Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.     

Stimulation of human B lymphocytes by phorbol esters reported to be selective in the protein kinase C isoforms they activate

It is reported here that B cells can be stimulated by two phorbol esters which, in cell free substrate phosphorylation assays, are selective in the PKC isoforms they activate: thymeleatoxin (Thy) stimulates all of the classical (c) or Group A PKCs (alpha, beta 1, beta 2 and gamma) but not PKC delta and epsilon which belong to the novel (n) or Group B PKCs, while 12-deoxyphorbol-13-O-phenylacetate-20-acetate (dPPA) is a specific activator of PKC beta 1. By itself, phorbol 12-myristate, 13-acetate (PMA)--which activates all cPKC and nPKC--was, on a molar basis, some 40-times more potent than either Thy or dPPA which were themselves equipotent at promoting DNA synthesis in resting B cells: the peak response achieved with Thy and dPPA was higher (1.4 x) than that obtained with PMA. In the presence of calcium ionophore, PMA, Thy and dPPA all stimulated a higher (and equivalent) peak response which was achieved at a lower phorbol ester concentration in each case: however, whereas Thy now approached PMA in potency, dPPA remained some 40-times less potent.     

Persistent membrane translocation of protein kinase C alpha during 12-0-tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells

Others have reported that the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator and down-regulator of most protein kinase C (PKC) isozymes, can induce apoptotic cell death of androgen-sensitive LNCaP but not androgen-insensitive PC-3 or DU 145 human prostate cancer cells. As a first step toward uncovering the mechanism by which TPA induces apoptosis of LNCaP cells, we quantified expression of PKC isozyme mRNAs in unmodified and TPA-resistant LNCaP cells and in naturally TPA-resistant PC-3, PC-3M, and DU 145 cells. All of the cell lines and normal prostate expressed RNAs for PKC alpha, delta, epsilon, eta, and mu; only DU 145 cells and normal prostate expressed PKC beta and theta RNAs, and none expressed PKC gamma. The amount of PKC alpha RNA and protein was 6- to 38-fold lower, and PKC mu RNA was 4.5- to 16.5-fold higher in unmodified and TPA-resistant LNCaP cells than in the androgen-independent cells. We examined the effects of TPA on PKC alpha and mu mRNA levels and on membrane translocation of PKC alpha. Incubation with TPA for 6 h or more induced 95% inhibition of cell growth, a transient 12-fold increase and 5-fold decrease in PKC alpha and mu mRNA levels, respectively, and prolonged translocation of PKC alpha to non-nuclear membranes in unmodified LNCaP cells and in TPA-resistant LNCaP cells from which TPA had been removed for 10 days. TPA-resistant LNCaP cells in the continuous presence of TPA, or 24 h after removal of TPA, had down-regulated PKC alpha and remained resistant to re-addition of TPA. These data demonstrate a strong correlation of the presence and absence of membrane PKC alpha with apoptosis and resistance to apoptosis, respectively.     

The bryostatins inhibit growth of B16/F10 melanoma cells in vitro through a protein kinase C-independent mechanism: dissociation of activities using 26-epi-bryostatin 1

Bryostatin 1 is a potential cancer chemotherapeutic agent in Phase II clinical trials, with positive responses observed for malignant melanoma, among other tumors. The bryostatins are known to be potent ligands for protein kinase C (PKC), functioning as partial antagonists. In the present study, we explore the mechanism by which the bryostatins inhibit growth to B16/F10 mouse melanoma cells in vitro. Three experimental approaches suggest that the growth inhibition is independent of PKC. First, we characterized in detail the translocation and down-regulation of the PKC isozymes alpha, delta, and epsilon in response to phorbol ester and bryostatin 1 in these cells. Although the dose-response curves obtained for the translocation-activation of PKC isozymes showed good correlation with the growth-enhancing activity of phorbol 12-myristate 13-acetate, for no PKC isozyme was there a good correlation with the growth-inhibitory activity of bryostatin 1. Second, inhibition PKC, inhibited the growth of the B16/F10 melanoma cell lines with potency similar to that of bryostatin 1. We confirmed here that 26-epi-bryostatin 1 showed 60-fold reduced affinity for PKC and 30-60-fold reduced potency to translocate and downregulate PKC isozymes compared with bryostatin 1. We presumed that the principal toxicity of bryostatin 1 reflects its interaction with PKC, and we would thus predict that epi-bryostatin 1 would be less toxic. Indeed, we found at least 10-fold reduced toxicity of 26-epi-bryostatin 1 in C57BL/6 mice compared with bryostatin 1. We conclude that the growth inhibition of the bryostatins, at least in this system, does not result from interaction with PKC. As exemplified by 26-epi-bryostatin 1, this insight permits the design of analogues with comparable growth inhibition to bryostatin 1 but with reduced toxicity.     

