Altered erythrocyte protein kinase C activity and membrane protein phosphorylation in chronic myelogenous leukemia

The membrane protein kinase C (PKC) content was found to be higher in erythrocytes form patients suffering from chronic myelogenous leukemia (CML) compared to normal erythrocytes. PKC activity was also higher in the cytosol and after translocation to the membrane, as assessed by histone phosphorylation. The increased PKC activity in CML erythrocytes was associated with abnormal phosphorylation of protein 4.1. Since phosphorylation-dephosphorylation mechanisms are likely candidates for controlling membrane protein associations, the altered PKC activity may be one of the factors responsible for altered thermal sensitivity and mechanical stability of CML erythrocytes.     

Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.

In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.     

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.     

Thrombin-induced translocation of protein kinase C in human megakaryoblastic leukemia cells (MEG-01)

Our previous immunocytochemical study showed that Ca2+ ionophore-induced translocation of protein kinase C (PKC) in human megakaryoblastic leukemia cells (MEG-01) was potentiated by a synthetic diacylglycerol (T. Ito, T. Tanaka, T. Yoshida, K. Onoda, H. Ohta, M. Hagiwara, Y. Itoh, M. Ogura, H. Saito, and H. Hidaka, 1988, J. Cell Biol. 107, 929). In the present study, we analyzed the roles of the intracellular Ca2+ levels ([Ca2+]i) and diacylglycerol (DG) levels in thrombin-induced translocation of PKC using MEG-01 cells. When the cells were treated with thrombin (0.5 U/ml), PKC was translocated from the cytosol to the plasma membrane after 15 s, and the maximal membrane association was observed after 90 s. The [Ca2+]i of the cells rapidly increased (15 s) and reached a maximum level at 60 s which was sustained for a total of 600 s after thrombin addition. The increase in DG was biphasic with the first phase occurring in the first 15 s and the increase during the second phase lasting less than 600 s. The experiments without extracellular Ca2+ indicated that Ca2+ efflux accompanied by DG in the first phase was sufficient to initiate the membrane association of the PKC and that the large Ca2+ influx enhanced the binding. PKC returned to the cytosol within 600 s despite high levels of both [Ca2+]i and DG. We found that a relatively selective PKC inhibitor, H-7, enhanced thrombin-induced translocation of PKC without modulating [Ca2+]i or DG levels. These results indicate that certain protein phosphorylation events, potentially those mediated by PKC, may be responsible for, at least in part, inhibiting membrane association and further activation of the enzyme.     

Chronic exposure to ammonia induces isoform-selective alterations in the intracellular distribution and NMDA receptor-mediated translocation of protein kinase C in cerebellar neurons in culture

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.     

Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.     

Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

Activation of protein kinase C in lipid monolayers

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.     

Participation of cyclic nucleotides, protein kinase A and C in realization of n-acylethanolamine action in human adrenocortical cells

The messenger mechanisms mediating N-acylethanolamines (NAE) regulatory signals in the adrenal cortex were studied. An analysis of the mechanisms of realization of NAE effects in the post-operation human adrenal cortex was carried out in vitro. Influence of NAE mix on cAMP and cGMP level, protein kinase A and C activity in sub-cellular fraction of adrenocorticocytes and homogenates of conditionally normal adrenal cortex tissues was investigated. It was shown, that N-acylethanolamines treatment resulted in a decrease of cAMP level in adrenocortical cells. cGMP level is not changed in these conditions. The rise of protein kinase C activity was obtained in the membrane fraction after N-acylethanolamines in vitro treatment (3.3 microg/ml). Activity of cAMP-dependent protein kinase A significantly decreased in cytosol fraction of adrenocorticocytes. It was concluded, that steroid genesis activation is determined by protein kinase C activation, inhibition is determined by cAMP-dependent messenger system.     

