Protein Kinase C Gamma Research Articles

Protein kinase C isoforms alpha, delta and theta require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells).

Protein kinase C (PKC) isoforms are potentially important as modulators of the insulin signalling chain and could be involved in the pathogenesis of cellular insulin resistance. We have previously shown that phorbol ester stimulated PKC beta1 and beta2 as well as tumor necrosis factor-alpha (TNFalpha) stimulated PKC epsilon inhibit human insulin receptor (HIR) signalling. There is increasing evidence that the insulin receptor substrate-1 (IRS-1) is involved in inhibitory signals in insulin receptor function. The aim of the present study was to elucidate the role of IRS-1 in the inhibitory effects of protein kinase C on human insulin receptor function. HIR, PKC isoforms (alpha, beta1, beta2, gamma, delta, epsilon, eta, theta and zeta) and IRS-1 were coexpressed in human embryonic kidney (HEK) 293 cells. PKCs were activated by preincubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (CTPA) (10(-7) mol/l) following insulin stimulation. While PKCs alpha, delta and theta were not inhibitory in HEK 293 cells which were transfected only with HIR and PKC, additional transfection of IRS-1 induced a strong inhibitory effect of these PKC isoforms being maximal for PKC theta (99 +/- 1.8% inhibition of insulin stimulated receptor autophosphorylation, n = 7, p < 0.001). No effect was seen with PKC gamma, epsilon, zeta and eta while the earlier observed insulin receptor kinase inhibition of PKC beta2 was further augmented (91 +/- 13%, n = 7, p < 0.001 instead of 45% without IRS-1). The strong inhibitory effect of PKC theta is accompanied by a molecular weight shift of IRS-1 (183 kDa vs 180 kDa) in the sodium dodecyl sulphate polyacrylamide gel. This can be reversed by alkaline phosphatase treatment of IRS-1 suggesting that this molecular weight shift is due to an increased phosphorylation of IRS-1 on serine or threonine residues. In summary, these data show that IRS-1 is involved in the inhibitory effect of the PKC isoforms alpha, beta2, delta and theta and it is likely that this involves serine/threonine phosphorylation of IRS-1.

Down-regulation of protein kinase C isoforms in irritant contact dermatitis.

Protein kinase C (PKC) isoforms are important in cell signal transduction associated with regulation of cell proliferation and differentiation. In this study, alterations of PKC isoform levels in irritant patch test reactions were detected by Western immunoblots. 4 chemically and structurally different irritants, 4% and 8% hydrochloric acid (HCl); 1% and 2% sodium hydroxide (NaOH); 20% and 40% nonanoic acid (NON) in 1-propanol; and 5% and 10% sodium dodecyl sulfate (SDS) were applied on the backs of mice in Finn Chambers and fixed with surgical dressings. The patches were kept on the skin for 24 h and removed. 24 h after removal, mild erythema with thickening of skin without vesicles was observed in all irritated skin, except that 4% HCl and 1% NaOH treated skin showed unremarkable skin reaction. No visible skin reaction was detected in vehicle-treated skin. PKC isoform alpha, beta, gamma, and delta levels in irritated skin revealed a 10% to 65% decrease compared to vehicle treated skin. These results indicate that in HCl, NaOH, NON and SDS-induced irritation, activation of the PKC related cell signal transduction cascade may be involved, and that PKC mediated events may be a common phenomenon in irritant contact dermatitis.

Tissue-specific protein kinase C isoforms differentially mediate macrophage TNFalpha and IL-1beta production.

