Independent Protein Kinase C Isoforms Research Articles

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors

Activation of Gq protein-coupled receptors (GqPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the Gq signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of Gq We quantified signaling events downstream of GqPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (IKs) were measured as a functional readout of receptor-specific activation. Activation of muscarinic M1 receptors (M1-Rs) caused robust and reversible inhibition of IKs. In contrast, activation of α1B-adrenergic receptors (α1B-ARs) induced transient inhibition of IKs, which turned into delayed facilitation after agonist withdrawal. As a novel finding, we demonstrate that GqPCR-specific kinetics of IKs modulation are determined by receptor-specific desensitization, evident at the level of Gαq activation, phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, and diacylglycerol production. Sustained IKs inhibition during M1-R stimulation is attributed to robust membrane PIP2 depletion, whereas the rapid desensitization of α1B-AR delimits PIP2 reduction and augments current activation by protein kinase C (PKC). Overexpression of Ca2+-independent PKCδ did not affect the time course of α1B-AR-induced diacylglycerol formation, excluding a contribution of PKCδ to α1B-AR desensitization. Pharmacological inhibition of Ca2+-dependent PKC isoforms abolished fast α1B receptor desensitization and augmented IKs reduction, but did not affect IKs facilitation. These data indicate a contribution of Ca2+-dependent PKCs to α1B-AR desensitization, whereas IKs facilitation is induced by Ca2+-independent PKC isoforms. In contrast, neither inhibition of Ca2+-dependent/Ca2+-independent isoforms nor overexpression of PKCδ induced M1 receptor desensitization, excluding a contribution of PKC to M1-R-induced IKs modulation.

Protein Kinase C Modulates Aurora-kinase Inhibition Induced by CCT129202 in HMC-1560,816 Cell Line

The human mast cell line HMC-1⁵⁶⁰,⁸¹⁶ carries activating mutations in the proto-oncogene of c-kit that cause autophosphorylation and permanent c-kit receptor activation. The compound CCT129202 is a new and selective inhibitor of Aurora kinase A and B that decreases the viability of a variety of human tumor cell lines. The effect of Aurora kinase inhibition was assessed in the HMC-1⁵⁶⁰,⁸¹⁶ line in order to find a suitable tool for mastocytosis treatment. CCT129202 treatment induces a significant decrease in cell viability in HMC-1⁵⁶⁰,⁸¹⁶ cells after 48 hours of treatment. Moreover, caspase-3 and caspase-8 activation was induced after incubation of HMC-1⁵⁶⁰,⁸¹⁶ cells in the presence of CCT129202. It has been demonstrated that Protein Kinase C (PKC) plays a crucial role in mast cell activation as well as cell migration, adhesion and apoptotic cell death. Co-treatment of Ca²⁺-independent PKCs (δ ε and θ) inhibitor GF109203X with CCT129202, reduces caspase-3 activation which controls cell levels. In contrast, Go6976, an inhibitor of Ca²⁺-dependent PKCs, increases caspase-3 activation. Oppositely, GF109203X does not modify CCT129202-induced apoptosis through the caspase-8 pathway whereas Go6976 treatment abolishes the increase on caspase-8 activity due to CCT129202. This implies that Ca²⁺-independent PKC isoforms seems to be related with CCT129202-induced apoptosis through the caspase- 3 pathway, whereas Ca²⁺-dependent PKC isoforms are related with the CCT129202 effect on the caspase-8 pathway. Interestingly, CCT129202 cytotoxic effect remains even though Ca²⁺-dependent PKCs are inhibited, which shows that the Aurora kinase inhibitor effect is acting through the caspase-3 pathway. On the other hand, Ca²⁺-independent PKCs inhibition does not affect the final apoptotic CCT129202 effect because this seems to be mediated by the caspase-8 pathway. Moreover, CCT129202 does not affect PKCδ and Ca²⁺-dependent PKC translocation, which indicates that PKC translocation pivots on its activation. This demonstrates that Aurora kinase inhibition is not related to this process. Finally, when PKC is silenced in HMC-1⁵⁶⁰,⁸¹⁶ cells, the effect of CCT129202 on the caspase-3 pathway disappears, which indicates that the CCT129202 effect is clearly PKC-dependent.

