Atypical Protein Kinase C Isozymes Research Articles

Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials.

Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinase, is classed into three subfamilies based on their structural and activation characteristics: conventional or classic PKC isozymes (cPKCs; α, βI, βII, and γ), novel or non-classic PKC isozymes (nPKCs; δ, ε, η, and θ), and atypical PKC isozymes (aPKCs; ζ, ι, and λ). PKC inhibitors and activators are used to understand PKC-mediated intracellular signaling pathways and for the diagnosis and treatment of various PKC-associated diseases, such as cancers, neurological diseases, cardiovascular diseases, and infections. Many clinical trials of PKC inhibitors in cancers showed no significant clinical benefits, meaning that there is a limitation to design a cancer therapeutic strategy targeting PKC alone. This review will focus on the activators and inhibitors of PKC and their applications in clinical trials.

Diacylglycerol promotes GLUT4 translocation to the cell surface in a PKCε-dependent and PKCλ/ι and -ζ-independent manner

AimEmerging evidence has pointed to the participation of protein kinase C (PKC) in insulin-regulated trafficking of the glucose transporter GLUT4. The present study investigated the effect of the PKC activator diacylglycerol (DAG) on GLUT4 trafficking and glucose uptake. Main methods3T3L1-GLUT4myc fibroblast cells expressing GLUT4myc were differentiated into adipocytes. Western blotting, glucose assay, and real-time RT-PCR were carried out in 3T3L1-GLUT4myc adipocytes. PKCλ/ι, -ζ, -ε, and -γ were knocked-down by transfecting each siRNA. Activity of PKC isozymes was assayed under the cell-free conditions. Key findingsInsulin increased cell surface localization of GLUT4 in 3T3L1-GLUT4myc adipocytes, and a similar effect was obtained with 1,2-dioleoyl-sn-glycerol (DO-DAG), 1-oleoyl-2-acetyl-sn-glycerol (OA-DAG), or 1,2-dipalmitoyl-sn-glycerol (DP-DAG). Like insulin, DO-DAG stimulated glucose uptake into adipocytes, but no significant synergistic increase in the glucose uptake was found with co-treatment with insulin and DO-DAG. Insulin activated Akt in adipocytes, but no Akt activation was induced by any investigated DAG. In the cell-free PKC assay, DAGs examined here activated PKCα, -βI, -βII, -γ, -δ, and -ε, but the atypical PKC isozymes PKCλ/ι and -ζ were not activated. Insulin-induced GLUT4 translocation to the cell surface was inhibited by knocking-down PKCλ/ι and -ζ, but not PKCγ or -ε. In contrast, DO-DAG-induced GLUT4 translocation to the cell surface was clearly prevented by knocking-down PKCε. SignificanceThe results of the present study indicate that DAG stimulates GLUT4 translocation to the cell surface by activating PKCε, regardless of PKCλ/-ι and -ζ.

Protein Kinase C Isoforms ζ and ι Mediate Collagenase Expression and Cartilage Destruction via STAT3- and ERK-dependent c-fos Induction

The protein kinase C (PKC) signaling pathway is a major regulator of cellular functions and is implicated in pathologies involving extracellular matrix remodeling. Inflammatory joint disease is characterized by excessive extracellular matrix catabolism, and here we assess the role of PKC in the induction of the collagenases, matrix metalloproteinase (MMP)-1 and MMP-13, in human chondrocytes by the potent cytokine stimulus interleukin-1 (IL-1) in combination with oncostatin M (OSM). IL-1 + OSM-stimulated collagenolysis and gelatinase activity were ameliorated by pharmacological PKC inhibition in bovine cartilage, as was collagenase gene induction in human chondrocytes. Small interfering RNA-mediated silencing of PKC gene expression showed that both novel (nPKC delta, nPKC eta) and atypical (aPKC zeta, aPKC iota) isoforms were involved in collagenase induction by IL-1. However, MMP1 and MMP13 induction by IL-1 + OSM was inhibited only by aPKC silencing, suggesting that only atypical isoforms play a significant role in complex inflammatory milieus. Silencing of either aPKC led to diminished IL-1 + OSM-dependent extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) 3 phosphorylation, and c-fos expression. STAT3 gene silencing or ERK pathway inhibition also resulted in loss of IL-1 + OSM-stimulated c-fos and collagenase expression. Silencing of c-fos and c-jun expression was sufficient to abrogate IL-1 + OSM-stimulated collagenase gene induction, and overexpression of both c-fos and c-jun was sufficient to drive transcription from the MMP1 promoter in the absence of a stimulus. Our data identify atypical PKC isozymes as STAT and ERK activators that mediate c-fos and collagenase expression during IL-1 + OSM synergy in human chondrocytes. aPKCs may constitute potential therapeutic targets for inflammatory joint diseases involving increased collagenase expression.