Macrophage subpopulations are differentially activated during sepsis, shock, or trauma; however, it is unknown whether inherent mechanistic and phenotypic differences exist between macrophage subpopulations that may account for region-specific inflammation. We hypothesized that macrophage expression/function of protein kinase C (PKC) isoforms is tissue specific (alveolar versus peritoneal). Rat alveolar and peritoneal macrophages were each probed for the expression of PKC isoforms alpha, beta1, beta2, gamma, delta, epsilon, zeta, and theta by immunoblot. PKC isoforms alpha, beta1, beta2, and zeta were detected in both populations; however, isoforms epsilon, gamma, and eta were found in alveolar macrophages only. To investigate the functional role of the Ca2+-dependent PKC (cPKC) versus Ca2+-independent PKC (nPKC) isoforms, pan-PKC isoform inhibition (cPKC and nPKC), or cPKC isoform selective inhibition (alpha, beta1, beta2, gamma) was performed before endotoxin (lipopolysaccharide, Salmonella minnesota, 100 ng/mL) stimulation in vitro. Pan-PKC isoform inhibition attenuated TNFalpha and IL-1beta production by each population; however, selective cPKC (alpha, beta1, beta2, gamma) inhibition decreased peritoneal, but not alveolar, macrophage TNFalpha production. IL-1beta production was not affected by cPKC inhibition in either population. 1) alveolar and peritoneal macrophages constitutively express different PKC isoforms; 2) alveolar macrophages uniquely express isoforms epsilon, gamma, eta; 3) TNFalpha production is regulated by cPKCs in peritoneal macrophages, but by nPKCs in alveolar macrophages; 4) nPKCs regulate IL-1beta production in both populations. These results suggest that tissue-specific PKC isoforms differentially mediate macrophage function, which may have important regulatory implications in the compartmentalization of immune function. Further understanding may allow region-specific manipulation of inflammation.

Down-regulation of protein kinase C isoforms in irritant contact dermatitis

Protein kinase C (PKC) isoforms are important in cell signal transduction associated with regulation of cell proliferation and differentiation. In this study, alterations of PKC isoform levels in irritant patch test reactions were detected by Western immunoblots. 4 chemically and structurally different irritants, 4% and 8% hydrochloric acid (HCl); 1% and 2% sodium hydroxide (NaOH); 20% and 40% nonanoic acid (NON) in 1-propanol; and 5% and 10% sodium dodecyl sulfate (SDS) were applied on the backs of mice in Finn Chambers and fixed with surgical dressings. The patches were kept on the skin for 24 h and removed. 24 h after removal, mild erythema with thickening of skin without vesicles was observed in all irritated skin, except that 4% HCl and 1% NaOH treated skin showed unremarkable skin reaction. No visible skin reaction was detected in vehicle-treated skin. PKC isoform alpha, beta, gamma, and delta levels in irritated skin revealed a 10% to 65% decrease compared to vehicle treated skin. These results indicate that in HCl, NaOH, NON and SDS-induced irritation, activation of the PKC related cell signal transduction cascade may be involved, and that PKC mediated events may be a common phenomenon in irritant contact dermatitis.

Translocation of protein kinase Cgamma occurs during the early phase of acquisition of food rewarded spatial learning.

This study describes the translocation of the brain specific protein kinase C gamma isoenzyme (PKCgamma) in the hippocampus during food rewarded spatial learning. The holeboard test was used for spatial orientation, and immunoblot analysis was used for assessment of PKCgamma in cytosolic, membrane-inserted and membrane-associated fractions. Membrane-associated PKCgamma was increased during early acquisition of spatial learning, but not in a later phase of training. This transient and apparently temporary intracellular PKCgamma translocation was only observed in the posterior but not in the anterior hippocampus, and was only detected within 10 min after termination of the learning trial. This study supports the idea that PKCgamma is significantly involved in the biochemical events underlying learning and memory, notably during the period of novel information processing. The results further promote the hypothesis that the hippocampus is specifically involved in temporal information processing, which requires the engagement of PKCgamma.

An apparently novel protein of human leukocytes, reactive with an antibody to protein kinase C-gamma, is rapidly modified upon cell activation: initial characterization in neutrophils and their cytoplasts.

On immunoblots of human neutrophil cytoplasts (U-CYT), a previously undescribed 97 kDa protein was revealed by intense and selective reaction with an antibody that was initially raised to recognize PKC-gamma. Denoted "gamma-rp" for gamma-related protein, this acidic cytosolic protein somewhat resembled the classic forms of PKC in several biochemical respects. Appearing as a doublet on low-percentage SDS-PAGE gels, both its mobility and staining pattern were rapidly altered by treatment of U-CYT with either phorbol ester or chemotactic peptide. Whole neutrophil gamma-rp was detectable only after TCA precipitation of intact cells. It was also detectable in human platelets, lymphocytes, and neutrophil-like differentiated HL60 cells, but not in fibroblasts, erythrocytes, monocytes, or monocyte-like differentiated HL60 cells. Our data suggest that gamma-rp merits further study as a potential participant in cellular activation, and as a possible structural or functional relative of PKC.

