Translocation Of Protein Kinase C Delta Research Articles

Secreted PLA2 induces proliferation in astrocytoma through the EGF receptor: another inflammation-cancer link

We have investigated mechanisms that contribute to reinforce the relationship between inflammation and cancer. Secreted phospholipase A(2) group IIA (sPLA(2)-IIA) is a molecule relevant in inflammatory events and has been proposed as a marker for some of these. Previously, we reported the mitogenic properties of this sPLA(2) in the human astrocytoma cell line 1321N1. Here, we go deeper into the mechanisms that link this inflammatory protein with proliferation in one of the most aggressive types of tumors. We found that phosphorylation of the extracellular regulated kinase (ERK) was preceded by the activation of the small GTPase Ras, and both failed to be activated by inhibiting protein kinase C (PKC). Fractionation and immunofluorescence studies revealed translocation of PKC alpha, delta, and epsilon to the membrane fraction upon stimulation with sPLA(2)-IIA. Immunoprecipitation analysis showed that sPLA(2)-IIA induces phosphorylation of the epidermal growth factor receptor (EGFR) through a PKC-dependent pathway. We found that phosphorylation of this receptor contributed to Ras and ERK activation and that inhibition of ERK, PKC, and EGFR blocked the mitogenic response induced by sPLA(2)-IIA. This study showed that sPLA(2)-IIA is able to bring into play EGFR to trigger its signaling and that PKC leads the distribution of resources. Interestingly, we found that this is not a cell-specific response, because sPLA(2)-IIA was also able to transactivate EGFR in MCF7 human breast cancer cells. Therefore, this mechanism could contribute to worsen the prognosis of a tumor in an inflammatory microenvironment. We also present more links of the tumor chain possibly susceptible to targeting.

Involvement of protein kinase C isoenzymes in Trypanosoma cruzi metacyclogenesis induced by oleic acid

Previously, we showed that oleic acid (OA) induces Trypanosoma cruzi metacyclogenesis through a signaling pathway involving de novo diacylglycerol biosynthesis and simultaneous protein kinase C (PKC) activation. Herein, we demonstrated that OA also triggers a transient Ca(2+) signal in epimastigotes, necessary for parasite differentiation, that could account for PKC activation. In addition, we found that this free fatty acid (FFA) directly stimulated in vitro the activity of T. cruzi PKC in a dose-response way. We determined the presence of classical and novel PKC isoenzymes that were differentially expressed in the infective amastigotes (alpha and delta) and tripomastigotes (alpha, beta, and gamma) and in the non-infective epimastigotes (alpha, beta, gamma, and delta). We also demonstrated that OA induced in epimastigotes the translocation of PKC alpha, beta, gamma, and delta to the membrane, indicating a selective effect of this FFA. To establish a correlation between T. cruzi metacyclogenesis induced by OA and the activation of a particular PKC isoenzyme, the specific PKC inhibitors Ro 32-0432 and Rottlerin (9-30 nM and 5-35 microM, respectively) were employed. These compounds, even at the lowest concentrations assayed, abrogated both epimastigote differentiation and membrane translocation of PKC beta, gamma, and delta. These findings strongly support a key role for classical and novel PKC isoenzymes in the signaling pathways involved in T. cruzi metacyclogenesis induced by OA.

Low concentrations of sphingosylphosphorylcholine enhance pulmonary artery vasoreactivity: the role of protein kinase C delta and Ca2+ entry.

Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC(50) is approximately 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (<or=1 micromol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca(2+)]. SPC (1 micromol/L) had no effect on force or intracellular [Ca(2+)] on its own, but dramatically potentiated constrictions induced by approximately 25 mmol/L [K(+)], such that at 40 minutes, force and intracellular [Ca(2+)] (Fura PE3 340/380 ratio) were increased by 429+/-96% and 134+/-26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane-sensitive Ca(2+) entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC delta inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC delta. The potentiation could be attributed to enhancement of Ca(2+) entry. SPC also potentiated the responses to prostaglandin F(2 alpha) and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 micromol/L) caused translocation of PKC delta to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca(2+) entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC delta. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Analysis of protein kinase C delta (PKC δ) expression in endometrial tumors