Impaired protein kinase C activation/translocation in Epstein-Barr virus-infected monocytes

Infection of human monocytes by Epstein-Barr virus (EBV) has been linked to a decrease in the production of proinflammatory mediators as well as an impairment of phagocytosis. Considering the key role of protein kinases C (PKCs) in many biological functions of monocytes, including phagocytosis, we investigated the effects of EBV on the PKC activity in infected monocytes. Our results indicate that infection of monocytes by EBV impairs both phorbol 12-myristate 13-acetate (PMA)-induced translocation of PKC isozymes alpha and beta from cytosol to membrane as well as the PKC enzymatic activity. Similarly, the subcellular distribution of the receptor for activated C kinase (RACK), an anchoring protein essential to PKC translocation, was also found to be reduced in EBV-infected monocytes. Transfection of 293T cells with an expression vector coding for the immediate-early protein ZEBRA of EBV resulted in impaired PMA-induced translocation and activity of PKC. Using co-immunoprecipitation assays, the ZEBRA protein was found to physically interact with the RACK1 protein. Thus interaction of ZEBRA with RACK likely results in the inhibition of PKC activity, which in turn affects functions of monocytes, such as phagocytosis.     

Regulation of protein kinase C in Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Escherichia coli is one of the most important pathogens involved in the development of neonatal meningitis in many parts of the world. Traversal of E. coli across the blood-brain barrier is a crucial event in the pathogenesis of E. coli meningitis. Our previous studies have shown that outer membrane protein A (OmpA) expression is necessary in E. coli for a mechanism involving actin filaments in its passage through the endothelial cells. Focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) have also been activated in host cells during the process of invasion. In an attempt to elucidate the mechanisms leading to actin filament condensation, we have focused our attention on protein kinase C (PKC), an enzyme central to many signaling events, including actin rearrangement. In the current study, specific PKC inhibitors, bisindolmaleimide and a PKC-inhibitory peptide, inhibited E. coli invasion of human brain microvascular endothelial cells (HBMEC) by more than 75% in a dose-dependent manner, indicating a significant role played by this enzyme in the invasion process. Our results further showed that OmpA+ E. coli induces significant activation of PKC in HBMEC as measured by the PepTag nonradioactive assay. In addition, we identified that the PKC isoform activated in E. coli invasion is a member of the conventional family of PKC, PKC-alpha, which requires calcium for activation. Immunocytochemical studies have indicated that the activated PKC-alpha is associated with actin condensation beneath the bacterial entry site. Overexpression of a dominant negative mutant of PKC-alpha in HBMEC abolished the E. coli invasion without significant changes in FAK phosphorylation or PI3K activity patterns. In contrast, in HBMEC overexpressing the mutant forms of either FAK or PI3K, E. coli-induced PKC activation was significantly blocked. Furthermore, our studies showed that activation of PKC-alpha induces the translocation of myristoylated alanine-rich protein kinase C substrate, an actin cross-linking protein and a substrate for PKC-alpha, from the membrane to cytosol. This is the first report of FAK- and PI3K-dependent PKC-alpha activation in bacterial invasion related to cytoskeletal reorganization.     

Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca(2+) handling of human small arteries

The mechanisms of Ca(2+) handling and sensitization were investigated in human small omental arteries exposed to norepinephrine (NE) and to the thromboxane A(2) analog U-46619. Contractions elicited by NE and U-46619 were associated with an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), an increase in Ca(2+)-independent signaling pathways, or an enhancement of the sensitivity of the myofilaments to Ca(2+). The two latter pathways were abolished by protein kinase C (PKC), tyrosine kinase (TK), and Rho-associated protein kinase (ROK) inhibitors. In Ca(2+)-free medium, both NE and U-46619 elicited an increase in tension that was greatly reduced by PKC inhibitors and abolished by caffeine or ryanodine. After depletion of Ca(2+) stores with NE and U-46619 in Ca(2+)-free medium, addition of CaCl(2) in the continuous presence of the agonists produced increases in [Ca(2+)](i) and contractions that were inhibited by nitrendipine and TK inhibitors but not affected by PKC inhibitors. NE and U-46619 induced tyrosine phosphorylation of a 42- or a 58-kDa protein, respectively. These results indicate that the mechanisms leading to contraction elicited by NE and U-46619 in human small omental arteries are composed of Ca(2+) release from ryanodine-sensitive stores, Ca(2+) influx through nitrendipine-sensitive channels, and Ca(2+) sensitization and/or Ca(2+)-independent pathways. They also show that the TK pathway is involved in the tonic contraction associated with Ca(2+) entry, whereas TK, PKC, and ROK mechanisms regulate Ca(2+)-independent signaling pathways or Ca(2+) sensitization.     