Protein kinase C isoforms beta 1 and beta 2 inhibit the tyrosine kinase activity of the insulin receptor.

Downregulation of insulin receptor tyrosine kinase (IRK) activity yields to impaired insulin signalling and contributes to the pathogenesis of cellular insulin resistance. Activation of protein kinase C (PKC) by different agents is associated with an inhibition of IRK activity in various cell types. There is evidence that this effect on IRK activity might be mediated through phosphorylation of specific serine residues of the insulin receptor beta-subunit. Neither the domains of the IRK where inhibiting serine phosphorylation occurs nor the PKC isoform responsible for IRK inhibition have been identified. PKC consists of a family of at least 12 isoforms. The aim of the present study was to determine which PKC isoform might be capable of IRK inhibition. The human insulin receptor and the PKC isoforms alpha, beta 1, beta 2, gamma, delta, epsilon, eta, theta and zeta were overexpressed in human embryo kidney fibroblasts (HEK 293 cells) in order to answer this question. PKCs were activated by preincubation with the phorbolester (TPA) (10(-7) mol/l) following insulin stimulation of the cells. When the IRK was coexpressed with the PKC isoforms beta 1 and beta 2, a 50 +/- 15.7 and 45 +/- 10.1% inhibition of tyrosine autophosphorylation of IRK was observed while coexpression with the other isoforms did not significantly modify IRK autophosphorylation. The data suggest that the PKC isoforms beta 1 and beta 2 might be candidates for insulin receptor inhibition.

Effects of depolarization and NMDA antagonists on the role survival of cerebellar granule cells: a pivotal role for protein kinase C isoforms.

Primary cultures of cerebellar granule cells (CGCs) grown in high-K+ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3-4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC alpha, gamma, mu, lambda, and iota from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC epsilon and betaII, respectively, whereas no significant change was observed for betaI. The isoforms of delta, theta, eta, and zeta were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC iota and epsilon were decreased, those of PKC alpha, betaI, betaII, gamma, and lambda were unchanged, whereas that of PKC mu was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2-4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K+ cultures, levels of PKC alpha, mu, lambda, and iota, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC gamma, lambda, iota, and mu at 10 and 14 DIV. In addition, the PKC epsilon level was increased at 14 DIV but decreased at early stages, whereas PKC alpha, betaI, and betaII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.

The in vitro phosphorylation of p53 by calcium-dependent protein kinase C--characterization of a protein-kinase-C-binding site on p53.

We show that, in vitro, Ca2+-dependent protein kinase C (PKC) phosphorylates recombinant murine p53 protein on several residues contained within a conserved basic region of 25 amino acids, located in the C-terminal part of the protein. Accordingly, synthetic p53-(357-381)-peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53-(357-381)-peptide at micromolar concentrations has the ability to stimulate sequence-specific DNA binding by p53. That stimulation is lost upon phosphorylation by PKC. To further characterise the mechanisms that regulate PKC-dependent phosphorylation of p53-(357-381)-peptide, the phosphorylation of recombinant p53 and p53-(357-381)-peptide by PKC were compared. The results suggest that phosphorylation of full-length p53 on the C-terminal PKC sites is highly dependent on the accessibility of the phosphorylation sites and that a domain on p53 distinct from p53-(357-381)-peptide is involved in binding PKC. Accordingly, we have identified a conserved 27-amino-acid peptide, p53-(320-346)-peptide, within the C-terminal region of p53 and adjacent to residues 357-381 that interacts with PKC in vitro. The interaction between p53-(320-346)-peptide and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53-(357-381)-peptide, neurogranin and histone H1. Conventional Ca2+-dependent PKC alpha, beta and gamma and the catalytic fragment of PKC (PKM) were nearly equally susceptible to inhibition by p53-(320-346)-peptide. The Ca2+-independent PKC delta was much less sensitive to inhibition. The significance of these findings for understanding the in vivo phosphorylation of p53 by PKC are discussed.

