Phorbol esters increase dopamine transporter phosphorylation and decrease transport Vmax

Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport Vmax in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK1, cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport Vmax by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter Vmax suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.     

Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells

Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i extrusion.     

Opposite paracrine effects of 5-HT and dopamine on Na(+)-Pi cotransport in opossum kidney cells

The effect of insulin on protein kinase activity and plasma membrane translocation of the glucose transporter GLUT 4 has been studied in adipocytes permeabilized by Streptolysin-O. Insulin increased protein kinase activity, and this was completely inhibited by the PKC pseudosubstrate inhibitor peptide (PKC19-36). Insulin-mediated translocation of GLUT 4 was also inhibited by the PKC inhibitor peptide. Both these insulin effects were blocked by a PKCbeta neutralizing antibody. Our results are consistent with the hypothesis that insulin activates PKCbeta activity in adipocytes in situ, and that this PKC activation is a component of the system whereby insulin regulates translocation of GLUT 4 to the plasma membrane.     

Interaction of urokinase-type plasminogen activator with its receptor rapidly induces activation of glucose transporters

The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present. u-PAR stimulation was followed by translocation of GLUT1 from the microsomal to the membrane compartment, as shown by both immunoblotting and immunofluorescence of sonicated plasma membrane sheets and by activation of GLUT2 on the cell surface. Both translocation and activation resulted inhibitable by protein-tyrosine kinase inhibitors and independent of downregulation of protein kinase C (PKC). The increase of intracellular glucose was followed by neosynthesis of diacylglycerol (DAG) from glucose, as previously shown. Such neosynthesis was completely inhibited by impairment of facilitative GLUT transport by cytochalasin B. DAG neosynthesis was followed by activation of PKC, whose activity translocated into the intracellular compartment (PKM), where it probably phosphorylates substrates required for u-PAR-dependent chemotaxis. Our data show that u-PAR-mediated signal transduction, related with u-PA-induced chemotaxis, involves activation of tyrosine kinase-dependent glucose transporters, leading to increased de novo DAG synthesis from glucose, eventually resulting in activation of PKC.     

Suppression of insulin-stimulated phosphatidylinositol 3-kinase activity by the beta3-adrenoceptor agonist CL316243 in rat adipocytes

Insulin increased 2-deoxyglucose (2-DG) uptake via the translocation of glucose transporter (GLUT) 4 to the plasma membrane fraction in rat adipocytes. The stimulatory actions of insulin were accompanied by both an increase in the immunoreactive p85 subunit of phosphatidylinositol (PI) 3-kinase in the plasma membrane fractions and PI 3-kinase activation by tyrosine phosphorylation of the p85 subunit. The beta3-adrenoceptor agonist CL316243 (CL) suppressed all the insulin actions in adenosine deaminase (ADA)-treated cells, but was without effect in non-ADA-treated cells. The inhibitory effects of CL on GLUT 4 translocation and PI 3-kinase activation were abolished by the addition of N6-phenylisopropyl adenosine. Cholera toxin treatment, which markedly increased intracellular cAMP levels, suppressed increases in the levels of GLUT 4 and PI 3-kinase in the plasma membrane fractions in response to insulin. In addition, dibutyryl (Bt2) cAMP also impaired the activation of PI 3-kinase by insulin. These results indicated that CL suppressed insulin-stimulated glucose transport under conditions where cAMP levels were markedly increased (approximately 12-fold). The inhibitory actions of PI 3-kinase activation by insulin were exerted even when cAMP, 8-bromo-cAMP, or Bt2 cAMP was added to immunoprecipitates of the p85 subunit of PI 3-kinase, after treating the cells with insulin. These results suggest that CL suppressed insulin-stimulated PI 3-kinase activity via a cAMP-dependent mechanism, at least in part, direct cAMP action in ADA-treated adipocytes, by which PI 3-kinase activation was inhibited, resulting in the decrease in GLUT 4 translocation and subsequent 2-DG uptake in response to insulin.     

The ras signaling pathway mimics insulin action on glucose transporter translocation

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.     

Comparative effects of GTPgammaS and insulin on the activation of Rho, phosphatidylinositol 3-kinase, and protein kinase N in rat adipocytes. Relationship to glucose transport

Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.     

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.     

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.     

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.     

An inhibitory fragment derived from protein kinase Cepsilon prevents enhancement of nerve growth factor responses by ethanol and phorbol esters

We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKCdelta and PKCepsilon, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKCepsilon (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKCepsilon in cardiac myocytes. Phorbol ester-stimulated translocation of PKCepsilon was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKCdelta. These results demonstrate that PKCepsilon mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells.     

Activation of protein kinase C triggers its ubiquitination and degradation

Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms alpha, delta, and epsilon in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC delta. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC alpha, delta, and epsilon were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC alpha could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.     

Activation of protein kinase C-alpha and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells

The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.     

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.     

Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.     

Gq-coupled receptors transmit the signal for GLUT4 translocation via an insulin-independent pathway

Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces the translocation of glucose transporter type 4 (GLUT4) from an intracellular pool to the cell surface and increases glucose uptake in adipocytes. The GTP-binding protein(s) responsible for the translocation has remained to be identified. Using a sensitive and quantitative method to assess the translocation of c-MYC epitope-tagged GLUT4, we obtained evidence that the activation of receptor-coupled Gq (neither Gi nor Gs) triggered GLUT4 translocation in cells, independently of insulin signaling pathway(s). Platelet-activating factor (PAF) induced GLUT4 translocation in the cells expressing the Gi- and Gq-coupled PAF receptor, but the translocation was induced even after pretreatment with wortmannin, an islet-activating protein and phorbol 12, 13-dibutyrate. Norepinephrine triggered GLUT4 translocation in cells expressing the Gq-coupled alpha1-adrenergic receptor, but prostaglandin E2 did not cause GLUT4 translocation in cells expressing the Gs-coupled EP4 receptor or the Gi-coupled EP3alpha receptor. The norepinephrine-stimulated GLUT4 translocation and glucose uptake via Gq may possibly contribute to the fuel supply required for thermogenesis in brown adipocytes and for the enhanced contractility in cardiomyocytes, both of which have an abundant endogenous GLUT4.     

Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostiridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network.     

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.     

Protein kinase C activation potentiates the rapid secretion of the amyloid precursor protein from rat cortical synaptosomes

In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AbetaPP) by the alpha-secretase pathway within the betaA4 domain to generate a soluble secreted N-terminal fragment (AbetaPPs). AbetaPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform beta1 and novel PKCepsilon from cytosol to membrane fractions, but there was no alteration in the proportion of AbetaPP associated with the Triton-soluble and -insoluble fractions. AbetaPPs was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AbetaPPs was only partially blocked by the PKC inhibitor GF109203X (2.5 microM), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AbetaPPs secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the alpha-secretase activity leading to the secretion of AbetaPPs can occur at the level of the presynaptic terminal.