Expression of protein kinase C isozymes in human basophils: regulation by physiological and nonphysiological stimuli

The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) +/- ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCbetaI, betaII, delta, and were expressed, PKCalpha was difficult to detect, and PKCgamma and eta were undetectable. In unstimulated basophils, PKCbetaI and betaII were found primarily in the cytosol fraction (95% +/- 3% of total and 98% +/- 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCbetaI and betaII were translocated to the membrane fraction (85% +/- 4% and 83% +/- 6%, respectively). In resting cells, 48% +/- 3% and 61% +/- 10% of PKCdelta and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% +/- 6% of PKC was found in the membrane fraction, however, no translocation of PKCdelta was apparent. Stimulation with FMLP caused modest translocation ( approximately 20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti-IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.     

Different isozymes of protein kinase C mediate feedback inhibition of phospholipase C and stimulatory signals for exocytosis in rat RBL-2H3 cells

Previous studies indicated that rat basophilic RBL-2H3 cells contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C (PKC); of these, PKC beta and delta were the most potent transducers of signals for exocytosis in antigen-stimulated permeabilized cells. Exocytosis, nevertheless, was still dependent on an elevated free Ca2+. (Ozawa, K., Szallasi, Z., Kazanietz, M. G., Blumberg, P. M., Mischak, H., Mushinski, J. F., and Beaven, M. A. (1993) J. Biol. Chem. 268, 1749-1756). We now demonstrate that PKC alpha and epsilon, exclusively, inhibit antigen-induced hydrolysis of inositol phospholipids in the same permeabilized RBL-2H3 cells. Unlike secretion, the inhibitory actions occurred at a basal concentration (0.1 microM) of free Ca2+. The inhibitory actions of the two isozymes were potentiated by 20 nM phorbol 12-myristate 13-acetate. As indicated by the effects of the phorbol ester, the probable mechanism was reduced tyrosine phosphorylation of phospholipase C gamma 1. The negative regulation of phospholipase C was apparent in intact cells, because the PKC inhibitor Ro31-7549 or down-regulation of PKC with phorbol ester enhanced antigen-induced hydrolysis of inositol phospholipids. The concentrations of the various isozymes of PKC in RBL-2H3 cells, as estimated by immunoblotting studies, were sufficient for promotion of exocytosis (i.e. beta and delta) and inhibition of phospholipid hydrolysis (i.e. alpha and epsilon).     

Role of nitric oxide and vasoactive intestinal polypeptide in vagally mediated relaxation of the gastric corpus in the anaesthetized ferret

We have examined the role of protein kinase C (PKC) in the stimulation of protein synthesis by insulin using two complementary approaches. In the first, fibroblasts were pretreated with phorbol esters to down-regulate PKC. In these cells, the effects of insulin and of phorbol esters on protein synthesis were completely abolished, although serum still elicited an effect approaching that seen in control cells. Secondly, we used newly developed inhibitors of PKC which, again, blocked the effects of insulin and phorbol esters without greatly reducing the response to serum. Thus PKC apparently plays an important role in the stimulation of translation by insulin.     