Inhibition of protein kinase C in multidrug-resistant cells by modulators of multidrug resistance.

We have evaluated the protein kinase C (PKC) activity in two series of cultured cell lines presenting the multidrug-resistance (MDR) phenotype and in the corresponding wild-type cells: the human KB 3.1, KB A1 and KB 8.5 cell lines, and the rat C6, C6 0.5 and C6 1V cell lines. We have observed an increase in PKC activity in the MDR cell lines of the KB cell lineage, proportional to their degree of resistance to doxorubicin. In contrast, the MDR cell lines of the C6 cell lineage presented no change (C6 0.5) or even decrease (C6 1V) in PKC activity; the basal level of PKC activity in C6 cells was, however, 50-fold higher than in KB 3.1 cells. We have tested, in these lines, the effect of four modulators of MDR: verapamil, cyclosporin A, quinine and S-9788, on doxorubicin acytotoxicity and on PKC activity. We observed that cyclosporin A and S-9788, which were the most active on MDR reversal, were able to inhibit PKC activity in the KB resistant lines as well as in all C6 lines, whereas verapamil and quinine had only marginal effects on PKC activity. The distribution of PKC isoenzymes was studied by Western blots. The PKC alpha, gamma and delta isoforms were increased in the KB resistant lines as compared to wild-type cells, which could account for the increase PKC activity we observed. In contrast, PKC alpha and gamma were decreased in C6 1V cells, as expected from the results obtained for total PKC activity, but we also noticed an important decrease in PKC delta in the C6 0.5 line. Our results suggest that an increase in PKC activity is not an absolute requirement for expression of MDR, provided that the basal level be high enough; and that some modulators may act on MDR, not only through direct P-glycoprotein interaction, but also through P-glycoprotein phosphorylation or expression. The distribution of PKC isoenzymes revealed that the modifications encountered between sensitive and resistant cells mainly concerned alpha, gamma and delta isoenzymes of PKC.

Effect of halothane on conventional protein kinase C translocation and down-regulation in rat cerebrocortical synaptosomes

Protein kinase C (PKC) is a key regulatory enzyme that has been implicated as a molecular target for the action of general anaesthetics. We have determined the effects of halothane on the translocation and down-regulation of conventional PKC (cPKC) by analysing the subcellular distribution of PKC activity, [3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding and PKC immunoreactivity in intact rat cerebrocortical synaptosomes, a subcellular fraction that contains functional nerve terminals. Halothane alone (2.4 vol%) reduced membrane-associated (P < 0.05) and increased cytosol (P < 0.01) PKC activity, while phorbol-12-myristate, 13-acetate (PMA) 0.1 mumol litre-1, a metabolically stable activator of PKC, reduced membrane (P < 0.01) without altering cytosol PKC activity. Halothane and PMA in combination reduced membrane PKC activity to undetectable levels and reduced cytosol PKC activity (P < 0.01). Halothane alone had no significant effects on the distribution of [3H]PDBu binding, while PMA alone significantly reduced both membrane and cytosol [3H]PDBu binding (P < 0.01). Halothane and PMA in combination reduced membrane and cytosol [3H]PDBu binding further, but this effect was not significantly different from the effect of PMA alone. Experiments using isoform-selective antibodies to PKC alpha, PKC beta or PKC gamma demonstrated synergistic interactions between halothane and PMA in promoting translocation of the three conventional PKC isoforms from the cytosol to the membrane fraction of synaptosomes and down-regulation of their immunoreactivity. Halothane and PMA together reduced cytosol PKC alpha/beta/gamma immunoreactivity significantly more (P < 0.05) than PMA alone. Halothane thus has two distinct actions on PKC in synaptosomes: activation of endogenous PKC activity and potentiation of activation-induced cPKC translocation and down-regulation. These potentially competing effects may underlie some of the conflicting results obtained with halothane on PKC-mediated processes in intact cells.