Endometrial cancer is the most common gynecologic malignancy in the United States. However, its underlying molecular mechanisms are poorly understood; and few prognostic indicators have been identified. The protein kinase C (PKC) family has been shown to regulate pathways critical to malignant transformation; and in endometrial tumors, changes in PKC expression and activity have been linked to a more aggressive phenotype and poor prognosis. We have recently shown that PKC delta is a critical regulator of apoptosis and cell survival in endometrial cancer cells; however, PKC delta levels in endometrial tumors had not been determined. We used immunohistochemistry to examine PKC delta protein levels in normal endometrium and endometrioid carcinomas of increasing grade. Normal endometrium exhibited abundant nuclear and cytoplasmic staining of PKC delta confined to glandular epithelium. In endometrial tumors, decreased PKC delta expression, both in intensity and fraction of epithelial cells stained, was observed with increasing tumor grade, with PKC delta being preferentially lost from the nucleus. Consistent with these observations, endometrial cancer cell lines derived from poorly differentiated tumors exhibited reduced PKC delta levels relative to well-differentiated lines. Treatment of endometrial cancer cells with etoposide resulted in a translocation of PKC delta from cytoplasm to nucleus concomitant with induction of apoptosis. Decreased PKC delta expression, particularly in the nucleus, may compromise the ability of cells to undergo apoptosis, perhaps conferring resistance to chemotherapy. Our results indicate that loss of PKC delta is an indicator of endometrial malignancy and increasing grade of cancer. Thus, PKC delta may function as a tumor suppressor in endometrial cancer.

Selective activation of protein kinase C delta in human neutrophils following ischemia reperfusion of skeletal muscle.

Circulatory neutrophils are known to be critical mediators of inflammation and oxidative stress during ischemia reperfusion (I/R) injury. Recent studies have shown an important role for protein kinase C (PKC) in neutrophil survival and function. Activation of specific isotypes of PKC are known to be involved in membrane alteration and motility, oxidative phosphorylation, and apoptosis modulation of neutrophils. However, the role of PKC in neutrophil responses to I/R in the clinical setting has not been studied. In this study, we examined the neutrophil activation of PKC induced by tourniquet-controlled I/R of skeletal muscle in humans. We found that I/R rapidly activates and translocates PKC delta, but not any of the classical forms of PKC (alpha or beta) from cytosol to the particulate fraction of neutrophils. Particulate translocation of PKC delta is sustained up to 4 h after reperfusion and is associated with kinase activity. Postreperfusion activation of PKC delta in neutrophils signals proapoptosis, but does not cause immediate cell death (as revealed by neutrophil morphology study and DNA-laddering assay). This study indicates that calcium-independent novel PKC delta (nPKC delta) might be predominantly involved in regulating membrane functions and survival of neutrophils associated with post-I/R-induced inflammatory oxidative stress.

Magnesium sulfate induces translocation of protein kinase C isoenzymes alpha and delta in myometrial cells from pregnant women

ObjectivesThe purpose of this study was to determine the effect of magnesium sulfate on protein kinase C (PKC) translocation in myometrial cells from pregnant women. Study designMyometrium was obtained at the time of cesarean delivery from women at term before labor. Cultured myometrial cells were treated with magnesium sulfate (3, 5, and 10 mmol/L), oxytocin (0.1 μmol/L), or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 μmol/L). The translocation of PKC isozymes alpha (calcium dependent) and delta (calcium independent) from cytosol to membrane fractions was assessed with use of Western blot analysis. ResultsIn unexposed control cells, the majority of PKC alpha and delta was located in the cytosol fraction. Exposure to magnesium sulfate for 60 minutes induced translocation of both PKC alpha and delta at concentrations as low as 5 and 3 mmol/L, respectively. The magnitude of magnesium sulfate induced translocation for PKC delta is similar to that seen after oxytocin or TPA exposure but less for PKC alpha. Exposure to oxytocin for 30 minutes and 60 minutes induced translocation of PKC alpha and delta, respectively. Exposure to TPA for 5 and 30 minutes induced translocation of PKC alpha and PKC delta, respectively. In calcium-free media, only TPA induced translocation of these two isoenzymes. ConclusionMagnesium sulfate stimulates PKC translocation in cultured myometrial cells from pregnant women. Magnesium sulfate and oxytocin require extracellular calcium to induce translocation of both PKC alpha and delta.