Protein Kinase C Inhibition Ameliorates Posttransplantation Preservation Injury in Rat Renal Transplants

Prolonged cold preservation frequently causes delayed renal graft function resulting from tubular epithelial injury. Inhibition of signal transduction downstream from protein kinase C (PKC) may reduce renal ischemia-reperfusion injury and confer renal graft protection. We therefore evaluated the effect of sotrastaurin, a small-molecule inhibitor of Ca²⁺-dependent and Ca²⁺-independent PKC isoforms, in comparison with mycophenolic acid (MPA) on rat renal transplants with prolonged cold preservation. Donor kidneys from male Lewis rats were cold stored in University of Wisconsin solution for 24 hr before syngeneic grafting. Recipients received sotrastaurin (30 mg/kg twice daily), MPA (20 mg/kg/day), or vehicle through gavage starting 1 hr after surgery. Renal function was evaluated by serum creatinine and histology on day 2 (acute injury) and day 7 (repair phase) after transplantation. Postreperfusion inflammation was determined by real-time polymerase chain reaction of proinflammatory genes and histology. Signaling mechanisms were studied by Western blotting and immunohistochemistry. Sotrastaurin enhanced immediate transplant function, attenuated epithelial injury, and accelerated renal function recovery compared with MPA. Despite the stronger anti-inflammatory capacity of MPA, only sotrastaurin treatment achieved significant cellular protection with persisting reduced apoptosis of tubular epithelial cells. Decreased phosphorylation of extracellular signal-regulated protein kinase and p66Shc adaptor protein, both involved in cellular stress and apoptosis, were likely the responsible mechanism of action. The PKC inhibitor sotrastaurin effectively ameliorated ischemia-reperfusion organ damage and promoted cytoprotection in a clinically relevant model of extended renal cold preservation followed by transplantation. Pharmacologic targeting of PKC may be beneficial for recipients receiving renal transplants at risk for delayed graft function.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Minocycline Down-regulates MHC II Expression in Microglia and Macrophages through Inhibition of IRF-1 and Protein Kinase C (PKC)α/βII

Minocycline Down-regulates MHC II Expression in Microglia and Macrophages through Inhibition of IRF-1 and Protein Kinase C (PKC)α/βII

Modulation of protein kinase C by curcumin; inhibition and activation switched by calcium ions

Previous studies have identified the natural polyphenol curcumin as a protein kinase C (PKC) inhibitor. In contrast, we found significant stimulation of PKC activity following curcumin treatment. Thus, the mechanism of curcumin interaction with PKC was investigated. We employed phosphorylation assays in the presence of soluble or membrane-bound PKC substrates, followed by SDS-PAGE, autoradiography and phosphorylation intensity measurements. Curcumin inhibited PKC in the absence of membranes whereas stimulation was observed in the presence of membranes. Further analysis indicated that curcumin decreased PKC activity by competition with Ca(2+) stimulation of the kinase, resulting in inhibition of activity at lower Ca(2+) concentrations and stimulation at higher Ca(2+) concentrations. The role of the membrane is likely to be facilitation of Ca(2+)-binding to the kinase, thus relieving the curcumin inhibition observed at limited Ca(2+) concentrations. Curcumin was found to mildly stimulate the catalytic subunit of PKC, which does not require Ca(2+) for activation. In addition, studies on Ca(2+)-independent PKC isoforms as well as another curcumin target (the sarcoplasmic reticulum Ca(2+)-ATPase) confirmed a correlation between Ca(2+) concentration and the curcumin effects. Curcumin competes with Ca(2+) for the regulatory domain of PKC, resulting in a Ca(2+)-dependent dual effect on the kinase. We propose that curcumin interacts with the Ca(2+)-binding domains in target proteins. To our knowledge, this is the first study that defines an interaction domain for curcumin, and provides a rationale for the broad specificity of this polyphenol as a chemopreventive drug.