Gamma Isoform-selective changes in PKC immunoreactivity after trace eyeblink conditioning in the rabbit hippocampus.

An immunocytochemical examination of the rabbit hippocampus was done to determine which of the Ca(2+)-dependent protein kinase C (PKC) isoforms (PKC alpha, -beta I, -beta II, or -gamma) are involved in associative learning. The hippocampally dependent trace eyeblink conditioning task was used for behavioral training, and pseudoconditioned and naive animals served as controls. Significant increases (P < 0.05) in staining intensity were found with antibodies reactive with the catalytic or the regulatory domain of PKC gamma in conditioned animals compared with naive and pseudoconditioned subjects at a 24-h post-conditioning time point. The increase was found in CA1 and CA3 pyramidal cell bodies, in apical dendrites and the proximal part of the basilar dendrites, and in cell bodies of dentate granule cells. In contrast, no conditioning-specific changes were found for PKC alpha, -beta I, or -beta II in in hippocampal neurons. The increase in PKC gamma immunoreactivity (ir) was significantly less (P < 0.05) in poor learners than in good learners. The correlation between the degree of PKC gamma-ir and the total number of conditioned responses across training sessions was both positive and significant. These results suggest that PKC gamma is the major Ca(2+)-dependent PKC isoform involved in hippocampal neurons during acquisition of associative memories. Immunoblots revealed no conditioning-induced increase in the total amount or translocation of PKC gamma at the 24-h time point, and no proteolytic PKC fragments were observed. In agreement with the Western blot data, PKC activity did not differ among naive, pseudoconditioned, and trace conditioned animals. The conditioning-induced increase in antibody binding to the gamma-isoform must therefore be due to an increased access to the antigenic site(s) as a result of alteration in the tertiary structure of PKC gamma or in quaternary interactions of PKC gamma in situ.

Protein kinase C isoenzymes in rat and human cardiovascular tissues.

1. We have compared the expression of protein kinase C (PKC) activity and immuno-detectable isoenzymes in cytosolic and membrane extracts of rat and human cardiovascular tissues (heart, kidney, aorta, saphenous vein). Experiments were performed in raw extracts and upon combined diethylaminoethylcellulose (DEAE) and phenylsepharose column chromatography. 2. PKC activity that bound to DEAE mostly eluted with 200 mM NaCl. DEAE-purified PKC from all tissues except rat kidney bound almost quantitatively to phenylsepharose and eluted with 0.5-0 M NaCl. 3. Immunoblots with an antibody against classical PKCs and the activator profile for phosphatidylserine, diolein and Ca2+ revealed that the PKC from rat kidney, which did not bind to phenylsepharose, was most probably due to a proteolytically-generated, constitutively active PKC which is not under the control of a regulatory subunit. 4. Studies in the reference tissue, rat brain, demonstrated that all PKC isoenzymes investigated (classical PKCs alpha, beta, gamma, new PKCs delta, epsilon, eta, theta, and atypical PKCs zeta, lambda, iota) have similar DEAE and phenylsepharose chromatography elution profiles. In the functional assay an inhibitor of all known PKC isoenzymes, bisindolylmaleimide, and a specific inhibitor of classical PKCs, Gö 6976, both inhibited PKC from rat brain completely and with high potency indicating that the functional assay preferentially detects classical PKC isoenzymes. 5. Each PKC isoenzyme had a tissue-specific expression profile which was similar in rat and man. The classical PKC alpha, the new PKCs delta and epsilon and all atypical PKCs were detectable in most tissues, whereas the PKC beta and PKC gamma were not detected in any pheripheral tissue; PKC eta and PKC theta were found in some tissues. 6. We conclude that combined DEAE and phenylsepharose chromatography is useful to enrich and detect PKC isoenzymes; no major species differences in tissues-specific expression patterns appear to exist between rat and man.

Fumonisin B1 induces protein kinase C translocation via direct interaction with diacylglycerol binding site.