Subcellular Alterations of Protein Kinase C Isozymes in the Rat Brain after Organophosphate Poisoning

The protein kinase C (PKC) signaling pathway has been associated with modulation of N-metyl-D-aspartate receptor activity, motor behavior, learning, and memory, all of which are severely impaired in organophosphate (OP) intoxication. Nevertheless, the role of PKC in OP intoxication is largely unknown. The present study attempted to characterize alterations in the immunoreactivity levels of PKC isozymes expressed in different brain areas in the rat following exposure to the nerve agent sarin (1x LD(50)). Furthermore, possible neuroprotective effect of selective PKC regulating peptide after such insult was evaluated. The results indicated that a significant reduction in the immunoreactivity level of the conventional betaII-PKC and the atypical zeta-PKC was observed in frontal cortex up to 24 h postsarin and in the striatum up to 5 days postsarin exposure. This reduction was in contrast to the increase in the immuno-reactivity level of both isozymes seen in the hippocampus or thalamus. Treatment with the anticonvulsant midazolam (0.5 mg/kg) 10 min postsarin exposure markedly reduced zeta-PKC immunoreactivity level and betaII-PKC in the membrane fractions in the hippocampus. betaII-PKC peptide (380 ng/kg), known to inhibit PKC translocation and activation, attenuated sarin-induced neuropathology. These observations suggest a role for both conventional and atypical PKC isozymes in OP-induced neuropathy in the rat and further support their involvement in cell death.

Opposing effects of δ- and ζ-protein kinase C isozymes on cardiac fibroblast proliferation: use of isozyme-selective inhibitors

Neonatal primary cardiac fibroblasts in defined medium continue to proliferate. Here, we show that phorbol ester inhibited and transforming growth factor-β1 (TGFβ1) stimulated this fibroblast proliferation. Cardiac fibroblasts contain six protein kinase C (PKC) isozymes: α-, δ-, ϵ-, βI-, βII-, and ζ-PKC. To evaluate the effect of different PKC isozymes on the proliferation of these cells, we used isozyme-selective PKC inhibitors. Inhibition of endogenous δ-PKC with δV1-1, an isozyme-selective translocation inhibitor, resulted in increased basal thymidine incorporation by 58 ± 12% of control cells, but did not affect TGFβ1-induced cell growth. Inhibition of endogenous ζ-PKC in neonatal rat cardiac fibroblasts with ζ-pseudosubstrate, a selective inhibitor for the atypical PKC isozymes, revealed an opposite effect; this inhibitor reduced basal growth to 45 ± 11% and TGFβ1-induced growth to 61 ± 10%. Other isozyme-specific inhibitors used in this study did not alter basal or TGFβ1-stimulated fibroblast growth. Taken together, our data provide evidence that δ-PKC inhibits and ζ-PKC stimulates proliferation of neonatal rat cardiac fibroblasts.

Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells

Background & Aims:Protein kinase C (PKC) is a key signaling molecule in excitation-contraction coupling in several types of smooth muscle cells. We investigated whether the attenuated contraction in inflamed colon cells is caused by alterations in the expression, distribution, and activation of specific PKC isorymes.Methods:Kinase assays, immunofluorescence imaging, and Western immunoblotting were performed on single circular smooth muscle cells obtained from the normal dog colon as well as from colon with experimental colitis induced by mucosal exposure to ethanol and acetic acid, to determine the distribution, expression, and activation of PKC isorymes.Results:Classical (α, β, and γ), novel (σ and ε), and the atypical PKC (ι, λ, and ζ) isozymes were detected in coIonic circular muscle cells. The expression of PKC α, (3, and ε isozymes was down-regulated, whereas that of PKC ι and λ isozymes was up-regulated; other isozymes were not affected by inflammation. Acetylcholine (ACh) treatment translocated only the PKC α, β, and ε isozymes from the cytosol to the membrane in normal cells; this translocation was absent in inflamed colon cells. Immunofluorescence imaging confirmed the translocation of PKC α from the cytosol to the membrane in response to ACh in normal cells. PKC inhibitors, chelerythrine, and myristoylated peptides to α, β, and ε isozymes inhibited the contractile response to ACh in normal, but not in inflamed, cells. PKC ι and λ did not participate in the contractile response to ACh. Conclusions: ACh-induced contraction is mediated by PKC α, (3, and ε isozymes in normal colonic circular muscle cells. Contractile dysfunction in inflamed colon cells is, in part, caused by decreased expression and impaired activation of specific PKC isozymes.

Protein kinase C activation: isozyme-specific effects on metabolism and cardiovascular complications in diabetes.

Protein kinase C (PKC) is a family of multifunctional isoenzymes, activated by diacylglycerols (DAGs), which play a central role in signal transduction and intracellular crosstalk by phosphorylating at serine/threonine residues an array of substrates, including cell-surface receptors, enzymes, contractile proteins, transcription factors and other kinases. Individual isozymes vary in their pattern of tissue and subcellular distribution, function and Ca2+/phospholipid cofactor requirements, and in diabetes there is widespread activation of the DAG-PKC pathway in metabolic, cardiovascular and renal tissues. In liver, muscle and adipose tissue, PKC isozymes have been implicated both as mediators and inhibitors of insulin action. Activation of DAG-sensitive PKC isoforms, such as PKC-theta and PKC-epsilon, down-regulates insulin receptor signalling and could be an important biochemical mechanism linking dysregulated lipid metabolism and insulin resistance in muscle. On the other hand, atypical PKC isozymes, such as PKC-zeta and PKC-lambda, have been identified as downstream targets of PI-3-kinase involved in insulin-stimulated glucose uptake, especially in adipocytes. Glucose-induced de novo synthesis of (palmitate-rich) DAG and sustained isozyme-selective PKC activation (especially but not exclusively PKC-beta) has been strongly implicated in the pathogenesis of diabetic microangiopathy and macroangiopathy through a host of undesirable effects on endothelial function, VSM contractility and growth, angiogenesis, gene transcription (in part by MAP-kinase activation) and vascular permeability. Interventions that increase DAG metabolism (e. g. vitamin E) and/or inhibit PKC isozymes (e. g. the beta-selective inhibitor LY333531) ameliorate the biochemical and functional consequences of DAG-PKC activation in experimental diabetes, for example improving retinal blood flow and albuminuria in parallel with reductions in membrane-associated PKC isozyme activities. Thus, a greater understanding of the functional diversity and pathophysiological regulation of PKC isozymes is likely to have important clinical and therapeutic benefits.