Pro-apoptotic protein kinase C delta is associated with intranuclear inclusions in a transgenic model of Huntington's disease.

In order to investigate any effect of truncated mutant huntingtin (tNhtt) aggregation on protein kinase C (PKC) signaling in Huntington's disease (HD), we studied a possible association of PKC isoforms with the aggregates using cellular and transgenic models of HD. In this report we describe an association of mutant tNhtt with at least three PKC isoforms (alpha, delta, zeta), as revealed by co-immunoprecipitation assays and immunocytochemistry in a cellular model of HD (Neuro2a cells expressing tNhtt-150Q-EGFP), as well as a specific association of PKC delta with intranuclear aggregates in a transgenic model (R6/2 mice). Immunoblot analysis of isolated nuclear fractions shows an elevation of nuclear PKC delta in transgenic brain tissue. The observed elevation has a strong similarity with the apoptotic translocation of PKC delta detected in experiments with the mouse neuroblastoma Neuro2a cells. Using a Neuro2a cell line expressing tNhtt with the nuclear localization signal, we demonstrate the association of PKC delta with intranuclear aggregates and present evidence that accumulation of PKC delta in cell nuclei does not depend on mutant htt nuclear translocation. Our results suggest that the association of PKC delta with intranuclear htt-aggregates may affect its apoptotic function in a transgenic model of HD.

Differential expression of protein kinase C isoforms in streptozotocin-induced diabetic rats

The cellular effects of hyperglycemia are mediated by protein kinase C (PKC). However, PKC consists of several distinct isoforms, and their contribution to the pathogenesis of diabetic complications in different organs is not clear. We investigated the expression and translocation of PKC isoforms alpha, betaI, betaII, delta, epsilon, and zeta in kidney, heart, and aorta from diabetic rats. Hyperglycemia was induced with streptozotocin (70 mg/kg) in the rat. After four weeks, PKC isoform expression was assessed by Western blot after tissue fractionation and by immunohistochemistry. Streptozotocin increased blood glucose from 117.0 +/- 3.6 to 510.0 +/- 19.4 mg/dl (N = 8, P < 0.01) and induced albuminuria. PKC isoforms alpha, beta, delta, epsilon, and zeta were all detected in control animals. Western blot showed increased PKC alpha expression in kidney and heart (160% and 170%, respectively). PKC betaI, betaII, and delta expression was not influenced by hyperglycemia. PKC zeta was decreased in diabetic animals in both tissues by 60%. The membrane association of PKC alpha and PKC epsilon was increased; however, the relative amount of PKC in the particulate fraction was not influenced by hyperglycemia. Immunohistochemistry revealed a marked increase in PKC alpha immunoreactivity in renal glomeruli and interstitial capillaries, cardiac capillaries, and skeletal muscle, as well as in the endothelial cells of larger arteries. PKC beta showed a small decrease in the glomeruli. PKC epsilon was increased in renal tubules in diabetic rats but was decreased in the myocardium. PKC zeta was expressed in both myocardial and glomerular cells but was decreased during hyperglycemia. Our results demonstrate that PKC isoforms are differentially regulated in kidney and heart in diabetes. High glucose increases PKC alpha expression, whereas PKC zeta is down-regulated. The finding that PKC alpha is mostly increased in endothelial cells supports a role for PKC alpha in functional endothelial disturbances observed in diabetes.

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes.