Fumonisins are carcinogenic to rats and are suspected human carcinogens. However, the mechanism(s) of carcinogenesis of fumonisn B1 (FB1) is poorly understood. Multiple signal transduction pathways such as protein kinase C (PKC) have been shown to play an important role in carcinogenesis. This study was undertaken to evaluate whether FB1 affects PKC activation. Similar to tumor-promoting phorbol ester, phorbol 12-myristate-13-acetate (PMA), PKC is also catalytically activated by FB1. Protein kinase C activity and its redistribution in response to FB1 were determined in rat cerebrocortical slices. Cytosolic and membranous PKC activities were determined by histone phosphorylation in the presence of [gamma-32P]ATP, phosphatidyl-L-serine, PMA, and Ca2+. Distribution of gamma PKC isozyme in the presence of FB1 was also assessed by immunoblotting using affinity purified anti-peptide antibodies. Similar to PMA, FB1 added in vitro to rat cerebrocortical slices facilitated PKC translocation from cytosol to membrane in a concentration-dependent manner. This FB1-induced PKC translocation was inhibited by incubation with the inactive 4 alpha-phorbol 12,13-didecanoate. The effects of FB1 and PMA were neither additive nor synergistic. In addition, PMA and FB1-induced PKC enzyme redistribution were inhibited by pretreating tissues with sphingosine. A concentration-related FB1 attenuation of specific phorbol dibutyrate, [3H]PDBu, binding was also observed when cortical membranes were incubated with either PMA or sphingosine. This is the first report of FB1-induced PKC translocation via a direct action on the diacylglycerol site that also binds phorbol esters. Because phorbol esters are well known tumor promoters, we provide a plausible cellular mechanism to explain the carcinogenicity of FB1.

Role of Protein Kinase C-β Isozyme in Activation of Latent Human Immunodeficiency Virus Type 1 in Promonocytic U1 Cells by Phorbol-12-Myristate Acetate

Protein kinase C (PKC) appears to play a role in replication of human immunodeficiency virus type 1 (HIV-1). PKC is a family of at least 12 isozymes. In this study, we investigated a role of Ca(2+)-dependent PKC isozymes (alpha, beta, and gamma) in activation of latent HIV-1 in U1, a chronically infected promonocytic cell line, using polyclonal rabbit anti-PKC isozyme antibodies as specific inhibitors. Antibodies were introduced intracellularly by electroporation and then cells were stimulated with PMA. HIV-1 production was measured as p24 antigen using ELISA and reverse transcriptase activity. Anti-PKC beta antibody significantly inhibited PMA-induced HIV-1 production, whereas antibodies against PKC alpha and gamma had no significant effect. Furthermore, anti-PKC beta antibody inhibited PMA-induced activation of NF-kappa B and HIV-1 LTR. Preincubation of anti-PKC beta antibody with its antigenic peptide reversed the inhibitory effect of anti-PKC beta antibody. This study suggest that PKC beta plays a role in PMA-induced activation of latent HIV-1.

Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance.

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

Protein Kinase C Isozymes Differentially Regulate Promoters Containing PEA-3/12-O-Tetradecanoylphorbol-13-acetate Response Element Motifs

To investigate the regulation of promoters containing classical phorbol ester response sequences (PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs) by protein kinase C (PKC) isozymes, co-transfections were performed in human dermal fibroblasts with a plasmid containing either the human collagenase promoter or the porcine urokinase plasminogen activator (uPA) promoter linked to the chloramphenicol acetyltransferase gene and a plasmid expressing an individual PKC isozyme. Using this experimental design, seven PKC isozymes were analyzed for their ability to trans-activate the collagenase and uPA promoters. Our results demonstrate that only PKC delta, epsilon, and eta trans-activated the collagenase promoter and that binding of Ap-1 family members to the collagenase 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was not responsible for the isozyme-specific trans-activation. In contrast, the uPA promoter was stimulated by all of the PKC isozymes examined (PKC alpha, betaII, gamma, delta, epsilon, zeta, and eta). These results indicate that PKC isozymes differentially regulate promoters containing PEA-3/TRE motifs and suggest that individual isozymes play unique roles within the cell.

Protein kinase C isozymes in human megakaryoblastic leukemia cell line, MEG-01: possible involvement of the isozymes in the differentiation process of MEG-01 cells.