Opposing effects of phorbol‐12‐myristate‐13‐acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

The 'cross-talk' between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP-dependent signaling by structurally related human D1-like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, followed by analysis of dopamine-mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist-evoked D1 receptor signaling, whereas constitutive and dopamine-mediated D5 receptor activation were rapidly blunted. RT-PCR and immunoblotting analyses showed that phorbol ester-regulated PKC isozymes (conventional: alpha, betaI, betaII, gamma; novel: delta, epsilon, eta, theta) and protein kinase D (PKCmicro) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca2+-independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross-talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1-like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.

Inhibition of Ca2+-dependent PKC isoforms unmasks ERK-dependent hypertrophic growth evoked by phenylephrine in adult ventricular cardiomyocytes.

The duration of extracellular signal-regulated kinase (ERK) activation and the ERK-dependency of hypertrophic growth differ between stimulation of alpha-adrenoceptors or angiotensin II receptors. As both receptor systems activate different protein kinase C (PKC) isoforms, we hypothesized that PKC isoforms contribute to the specific effect of alpha-adrenoceptor stimulation. Isolated adult ventricular cardiomyocytes from rats were used. Different PKC isoforms were inhibited either pharmacologically by six different PKC inhibitors or specifically downregulated by antisense oligonucleotides. ERK activation was determined by phosphorylation relative to total ERK. The rate of protein synthesis was determined by 14C-phenylalanine incorporation. The hypertrophic response of phenylephrine was inhibited in a concentration-dependent fashion by three different inhibitors of Ca2+-independent PKC isoforms (Gö6983, rottlerin, Gö6850), but not by three distinct PKC inhibitors directed preferentially against Ca2+-dependent PKC isoforms (Ro32-0432, HBDDE, Gö6976). Antisense oligonucleotides directed against PKC-alpha, -delta, or -epsilon downregulated their specific isoforms. Their corresponding sense oligonucleotides did not affect PKC isoform expression. The phenylephrine-induced increase in protein synthesis was blocked by antisense oligonucleotides directed against PKC-delta or PKC-epsilon but not PKC-alpha, confirming the pharmacological experiments. Inhibition of Ca2+-dependent PKC isoforms by HBDDE or Gö6976 converted a transient activation of ERK by phenylephrine into a sustained response. Under these conditions, phenylephrine increased protein synthesis in an ERK-dependent way. Inhibition of Ca2+-dependent PKC isoforms converts the ERK-independent effect of phenylephrine on protein synthesis into an ERK-dependent induction of protein synthesis. We conclude that co-activation of Ca2+-dependent PKC isoforms by phenylephrine contributes to the specific effect on adult ventricular cardiomyocytes from rat.

Ca2+-independent protein kinase C signalling in mouse eggs during the early phases of fertilization.

Protein kinase C (PKC), an enzyme playing a central role in signal transduction pathways, is activated in fertilized mouse eggs downstream of the fertilization Ca2+ signal, to regulate different aspects of egg activation. Given the presence of Ca2+-independent PKC isoforms within the egg, we investigated whether fertilization triggers PKC stimulation in mouse eggs by activating Ca2+-independent signalling pathways. An increase in PKC activity was detected as early as 10 min after the beginning of insemination, when about 90% of eggs had fused with sperm and the first Ca2+ rise was evident in most of the eggs. A similar level of activity was found 20 min later, when about 60% of eggs had resumed meiosis. When the Ca2+ increase was buffered by an intracellular Ca2+ chelating agent, PKC stimulation was not blocked but only slightly reduced. Confocal microscopy analysis revealed that the increase in PKC activity at fertilization coincided with the translocation of PKCdelta, a Ca2+-independent and diacylglycerol-dependent PKC isoform, to the meiotic spindle. When, in the absence of the Ca2+ signal, metaphase-anaphase transition was inhibited, PKCdelta moved to the meiotic spindle but still maintained a sustained cytoplasmic distribution. In summary, our results indicate that: 1) PKC activation is an early event of egg activation; 2) both Ca2+-dependent and Ca2+-independent pathways contribute to increased PKC activity at fertilization; 3) PKCdelta is one of the isoforms participating in this signalling process.

NF-kappaB is involved in regulation of CD40 ligand expression on Mycobacterium bovis bacillus Calmette-Guérin-activated human T cells.