Previous studies have demonstrated that cyclic strain induces keratinocyte proliferative and morphological changes. Since protein kinase C (PKC) is known to play an important role in the regulation of keratinocyte growth and differentiation, the objective of this study was to determine the role of the PKC signaling pathway as a mediator of strain modulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activity and translocation, (2) cyclic strain activates PKC in an isoform-specific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test these hypotheses, keratinocytes were subjected to vacuum-generated cyclic strain (10% average strain), followed by measurement of PKC activity, PKC isoform distribution by Western blot analysis and confocal microscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological changes. We observed stimulation of PKC activity (62.3 +/- 5.1% increase) coupled with translocation of PKC from the cytosolic to the membrane fraction in keratinocytes subjected to acute cyclic strain. Cyclic strain also caused translocation of PKC alpha and delta, but not zeta isoforms, from the cytosolic to the membrane fraction as demonstrated by both Western blot analysis and confocal microscopy. PKC beta was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and staurosporine (5 nM), inhibited strain-induced PKC activation and keratinocyte proliferation, but did not block the effects of strain on cellular morphology or alignment. We conclude that these data support our hypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreover, our studies with PKC inhibitors support the hypothesis that strain-induced changes in the keratinocyte phenotype may be selectively modulated by PKC.

Protein Kinase C Isozyme-mediated Cell Cycle Arrest Involves Induction of p21waf1/cip1 and p27kip1 and Hypophosphorylation of the Retinoblastoma Protein in Intestinal Epithelial Cells

The molecular mechanisms underlying protein kinase C (PKC) isozyme-mediated control of cell growth and cell cycle progression are poorly understood. Our previous analysis of PKC isozyme regulation in the intestinal epithelium in situ revealed that multiple members of the PKC family undergo changes in expression and subcellular distribution precisely as the cells cease proliferating in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC isozyme(s) in control of intestinal epithelial cell growth and cell cycle progression was examined directly using the IEC-18 immature crypt cell line as a model system. Treatment of IEC-18 cells with PKC agonists resulted in translocation of PKC alpha, delta, and epsilon from the soluble to the particulate subcellular fraction, cell cycle arrest in G1 phase, and delayed transit through S and/or G2/M phases. PKC-mediated cell cycle arrest in G1 was accompanied by accumulation of the hypophosphorylated, growth-suppressive form of the retinoblastoma protein and induction of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC alpha, delta, and epsilon. Differential down-regulation of individual PKC isozymes revealed that PKC alpha in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC alpha in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Regulation of protein kinase C isoform proteins in phorbol ester-stimulated Jurkat T lymphoma cells

Activation of protein kinase C (PKC) in T cells leads to a variety of responses including IL-2 production and IL-2 receptor expression. PKC consists of several isoforms that exhibit some different in vitro properties. We have set up a Western blotting system to explore the regulation of PKC isoforms during T cell activation. In Jurkat T lymphoma cells, PKC alpha, beta, delta, epsilon, and zeta were detected. PKC alpha and beta existed primarily in the cytosol, translocated to the membrane fraction after 10 minutes of treatment with PMA, and almost completely disappeared within 16 h. A larger fraction of PKC delta and epsilon existed in the membrane fraction compared to PKC alpha or beta, and PKC epsilon translocated to the membrane fraction rapidly. Translocation of PKC delta was not apparent after 1 h treatment with PMA, but total PKC delta protein was reduced within 4 to 6 h of treatment. Consistent with this, overnight treatment with PMA caused down-regulation of both PKC delta and epsilon, but to a lesser degree than was observed with PKC beta. Anti-PKC zeta antibody detected two bands at 82 and 75 kDa. The 75-kDa band existed mostly in the cytosol fraction and showed no translocation or down regulation after PMA. We present evidence that this 75-kDa band represents PKC zeta. Similar PMA-induced translocation responses were observed in murine thymocytes showing that the responses are not unique to PKC isoforms in Jurkat. These results demonstrate that it is possible for the PKC isoforms to be differentially regulated during T cell activation.

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.

Identification of protein kinase C and its multiple isoforms in FRTL-5 thyroid cells.