The expression of the different protein kinase C (PKC) isozymes in various states of differentiation of the human megakaryoblastic leukaemia cell line MEG-01 were analysed using thermocycle amplification of mRNA and immunoblotting. MEG-01 expressed mRNAs of PKC alpha, -beta I, -beta II, -delta, -epsilon, -eta, -theta and -zeta, but not PKC gamma. At the protein molecule level, MEG-01 was observed to express PKC alpha, -beta I, -beta II,- epsilon, -theta and -zeta, but lack -gamma, -delta and -eta. When differentiation of MEG-01 was induced by 100 nm 12-O-tetradecanoyl-phorbol-13-acetate (TPA), rapid translocation from cytosol to membrane fraction and down-regulation of PKC alpha, -epsilon and -theta was observed in 1-2h. On the other hand, PKC beta I and -beta II were observed to translocate not only to the membrane fraction but also to the cytoskeletal fraction in a different manner, and their down-regulation, especially beta II, was very slow. The myristoylated, alanine-rich C kinase substrate (MARCKS) in the membrane fraction of MEG-01 cells was observed to decrease gradually throughout the differentiation process. Additionally, time-course study of TPA treatment indicated that incubation of the cells for 30 min is sufficient for differentiation. These results strongly suggest that the activation of PKC alpha, -epsilon and -theta is involved in the initiation of differentiation, and that PKC beta I and -beta II have important roles in the maintenance of differentiation. Although PKC zeta was resistant to TPA treatment and its translocation was reduced, the amount of this isozyme in the cytosol fraction decreased throughout the differentiation process.

Characterization of IκB Kinases: IκB-α IS NOT PHOSPHORYLATED BY Raf-1 OR PROTEIN KINASE C ISOZYMES, BUT IS A CASEIN KINASE II SUBSTRATE

The NF-kappaB transcription factor is activated by a wide variety of stimuli, including phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate. In its inactive state, NF-kappaB is sequestered in the cytoplasm tethered to an inhibitor protein, IkappaB. Activation comprises the rapid phosphorylation of IkappaB-alpha at N-terminal sites, which presumably marks IkappaB-alpha for proteolytic degradation and leads to release of NF-kappaB into the nucleus. In addition, IkappaB-alpha is constitutively phosphorylated at the C terminus, which may be a prerequisite for proper IkappaB function. Protein kinase C (PKC) is activated by 12-O-tetradecanoylphorbol-13-acetate and has been previously reported to phosphorylate IkappaB-alpha in vitro. As PKC has turned out to constitute a multigene family encoding isozymes with different biological functions, we have reinvestigated IkappaB-alpha phosphorylation by PKC using recombinant PKC isozymes expressed in insect cells. While crude PKC preparations were efficient IkappaB-alpha kinases, highly purified PKC isozymes completely failed to phosphorylate IkappaB-alpha. Biochemical separation of porcine spleen yielded at least two fractions with IkappaB-alpha kinase activity, both of which were devoid of detectable PKC isozymes. One peak contained both Raf-1 and casein kinase II (CKII). Purified Raf-1 does not phosphorylate IkappaB-alpha directly, but associates with CKII, which efficiently phosphorylates the C terminus of IkappaB-alpha. Two-dimensional phosphopeptide mapping and high pressure liquid chromatography-mass spectroscopy analysis showed that all IkappaB-alpha kinases induced phosphorylation at the same prominent sites in the C terminus. Our results clearly indicate that PKC isozymes alpha, beta, gamma, delta, epsilon, eta, and zeta as well as Raf-1 are not IkappaB-alpha kinases. They furthermore demonstrate that IkappaB-alpha is targeted by several kinases, one of which appears to be CKII.

Role of protein kinase C alpha in nerve growth factor-induced arachidonic acid release from PC12 cells.

Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 microM) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs alpha, delta, epsilon, and zeta. PMA down-regulation depleted PKCs alpha, delta, and epsilon, and partially depleted zeta. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs alpha and beta specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs alpha, beta, and gamma, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC alpha plays a role in NGF-induced AA release.