolic fraction, a transient early 1.4-fold increase in PKC (beta II) and PKC (gamma) levels was seen, whereas no change in the levels PKC (alpha) was noted. PKC (gamma) levels then progressively declined, reaching 50% at 15 min of ischemia. At 5 min of ischemia, a 43% decrease in PKC activity was seen in the particulate fraction, reaching 50% at 15 min of ischemia concomitant with a 27% decrease in the cytosolic fraction. There was no change in the activator-independent PKC activity. Pretreatment with the ganglioside AGF2 prevented the redistribution of PKC (gamma) in the particulate fraction at 5 min, but not at 10 min of ischemia. The observed time course for the translocation of PKC (gamma) parallels the ischemia-induced release of neurotransmitters and increased levels of diacylglycerols, arachidonate, and increased levels of diacylglycerols, arachidonate, and intracellular calcium and delineates this subspecies as especially ischemia-sensitive. Ganglioside pretreatment delayed the translocation of PKC (gamma), possibly by counter-acting the effects of ischemia-induced factors that favor PKC binding to cell membranes.

Nonphorbol tumor promoters okadaic acid and calyculin-A induce membrane translocation of protein kinase C

The cell-permeable inhibitors of type 1 and 2A protein phosphatases, okadaic acid and calyculin-A, induced a redistribution of protein kinase C (PKC) activity and immunoreactivity (40 to 60%) from cytosol to membrane in some cell types. Calyculin-A was 100-fold more potent than okadaic acid and required only 5 to 10 nM concentrations to induce this PKC translocation. The concentration of these agents required to induce the redistribution of PKC correlated with the potency of these agents to inhibit both type 1 and 2A protein phosphatases. There was a lag period of 15 to 30 min before the onset of PKC translocation, as this process might have been induced by indirect cellular events triggered by inhibitions of protein phosphatases (1 and 2A). Taken together these results suggest that although the okadaic acid class of tumor promoters and phorbol ester-related agents bind to two different cellular receptors having counteracting enzymic activities, they share a common mechanism of action, namely the induction of cytosol to membrane translocation of PKC.

Translocation of protein kinase C in bovine tracheal smooth muscle strips: the effect of methacholine and isoprenaline

Investigations into the mechanisms involved in the contraction of smooth muscle have suggested that the generation of diacylglycerol and the activation of protein kinase C (PKC) may be important in the generation or maintenance of smooth muscle tone. The present study examined the possible role of PKC in the contraction of bovine tracheal smooth muscle. Methacholine (10 μM) induced a rapid elevation in PKC activity associated with the membrane fraction. PKC levels were significantly elevated in the membrane fraction 30 s after agonists addition, reached a maximum at 1 min and then declined to and remained at a lower level which was still elevated above basal. A concomitant decrease in cytosolic PKC activity of smaller magnitude was observed during this period of stimulation. This methacholine-induced re-distribution of PKC from the cytosol to the membrane was concentration-dependent and was blocked by atropine. Pre-treatment of tissues for 2 min with 100 μM isoprenaline prevented both the re-distribution of PKC and the contraction produced by 1 μM methacholine. Addition of 1 μM isoprenaline to tissues pre-contracted with 1 μM methacholine reversed the re-distribution of PKC produced during contraction and completely relaxed the tissues. Thus, under these conditions, translocation of PKC from the cytosol to the membrane seems to be well correlated with contractions of bovine tracheal smooth muscle. Whether the PKC translocation is responsible for the observed changes in muscle tone or whether the enzyme translocation is a result of drug-induced changes in [Ca 2+] i remains to be determined.

Modulation by staurosporine of phorbol-ester-induced effects on growth and protein kinase C localization in A549 human lung-carcinoma cells.

12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin 1 are activators of protein kinase C (PKC). TPA is a potent inhibitor of the growth of A549 cells, while bryostatin 1 exerts a weak antiproliferative effect upon this cell line. We tested the hypothesis that the PKC inhibitor staurosporine (STAU) can interfere with the effects of TPA or bryostatin 1 on A549 cells. STAU alone arrested A549 cell growth effectively with an IC50 of 0.65 nM as determined by cell counting after incubation for 96 hr. It also caused the release of lactate dehydrogenase from cells with an IC50 of 18.4 nM. On incubation with cells for up to 8 hr, STAU (100 nM) alone did not reduce thymidine incorporation into cells. However, it partially abrogated the inhibition of DNA synthesis caused by TPA or bryostatin 1 (10 nM). The IC50 for inhibition by STAU of the activity of PKC purified from A549 cells was 6.1 nM. Localization and levels of PKC were studied by Western blot and phorbol ester receptor binding analyses. STAU (100 nM) did not prevent the TPA-induced rapid redistribution of PKC to the cell membrane, but instead increased it by 25%. The PKC downregulation caused by TPA was not reduced in the presence of STAU. The results suggest that (i) PKC activation is involved in growth inhibition caused by TPA or bryostatin 1 in A549 cells, and (ii) subcellular localization or levels of PKC can be pharmacologically manipulated even under conditions of inhibited kinase function.