Protein kinase C (PKC) has been implicated as an important regulator of signal transduction in the FRTL-5 thyroid cell line, but little is known about its isoforms in this cell line. In the present investigation, we characterized the activation of PKC by measuring the enzyme activity and identifying its isoforms in both cytosol and membrane fractions. Phorbol 12-myristate 13-acetate (PMA) was used as a PKC activator in this study. PKC activity assay revealed that PMA (300 nM) induced a rapid translocation from cytosol to membrane within 1 min and led to an almost complete translocation within 15 min. Multiple PKC isoforms were examined by Western blot analysis with specific antibodies against alpha, beta, gamma, delta, epsilon, and zeta isoforms. PKC alpha, delta, epsilon, and zeta were identified in this cell line, but PKC beta and gamma were not. Exposure of the cells to PMA (300 nM) for 5 to 30 min led to the translocation of PKC alpha, delta, and epsilon from the cytosol to the membrane fraction, while PKC zeta was not affected. Treatment with PMA (300 nM) for 24 h resulted in the down-regulation of PKC alpha, delta, and epsilon, but not PKC zeta. This study demonstrates for the first time direct evidence for the activation of PKC, and expression and distribution of its isoforms in FRTL-5 thyroid cells.

Activation of Mitogen-activated Protein Kinase by Arachidonic Acid in Rat Liver Epithelial WB Cells by a Protein Kinase C-dependent Mechanism

Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 microM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, delta, and epsilon to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, delta and epsilon. In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.

Regulation of non-classical protein kinase C isoenzymes in a human T cell line.

We have examined the expression and responses to activation, of novel/atypical protein kinase C (PKC) isoforms epsilon, zeta, and delta in the T cell lymphoma cell line K-4. The effects of 1-h phorbol 12-myristate 13-acetate (PMA) and OKT3 activation of K-4 cells on PKC isoform distribution were examined. In addition, the effects of PMA-mediated down-regulation on the expression of PKC epsilon and zeta were determined using high concentrations of PMA over 24- and 48-h time periods in these cells. PKC zeta expression was not altered by incubation of K-4 cells with up to 200 ng/ml PMA over a 24- or 48-h period. PKC epsilon was down-regulated in a concentration-dependent manner by PMA after both 24- and 48-h of activation. Expression of PKC epsilon was not completely depressed, however, even at the highest concentration of the phorbol ester after 48-h incubation with PMA. The presence of PKC epsilon, zeta, and delta was confirmed by immunohistochemistry with distinct patterns of expression observed. PMA-induced PKC activation for a 1-h period resulted in a translocation of PKC delta from resting cytoplasmic/nuclear staining to a cytoplasmic aggregate. Following 1-h activation through the T cell receptor-associated complex CD3, PKC delta translocated from a peri-nuclear/cytoplasmic compartment to a putative cytoskeletal location in K-4 cells. This translocation was time dependent and redistributed to a cytoplasmic aggregate prior to the cytoskeleton. Similarly, following 1-h activation through the T cell receptor, PKC zeta redistributed directly to what is possibly a cytoskeletal cell compartment. The cytoplasmic distribution of PKC zeta was unaltered following activation with PMA over a 1-h time period. There was no apparent redistribution of PKC epsilon cytoplasmic staining pattern following a 1-h direct or indirect activation. These results underline the differences in individual PKC isoform distribution, and responses to different stimuli, thereby providing additional evidence for the use of discrete PKC isoform signaling pathways in T cells. Furthermore, this data underlines the differences in PMA-mediated PKC activation and activation through the T cell receptor.

Effects of insulin on protein kinase-C (PKC) in HIRC-B cells: specific activation of PKC epsilon and its resistance to phorbol ester-induced down-regulation.