Interaction between CD40L (CD154) on activated T cells and its receptor CD40 on antigen-presenting cells has been reported to be important in the resolution of infection by mycobacteria. However, the mechanism(s) by which Mycobacterium bovis bacillus Calmette-Guérin (BCG) up-regulates membrane expression of CD40L molecules is poorly understood. This study was done to investigate the role of the nuclear factor kappaB (NF-kappaB) signaling pathway in the regulation of CD40L expression in human CD4(+) T cells stimulated with BCG. Specific pharmacologic inhibition of the NF-kappaB pathway revealed that this signaling cascade was required in the regulation of CD40L expression on the surface of BCG-activated CD4(+) T cells. These results were further supported by the fact that treatment of BCG-activated CD4(+) T cells with these pharmacological inhibitors significantly down-regulated CD40L mRNA. In this study, inhibitor kappaBalpha (IkappaBalpha) and IkappaBbeta protein production was not affected by the chemical protease inhibitors and, more importantly, BCG led to the rapid but transient induction of NF-kappaB activity. Our results also indicated that CD40L expression on BCG-activated CD4(+) T cells resulted from transcriptional up-regulation of the CD40L gene by a mechanism which is independent of de novo protein synthesis. Interestingly, BCG-induced activation of NF-kappaB and the increased CD40L cell surface expression were blocked by the protein kinase C (PKC) inhibitors 1-[5-isoquinolinesulfonyl]-2-methylpiperazine and salicylate, both of which block phosphorylation of IkappaB. Moreover, rottlerin a Ca(2+)-independent PKC isoform inhibitor, significantly down-regulated CD40L mRNA in BCG-activated CD4(+) T cells. These data strongly suggest that CD40L expression by BCG-activated CD4(+) T cells is regulated via the PKC pathway and by NF-kappaB DNA binding activity.

Nitric Oxide Induces Degradation of the Neutral Ceramidase in Rat Renal Mesangial Cells and Is Counterregulated by Protein Kinase C

Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. In this study we show that the NO-triggered inhibition of neutral ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin II and ATP, resulting in a normalization of neutral ceramidase protein as well as activity. In vivo phosphorylation studies using (32)P(i)-labeled mesangial cells revealed that TPA, PDGF, angiotensin II, and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca(2+)-dependent isoforms, thus suggesting the involvement of a Ca(2+)-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-delta isoform and to a lesser extent the PKC-alpha isoform are efficient in directly phosphorylating neutral ceramidase. In summary, our data show that NO is able to induce degradation of neutral ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-delta isoenzyme, which can directly phosphorylate and thereby prevent neutral ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral ceramidase stability and subsequent ceramide accumulation.

Signalling components involved in the coupling of alpha 1-adrenoceptors to phospholipase D in neonatal rat cardiac myocytes.

Activation of phospholipase D (PLD) is assumed to be one major pathway by which alpha(1)-adrenoceptors (alpha(1)ARs) induce hypertrophic responses in cardiac myocytes. Heterotrimeric G proteins, protein kinase C (PKC) isoforms, protein tyrosine kinases, monomeric GTPases of the ADP-ribosylation factor (ARF) and Rho families, and as important cofactor phosphatidylinositol 4,5-bisphosphate (PIP(2)) seem to participate in the G protein-coupled receptor dependent regulation of PLD. We therefore studied the role of these components in the coupling of alpha(1)ARs to PLD in neonatal rat cardiac myocytes (NRCM). Stimulation of alpha(1)ARs, most likely of the alpha(1A) subtype, by noradrenaline increased PLD activity three- to fourfold concomitant with the stimulation of phospholipase C (PLC). In contrast, the partial agonist phenylephrine stimulated PLC, but failed to increase PLD activity. The PLC and PLD responses were pertussis toxin insensitive and treatment of the cells with the G(q)-activating toxin of Pasteurella multocida stimulated both phospholipases about fourfold. Over-expression of the G(q)-and G(i)-type-specific regulator of G protein signalling RGS4 blunted alpha(1)AR-induced PLC and PLD stimulation. Ro 31-8220, known to inhibit Ca(2+)-dependent and -independent PKC isoforms, strongly inhibited PLD activity, whereas Gö 6976, known to inhibit preferentially Ca(2+)-dependent PKC isozymes, was without effect. The ARF signalling inhibitor brefeldin A, protein tyrosine kinase inhibitors and the Rho-inactivating toxin B of Clostridium difficile blunted alpha(1)AR-induced PLD stimulation and largely reduced the cellular PIP(2) content. In membranes of toxin B-treated NCRM, PLD activity was similarly reduced, but was fully restored by addition of exogenous PIP(2). We conclude, that alpha(1A)ARs stimulate PLD activity via a G(q/11)-PLCbeta-novel PKC isoform-dependent pathway in NRCM. ARF and Rho GTPases as well as protein tyrosine kinases contribute to PLD stimulation in NRCM, most likely by regulating the supply of PIP(2).