Regulation of Conjunctival Goblet Cell Secretion by Ca2+and Protein Kinase C

Conjunctival goblet cells secrete mucus in response to cholinergic (muscarinic) agonists, but the underlying signaling pathways activated in this tissue are not well understood. Cholinergic agonists usually activate phospholipase C to produce inositol 1,4,5 trisphosphate and diacylglycerol. Inositol 1,4,5 trisphosphate increases the intracellular Ca2+concentration ([Ca2+]i) while diacylglycerol activates protein kinase C (PKC). PKC and Ca2+, either by itself or with calmodulin, activate cellular functions. Goblet cell glycoprotein secretion, our index of mucin secretion, was measured from pieces of rat conjunctiva with an enzyme-linked lectin assay using the lectin Ulex europaeus agglutinin I (UEA-I). UEA-I selectively recognizes high molecular weight glycoproteins secreted by the goblet cells. Increasing the [Ca+]iwith the Ca2+ionophore ionomycin stimulated glycoprotein secretion from conjunctival goblet cells. Cholinergic agonist-induced secretion was completely blocked by chelation of extracellular Ca2+and by the Ca2+/calmodulin-dependent protein kinase inhibitors KN93 and W7 as well as their inactive analogs KN92 and W5. Activation of classical and novel PKC isozymes by phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate stimulated goblet cell glycoprotein secretion. When ionomycin and PMA were added simultaneously, secretion was additive. PKC isozymes were identified by Western blotting analyses with antibodies specific to nine of the 11 PKC isozymes (PKCγ and ζ were not tested). All nine PKC isozymes were identified in the conjunctival epithelium. The cellular location of the PKC isozymes was determined by immunofluorescence microscopy. Goblet cells contained the classical PKC isozymes PKCα, -βI and -βII, the novel PKC isozymes PKCϵ, -θ, and -μ , and the atypical PKC isozyme PKCζ. We were unable to determine if PKC activation is required for cholinergic-agonist induced secretion because the PKC inhibitors chelerythrine and staurosporine alone greatly increased secretion. We conclude that Ca2+plays a major role in cholinergic agonist-induced conjunctival goblet cell secretion, but this agonist appears not to use Ca2+/calmodulin-dependent protein kinases. We also conclude that activated PKC can stimulate goblet cell secretion and that seven different PKC isoforms are present in the goblet cells.

Adrenergic regulation of translocation of protein kinase C isozymes in rat pinealocytes

Protein kinase C (PKC) is involved in the α 1-adrenergic-potentiation of β-adrenergic stimulated cyclic nucleotide responses in rat pinealocytes. In the present study, the PKC isozymes expressed in rat pinealocytes and their regulation by norepinephrine (NE) were investigated. Western blot analysis identified PKC α (a classical PKC isozyme), PKC δ and ε (novel PKC isozymes), and PKC ζ: (atypical PKC isozymes). NE caused an increase in PKC α, δ, and ε, but not PKC ζ, in the particulate fraction. BAPTA-AM, which clamps intracellular Ca 2+, reduced NE mediated translocation of PKC α, δ, and ε. Subjecting the animals to stimulus deprivation, which altered adrenergic-stimulated cyclic nucleotide responses, had no effect on the expression of PKC α, δ, ε, and ζ. Overnight treatment with 4 β-phorbol 12-myristate 13-acetate, an activator of PKC, down-regulated PKC α, δ, and ε, but not PKC ζ. Our results indicate that all three classes of PKC isozymes (PKC α, δ, ε, and ζ are expressed in the rat pineal gland. However, selective activation of these PKC isozymes does not appear to account for the differences in the pineal cAMP and cGMP responses to stimulation.

Role of protein kinase C (PKC) in agonist-induced mu-opioid receptor down-regulation: II. Activation and involvement of the alpha, epsilon, and zeta isoforms of PKC.

Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and mu-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.

Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells.

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.

Involvement of a Phorbol Ester-Insensitive Protein Kinase C in the α2-Adrenergic Inhibition of Voltage-Gated Calcium Current in Chick Sympathetic Neurons

alpha2-Adrenoceptors regulate the efficacy at the sympathoeffector junction by means of a feedback inhibition of transmitter release. In chick sympathetic neurons, the mechanism involves an inhibition of N-type calcium channels, and we now present evidence that this effect involves an atypical, phorbol ester-insensitive protein kinase C (PKC). The inhibition of voltage-gated Ca2+ currents by the specific alpha2-adrenergic agonist UK 14,304 was significantly attenuated when the PKC inhibitors PKC(19-36), staurosporine, or calphostin C were included in the internal solution used to fill the patch pipettes, or if staurosporine or calphostin C were applied extracellularly; however, phorbol esters as classical activators of PKC or oleoylacetylglycerol did not mimic the effect of UK 14,304, and chronic exposure to 4-beta-phorbol dibutyrate (PDBu) did not attenuate it, ever though PKCalpha and -epsilon isozymes were translocated to plasma membranes by PDBu. The atypical isozyme PKCzeta was translocated by 100 micrometer AA and this effect was attenuated when PKC(19-36) was added to the patch pipette solution. Our observations indicate that classical, new, and atypical PKC isozymes are present in chick sympathetic neurons and that an atypical, phorbol ester-insensitive PKC is involved in the inhibition of voltage-activated calcium currents by alpha2-adrenoceptor activation.