We evaluated the role of protein kinase-C (PKC) during insulin action in HIRC-B cells. Insulin provoked rapid increases in 1) diacylglycerol; 2) translocation of PKC epsilon, but not PKC alpha, PKC delta, or PKC zeta, from the cytosol to the membrane fraction; 3) membrane PKC enzyme activity; and 4) phosphorylation of immunoprecipitable 80-kilodalton (kDa) myristylated alanine-rich C-kinase substrate (MARCKS) protein and heat-stable 80-kDa protein (also probably MARCKS). Phorbol esters stimulated the translocation of PKC alpha and PKC delta as well as PKC epsilon, but not PKC zeta. The effects of phorbol esters on 80-kDa MARCKS phosphorylation were approximately 4 times as strong as those of insulin. Treatment of HIRC-B cells with phorbol esters for 20-24 h resulted in complete loss of immunoreactive PKC alpha and PKC delta in cytosol and membrane fractions, but substantial amounts of PKC epsilon were persistently translocated to the membrane fraction of down-regulated cells. This persistently translocated, residual PKC epsilon in down-regulated cells was associated with increased basal hexose uptake, but this was not due to PKC activation, as it was not inhibited by the PKC inhibitor, RO 31-8220. Acute insulin treatment, on the other hand, increased hexose uptake in down-regulated cells, and this insulin-stimulated uptake was inhibited by RO 31-8220 in down-regulated cells as well as in nondown-regulated cells. Insulin also stimulated the phosphorylation of the heat-stable 80-kDa protein in down-regulated cells, suggesting that the residual PKC epsilon in these cells can be activated by insulin.

Regulation of protein kinase C isoform proteins in phorbol ester-stimulated Jurkat T lymphoma cells.

Abstract Activation of protein kinase C (PKC) in T cells leads to a variety of responses including IL-2 production and IL-2 receptor expression. PKC consists of several isoforms that exhibit some different in vitro properties. We have set up a Western blotting system to explore the regulation of PKC isoforms during T cell activation. In Jurkat T lymphoma cells, PKC alpha, beta, delta, epsilon, and zeta were detected. PKC alpha and beta existed primarily in the cytosol, translocated to the membrane fraction after 10 minutes of treatment with PMA, and almost completely disappeared within 16 h. A larger fraction of PKC delta and epsilon existed in the membrane fraction compared to PKC alpha or beta, and PKC epsilon translocated to the membrane fraction rapidly. Translocation of PKC delta was not apparent after 1 h treatment with PMA, but total PKC delta protein was reduced within 4 to 6 h of treatment. Consistent with this, overnight treatment with PMA caused down-regulation of both PKC delta and epsilon, but to a lesser degree than was observed with PKC beta. Anti-PKC zeta antibody detected two bands at 82 and 75 kDa. The 75-kDa band existed mostly in the cytosol fraction and showed no translocation or down regulation after PMA. We present evidence that this 75-kDa band represents PKC zeta. Similar PMA-induced translocation responses were observed in murine thymocytes showing that the responses are not unique to PKC isoforms in Jurkat. These results demonstrate that it is possible for the PKC isoforms to be differentially regulated during T cell activation.

Protein kinase C-mediated phospholipase A2 activation, platelet-activating factor generation and prostacyclin release in spontaneously beating rat cardiomyocytes.

The expression of protein kinase C (PKC) isoenzymes and the effects of PKC activation on myocardial phospholipase A2 (PLA2) activity, platelet-activating factor (PAF) generation and eicosanoid release were studied in spontaneously beating cultured rat cardiomyocytes. Western blotting analysis indicated that these cells contain PKC alpha, beta, delta and zeta, but not PKC gamma or epsilon. Stimulation of cardiomyocytes with 4 beta-phorbol 12-myristate 13-acetate (PMA) led to a rapid increase in particulate-bound PKC activity, a response attributed to the activation of alpha-, delta- and zeta- type PKCs but not beta-type PKC. Translocation of PKC alpha, delta and zeta was accompanied by simultaneous increases in cellular lysophosphatidylcholine (lyso-PC), PAF, 15(S)-hydroxy-5,8,-11,13-eicosatetraenoic acid (15-HETE), prostaglandin E2 (PGE2) and prostacyclin (PGI2) generation, suggesting that one or more of these isoenzymes directly or indirectly activates a PLA2 in these cells. Confirming this, 4 beta-phorbol 12-monoacetate and 4 alpha-phorbol had no effect on cellular eicosanoid formation, while the PMA-induced response was fully abolished both in the presence of the PKC inhibitors staurosporine and CGP 41251 and in PKC-down-regulated cells. PKC alpha, delta and/or zeta therefore appear to play an important role in the PMA-mediated activation of cardiomyocyte PLA2, an event leading to subsequent production of PGI2, PGE2, 15-HETE, lyso-PC and PAF in this tissue.