The activation of the neutrophil respiratory burst by anti-neutrophil cytoplasm autoantibody (ANCA) from patients with systemic vasculitis requires tyrosine kinases and protein kinase C activation.

The ability of antineutrophil cytoplasm autoantibodies (ANCA) from patients with systemic vasculitis to stimulate protein kinase C (PKC) and tyrosine kinases was examined in human neutrophils. Using the superoxide dismutase-inhibitable reduction of ferricytochrome C, the kinetics of ANCA-induced superoxide (O2-) production were characterized and subsequently manipulated by specific inhibitors of PKC and tyrosine kinases. With this approach, ANCA IgG, but not normal IgG or ANCA F(ab')2 fragments caused a time and dose dependent release of O2- from TNF-alpha primed neutrophils. The kinetics of ANCA-induced O2- production showed an initial 10-15 min lag phase compared to the N-formyl-L-methionyl-L-leucyl-L-phenylalanine response, suggesting differences in the signalling pathways recruited by these two stimuli. Inhibitor studies revealed that ANCA-activation involved members of both the Ca2+-dependent and -independent PKC isoforms and also tyrosine kinases. ANCA IgG resulted in the translocation of the betaII isoform of PKC at a time corresponding to the end of the lag phase of O2- production, suggesting that PKC activity may be instrumental in processes regulating the activity of the NADPH oxidase in response to ANCA. Tyrosine phosphorylation of numerous proteins also peaked 10-15 min after stimulation with ANCA but not normal IgG. These data suggest that PKC and tyrosine kinases regulate O2- production from neutrophils stimulated with autoantibodies from patients with systemic vasculitis.

Protein kinase C isoform expression and activity in the mouse heart.

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.

Changes in Ca 2+-dependent protein kinase C isoforms induced by chronic ethanol treatment in mice

Protein kinase C (PKC) has been shown to regulate ethanol sensitivity. The goal of the present study was to ascertain whether chronic in vivo ethanol treatment could affect PKC isoforms in the mouse brain. We measured the protein level of membrane-bound PKC isoforms following chronic ethanol treatment using Western blotting. The protein level of membrane-bound PKCα and PKCγ isoforms, which are defined as Ca 2+-dependent PKC isoforms (cPKC), in the limbic forebrain during chronic ethanol treatment was significantly increased, whereas the levels of both were significantly decreased in the frontal cortex. By contrast, there was no change in PKCε, a Ca 2+-independent PKC isoform, in both areas. These findings suggest that the change in membrane-bound cPKC in the limbic forebrain and frontal cortex may play substantial roles for the development of ethanol dependence.

Oxidized-LDL enhances coronary vasoconstriction by increasing the activity of protein kinase C isoforms alpha and epsilon.