Protein kinase C is not necessary for transforming growth factor beta-induced growth-arrest in leukemia cell lines

Growth and differentiation of blood cell precursors are regulated by cytokines and hormones by mechanisms which are incompletely understood. Protein kinase C (PKC) isozymes are widely regarded as being important in signal transduction pathways. We have shown that one isozyme, PKC beta, is uniquely important in mediating phorbol ester-induced growth-arrest in the HL-60 myeloid cell line. 1,25-dihydroxyvitamin D3 induces differentiation and growth-arrest in many cells. It upregulates the expression of PKC beta, potentiating the action of phorbol ester. We tested the hypotheses that cytokines, which arrest the growth of hematopoietic cells, do so by activating PKC beta, and that differentiation and growth-arrest induced by 1,25-dihydroxyvitamin D3 is caused by upregulation of PKC beta isozyme gene expression. The influence on growth of combinations of five cytokines (TNF alpha, TGF beta 1, gamma-IFN, IL-1, and G-CSF) and 1,25-dihydroxyvitamin D3 on ten human leukemia cell lines (THP-1, HL-60 S, HL-60 PET, U937, K562, Jurkat, MOLT-4, RPM1 8402, KG-1, and KG-1a) was determined. Four cell lines (THP-1, HL-60 S and PET, and U937) exhibited total growth-arrest when incubated with 1,25-dihydroxyvitamin D3 followed by TGF beta 1. The expression by each cell line of mRNA encoding PKC alpha, beta, and delta, both before and after 24 or 48 h of incubation with 1,25-dihydroxyvitamin D3, was determined. Cell lines sensitive to TGF beta 1 each expressed PKC delta endogenously, or expression was up-regulated with 1,25-dihydroxyvitamin D3. U937 cells underexpressed PKC alpha, and HL-60 PET cells underexpressed PKC beta. These data suggested that PKC delta could be responsible for mediating growth-arrest by TGF beta 1. To test this hypothesis directly, we incubated the cells with two bisindolylmaleimide PKC inhibitors during the addition of 1,25-dihydroxyvitamin D3 and TGF beta 1. Surprisingly, the PKC inhibitors did not block the growth-arrest induced by 1,25-dihydroxyvitamin D3 and TGF beta 1. This experiment strongly suggests that neither growth-arrest induced by TGF beta 1 nor the potentiation of this growth-arrest by 1,25-dihydroxyvitamin D3 is mediated by a PKC isozyme which is inhibitable by the bisindolymaleimides.     

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.     

Protein kinase C isozyme expression and down-modulation in growing, quiescent, and transformed renal proximal tubule epithelial cells

Renal alpha-protein kinase C (PKC) is rapidly down-modulated modulated in animals treated with the renal toxin and tumor promoter, folic acid (Dong et al., Cancer Res., 53: 4542-4549, 1993). To further explore the role of PKC isozymes in renal growth and carcinogenesis, we compared phorbol ester receptor and PKC isozyme content, distribution, and regulation in primary and oncogene-altered rat renal proximal tubule epithelial cells (RPTE) in culture. Immunoblot analysis and RNase protection assays indicated that RPTE expressed at least four PKC isozymes, alpha, delta, epsilon, and zeta. Total phorbol ester receptors were decreased in primary proliferating, E1A-immortalized, and SV40-transformed RPTE compared to primary quiescent RPTE. The decrease in PDBu binding was largely due to a specific decrease in alpha-PKC protein content to approximately 50% of the level in quiescent RPTE. Degradation rates and message levels were compared to determine the mechanism for the decrease in alpha-PKC. Whereas alpha-PKC message levels in quiescent and proliferating primary RPTE were comparable, alpha-PKC degradation was increased in proliferating cells. These results indicate that the decreased alpha-PKC content was due largely to increased turnover. Phorbol ester stimulated the rate of degradation, thus demonstrating a link between degradation rate and PKC activation. These results suggest that the increased basal degradation rate in proliferating and oncogene-altered cells reflects an increase in activity of PKC in these cells.     