Oxidized low-density lipoprotein (ox-LDL) plays a critical role in the development of atherosclerotic coronary vasospasm; however, the cellular mechanisms involved are not fully understood. We tested the hypothesis that ox-LDL enhances coronary vasoconstriction by increasing the activity of specific protein kinase C (PKC) isoforms in coronary smooth muscle. Active stress was measured in de-endothelialized porcine coronary artery strips; cell contraction and [Ca(2+)](i) were monitored in single coronary smooth muscle cells loaded with fura-2; and the cytosolic and particulate fractions were examined for PKC activity and reactivity with isoform-specific anti-PKC antibodies with Western blots. Ox-LDL (100 microgram/mL) caused slow but significant increases in active stress to 1.3+/-0.4x10(3) N/m(2) and cell contraction (10%) that were completely inhibited by GF109203X (10(-6) mol/L), an inhibitor of Ca(2+)-dependent and -independent PKC isoforms, with no significant change in [Ca(2+)](i). 5-Hydroxytryptamine (5-HT; 10(-7) mol/L) and KCl (24 mmol/L) caused increases in cell contraction and [Ca(2+)](i) that were inhibited by the Ca(2+) channel blocker verapamil (10(-6) mol/L). Ox-LDL enhanced coronary contraction to 5-HT and KCl with no additional increases in [Ca(2+)](i). Direct activation of PKC by phorbol 12-myristate13-acetate (PMA; 10(-7) mol/L) caused a contraction similar in magnitude and time course to ox-LDL-induced contraction and enhanced 5-HT- and KCl-induced contraction with no additional increases in [Ca(2+)](i). The ox-LDL-induced enhancement of 5-HT and KCl contraction was inhibited by Gö6976 (10(-6) mol/L), an inhibitor of Ca(2+)-dependent PKC isoforms. Both ox-LDL and PMA caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and an increase in the particulate/cytosolic PKC activity ratio. Western blots revealed the Ca(2+)-dependent PKC-alpha and the Ca(2+)-independent PKC-delta, -epsilon, and -zeta isoforms. In unstimulated tissues, PKC-alpha- and -epsilon were mainly cytosolic, PKC-delta was mainly in the particulate fraction, and PKC-zeta was equally distributed in the cytosolic and particulate fractions. Ox-LDL alone or PMA alone caused translocation of PKC-epsilon from the cytosolic to particulate fraction, whereas the distribution pattern of PKC-alpha, -delta, and -zeta remained unchanged. 5-HT (10(-7) mol/L) alone and KCl alone did not change PKC activity. In tissues pretreated with ox-LDL or PMA, 5-HT and KCl caused additional increases in PKC-alpha activity. Native LDL did not significantly affect coronary contraction, [Ca(2+)](i), or PKC activity. These results suggest that ox-LDL causes coronary contraction via activation of the Ca(2+)-independent PKC-epsilon and enhances the contraction to [Ca(2+)](i)-increasing agonists by activating the Ca(2+)-dependent PKC-alpha. Activation of PKC-alpha and -epsilon may represent a possible cellular mechanism by which ox-LDL could enhance coronary vasospasm.

The calcium-independent protein kinase C participates in an early process of CD3/CD28-mediated induction of thymocyte apoptosis.

Thymocyte negative selection eliminates self-reactive clones and involves both a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. Anti-CD3/anti-CD28-triggered apoptosis in isolated CD4+CD8+ thymocytes in vitro provides a basic model for negative selection. Effects of isoform-selective and non-isoform-selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca2+-independent PKC isoforms during the first 2-3 hr of culture is essential for inducing apoptosis, and that Ca2+-dependent PKC isoforms may be influential, but not essential, for apoptosis. To assess the CD3/CD28-mediated activation of PKC in the apoptotic process, we prepared CD4+CD8+ thymocytes (without contamination with cells that had received negative or positive selection signals in vivo) by establishing TCR-transgenic mice with RAG-2-deficient and non-selecting major histocompatibility complex (MHC) backgrounds, in addition to a CD4+CD8+ thymocyte-enriched population from normal mice. Translocation of Ca2+-independent PKC from the cytosolic fraction to the particulate fraction of CD4+CD8+ thymocytes was induced by CD3/CD28-mediated stimulation, but not by CD3- or CD28-mediated stimulation alone, and peaked 2 hr after the start of culture. The kinase activity of the translocated Ca2+-independent PKC was dependent on cofactors in vitro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolytic PKC fragment, was responsible for the activity. Immunoblotting analysis indicated that the nPKC-theta isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC-alpha isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the theta isoform) in CD4+CD8+ thymocytes is involved in a pathway for negative selection.

Reversible protein kinase C activation in PC12 cells: effect of NGF treatment.

Although protein kinase C (PKC) is a key enzyme in the signal transduction process, there is little information on the mechanism leading to PKC activation in living cells. Using a new fluorescence imaging method, we studied this mechanism and correlated PKC conformational changes with intracellular Ca2+ concentration. PC12 cells were simultaneously loaded with Fura-2-AM and Fim-1, two fluorescent probes, which recognize Ca2+ and PKC, respectively. KCl and carbachol (an agonist to muscarinic receptors) applications induced dose-dependent increases of fluorescence for both probes. Both Ca2+ and PKC responses were observed within seconds following KCl or carbachol application, and were reversible upon stimulus withdrawal. PKC activation kinetics was slightly more rapid than the Ca2+ response after KCl application. After nerve growth factor (NGF) treatment of the cells, the amplitude of the KCl-induced PKC responses was larger indicating an increase in the activated PKC-pool in these cells. This difference between control and NGF-treated cells was not observed following carbachol application, suggesting the involvement of different PKC pools. While the Ca2+ response uniformly occurred in the cytosol, the PKC response displayed a patch pattern with higher intensities in the peripheral zone near the plasma membrane. This heterogeneous distribution of PKC activation sites was similar to the immunocytological localization of Ca2+-dependent and independent PKC isoforms, which suggested that at least several PKC isoforms interacted with intracellular elements. Upon repeated stimulation, the PKC response rapidly desensitized.

Protein Kinase Cθ Cooperates with Calcineurin to Induce Fas Ligand Expression During Activation-Induced T Cell Death

Abstract Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKCθ, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKCθ mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKCθ mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKCθ synergized with calcineurin to provide a potent stimulus for FasL promoter activation. Full activation of the promoter required its binding sites for the transcription factors NF-AT, AP-1, and NF-κB. The biological significance of these findings is implicated by the finding that rottlerin, a selective PKCθ inhibitor, blocked FasL induction by anti-CD3 or PMA plus ionomycin stimulation and, consequently, protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from activation-induced cell death.

Mitogenic Signaling by ATP/P2Y Purinergic Receptors in Astrocytes: Involvement of a Calcium-Independent Protein Kinase C, Extracellular Signal-Regulated Protein Kinase Pathway Distinct from the Phosphatidylinositol-Specific Phospholipase C/Calcium Pathway

Activation of ATP/P2Y purinergic receptors stimulates proliferation of astrocytes, but the mitogenic signaling pathway linked to these G-protein-coupled receptors is unknown. We have investigated the role of extracellular signal-regulated protein kinase (ERK) in P2Y receptor-stimulated mitogenic signaling as well as the pathway that couples P2Y receptors to ERK. Downregulation of protein kinase C (PKC) in primary cultures of rat cerebral cortical astrocytes greatly reduced the ability of extracellular ATP to stimulate ERK. Because occupancy of P2Y receptors also leads to inositol phosphate formation, calcium mobilization, and PKC activation, we explored the possibility that signaling from P2Y receptors to ERK is mediated by a phosphatidylinositol-specific phospholipase C (PI-PLC)/calcium pathway. However, neither inhibition of PI-PLC nor chelation of calcium significantly reduced ATP-stimulated ERK activity. Moreover, a preferential inhibitor of calcium-dependent PKC isoforms, Gö 6976, was significantly less effective in blocking ATP-stimulated ERK activity than GF102903X, an inhibitor of both calcium-dependent and -independent PKC isoforms. Furthermore, ATP stimulated a rapid translocation of PKCdelta, a calcium-independent PKC isoform, but not PKCgamma, a calcium-dependent PKC isoform. ATP also stimulated a rapid increase in choline, and inhibition of phosphatidylcholine hydrolysis blocked ATP-evoked ERK activation. These results indicate that P2Y receptors in astrocytes are coupled independently to PI-PLC/calcium and ERK pathways and suggest that signaling from P2Y receptors to ERK involves a calcium-independent PKC isoform and hydrolysis of phosphatidylcholine by phospholipase D. In addition, we found that inhibition of ERK activation blocked extracellular ATP-stimulated DNA synthesis, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y receptors.