Tissue equivalent vessel phantoms for intravascular ultrasound

The presence of the non-selective protein kinase C (PKC) inhibitors, staurosporine (100 nM) and polymyxin B (100 microM) in cultured human RPE cells for more than 24 h triggers apoptotic death. Apoptosis is characterized by a diminishing number of cells, a labelling of nuclei by the TUNEL method and by observable morphological changes. An inhibitor of PKC and cyclic nucleotide-dependent protein kinases, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H-7; 100 microM), was without effect, as was the specific PKC inhibitor, calphostin C (100 nM). The PKC-activating phorbol esters, phorbol-12-myristate-13-acetate (PMA; 1 microM) and phorbol-12,13-dibutyrate (PDB; 1 microM) and the non-tumour-promoting phorbol ester, 4 alpha-PMA (1 microM) were without effect, as was the diacyl glycerol analogue, 1,2-dioctanoyl-snglycerol (DOG; 10 microM). The PKC activators did not attenuate the apoptosis induced by staurosporine or polymyxin B. Furthermore, deprivation of glucose and oxygen (simulated ischemia) for 72 h induced apoptosis: this could be prevented by inclusion of 10% (v/v) foetal bovine serum (FBS) but not by a variety of PKC activators. Six PKC isoenzymes were shown to be present in RPE cells (alpha, beta 1, beta 2, delta, epsilon, E) and only the calcium-dependent cPKC levels changed after treatment with staurosporine or simulated ischaemia. Since only the less selective inhibitors of PKC induced apoptosis, it is suggested that PKC is not involved directly in the induction process of apoptosis in RPE cells. It is possible that the staurosporine and polymyxin B-induced effects of apoptosis in RPE cells are triggered by an unknown kinase-dependent pathway, but whether the 'ischaemia'-induced death is related to this same process remains to be elucidated.     

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.     

Role of protein kinase C (PKC) in agonist-induced mu-opioid receptor down-regulation: I. PKC translocation to the membrane of SH-SY5Y neuroblastoma cells is induced by mu-opioid agonists

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.     

The sevenfold way of PKC regulation

Protein kinase C (PKC) is a family of enzymes that are physiologically activated by 1,2-diacylglycerol (DAG) and other lipids. To date, 11 different isozymes, alpha, betaI, betaII, gamma, delta, epsilon, nu, lambda(iota), mu, theta and zeta, have been identified. On the basis of their structure and activators, they can be divided into three groups, two of which are activated by DAG or its surrogate, phorbol 12-myristate 13-acetate (PMA). PKC isozymes are remarkably different in number and prevalence in different cell lines and tissues. When activated, the isozymes bind to membrane phospholipids or to receptors that are located in and anchor the enzymes in a subcellular compartment. Some PKCs may also be activated in their soluble form. These enzymes phosphorylate serine and threonine residues on protein substrates, perhaps the best known of which are the myristoylated, alanine-rich C kinase substrate and nuclear lamins A, B and C. The enzymes clearly play a role in signal transduction, and, because of the importance of PMA as a tumor promoter, they are thought to affect some aspect of cell cycling. How PKC takes part in the regulation of cell transformation, growth, differentiation, ruffling, vesicle trafficking and gene expression, however, is largely unknown.     

Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.     

Modulation of dopamine release from rat striatum by protein kinase C: interaction with presynaptic D2-dopamine-autoreceptors

1. Interactions between dopamine receptors and protein kinase C (PKC) have been proposed from biochemical studies. The aim of the present study was to investigate the hypothesis that there is an interaction between protein kinase C and inhibitory D2-dopamine receptors in the modulation of stimulation-induced (S-I) dopamine release from rat striatal slices incubated with [3H]-dopamine. Dopamine release can be modulated by protein kinase C and inhibitory presynaptic D2 receptors since phorbol dibutyrate (PDB) and (-)-sulpiride, respectively, elevated S-I dopamine release. 2. The protein kinase C inhibitors polymyxin B (21 microM) and chelerythrine (3 microM) had no effect on stimulation-induced (S-I) dopamine release. However, when presynaptic dopamine D2 receptors were blocked by sulpiride (1 microM), an inhibitory effect of both PKC inhibitors on S-I dopamine release was revealed. Thus, sulpiride unmasks an endogenous PKC effect on dopamine release which suggests that presynaptic D2 receptors normally suppress endogenous PKC activity. This is supported by results in striatal slices which were pretreated with PDB to down-regulate PKC. In this case the facilitatory effect of sulpiride was completely abolished. 3. The inhibitory effect of the dopamine D2/D3 agonist quinpirole on S-I dopamine release was partially attenuated by PKC down-regulation. Since the effect of sulpiride was completely abolished under the same conditions, this suggests that exogenous agonists may target a PKC-dependent as well as a PKC-independent pathway. The inhibitory effect of apomorphine was not affected by either polymyxin B or PKC down-regulation, suggesting that it operated exclusively through a PKC-independent mechanism. 4. These results suggest that there are at least two pathways involved in the inhibition of dopamine release through dopamine receptors. One pathway involves dopamine receptor suppression of protein kinase C activity, perhaps through inhibition of phospholipase C activity and this is preferentially utilized by neuronally-released dopamine. The other pathway which seems to be utilized by exogenous agonists does not involve PKC.     

The hepatitis C virus NS2 protein generated by NS2-3 autocleavage is required for NS5A phosphorylation

Protein kinase C (PKC) is implicated in the regulation of a variety of important functions in small cell lung cancer (SCLC) cell lines, but the downstream signaling targets stimulated by PKCs in these cells remain poorly characterized. Here we report that treatment of the SCLC cell lines H 69, H 345, and H 510 with phorbol-12,13-dibutyrate (PDB) led to a rapid and striking activation of protein kinase D (PKD), a novel serine/threonine protein kinase distinct from all PKC isoforms. PKD activation induced by PDB in these SCLC cell lines was completely abrogated by treatment of the cells with the PKC inhibitor GF 109203X (GF I) at concentrations (0.5-2.5 microM) that did not inhibit PKD activity when added directly to the in vitro kinase assays. Treatment with the biologically active phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with membrane-permeable diacylglycerols also stimulated PKD activation, which was also completely prevented by prior exposure of the cells to GF I. The PKC inhibitors Ro 31-8220 and Go 7874 also blocked PKD activation in response to PDB. Addition of the autocrine growth factor bombesin to cultures of H 345 cells induced significant PKD activation that also was prevented by GF I. Our results demonstrate, for the first time, the existence of a PKC/PKD pathway in SCLC cells and raise the possibility that PKD may be an important mediator of some of the biological responses elicited by PKC activation in SCLC cells.     

Determination of the specific substrate sequence motifs of protein kinase C isozymes

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (alpha, betaI, betaII, gamma, delta, epsilon, eta, mu, and zeta) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position -3. All isozymes, except PKC mu, selected peptides with basic amino acids at positions -6, -4, and -2. PKC alpha, -betaI, -betaII, -gamma, and -eta selected peptides with basic amino acid at positions +2, +3, and +4, but PKC delta, -epsilon, -zeta, and -mu preferred peptides with hydrophobic amino acid at these positions. At position -5, the selectivity was quite different among the various isozymes; PKC alpha, -gamma, and -delta selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKC mu showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position -3 to +2 for PKC alpha, -betaI, -betaII, -gamma, -delta, and -eta were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKC alpha, -betaI,-delta, -zeta, and -mu were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.     

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.     

Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively. PMA at 1 microM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%. Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCbeta) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA. Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%. Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production. The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane. Western blot analysis revealed the presence of eight PKC isoforms (alpha, betaI, betaII, delta, epsilon, mu, lambda and xi) in RAW 264.7 cells and PMA was shown to induce the translocation of the alpha, betaI, betaII, delta, epsilon and mu isoforms from the cytosol to the cell membrane within 2 min. Pretreatment of cells with PMA for 2-24 h resulted in a time-dependent down-regulation of PKCalpha, betaI, betaII, and delta expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time-dependent down-regulation of PKC. These results indicate that PKCbeta has a crucial role in the mediation of cPLA2 activation by the phorbol ester PMA, whereas PMA utilizes PKC epsilon and/or mu to up-regulate AC activity.