Uneven distribution of protein kinase C-α and -β isozymes in human sarcomas and carcinomas

Protein kinase C (PKC) represents a family of structurally related Ser/Tre kinases which are involved in mitogenic signalling and may contribute to human neoplasia. To address this issue, the messenger RNA and protein levels of PKC isoenzymes α and β were analyzed in several human sarcoma- and carcinoma-derived cell lines. Carcinomas contained low or undetectable levels of either PKC-α or PKC-β. Sarcomas exhibited similar or increased PKC expression compared to human diploid fibroblasts. Moreover, sarcoma cell lines expressing one PKC isoform did not contain detectable levels of the other. When PKC was depleted from the tumor cells, we observed that the PKC overexpressing sarcomas had reduced their malignant properties as determined by their ability to grow in semisolid medium. In addition, epidermal growth factor-stimulated and erbB2-transformed fibroblasts exhibited enhanced cell growth in the absence of PKC. We propose a model for the effect of PKC as a negative regulator of proliferation in epithelial cells and a growth promoter in fibroblasts. © 1994 wiley-Liss, Inc.     

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.     

Subcellular distribution of protein kinase C of GH3 cells: quantitation and characterization by polyacrylamide gel electrophoresis

Quantitative estimation of cytosolic Ca2+- and phospholipid-dependent protein kinase (PKC) activity was performed by polyacrylamide gel electrophoresis under nondenaturating conditions (PAGE). With this method less than 50 micrograms of cytosol protein can be accurately quantitated for PKC activity. The amount of cytosolic PKC activity recovered after PAGE was comparable to the amount obtained by DEAE-cellulose chromatography. Homogenization of GH3 cells in the presence of 2 mM EGTA/EDTA revealed that 80% of the total cellular PKC activity resided in the cytosol. However, omission of the ion chelator during cell disruption followed by subcellular fractionation and extraction of subcellular fractions by EDTA/EGTA showed that 80% of the total PKC was found in the lysosomal-mitochondrial and microsomal extracts. Detailed analysis of PKC activities demonstrated that cytosolic PKC was identical with the PKC solubilized by EDTA/EGTA from subcellular fractions. In conclusion, GH3 cells appear to contain one species of PKC with an apparent molecular weight of 90,000 which seems to be associated with membranes via a calcium-dependent mechanism (or mechanisms).     

Inhibitory effect of calcium-binding protein regucalcin on protein kinase C activity in rat liver cytosol

Regucalcin, a calcium-binding protein isolated from rat liver cytosol, inhibited Ca2(+)- and phospholipid-dependent protein kinase (protein kinase C) activity in hepatic cytosol. With the increasing concentrations of Ca2+ or phosphatidylserine in the medium, regucalcin caused a remarkable inhibition of protein kinase C activity. Moreover, regucalcin significantly inhibited dioctanoylglycerol-activated protein kinase C. Regucalcin itself did not have protein kinase activity in either the presence or the absence of Ca2+ and phospholipids. These findings clearly indicate that regucalcin has an inhibitory effect on protein kinase C in hepatic cytosol. This inhibitory effect of regucalcin may be due to the regucalcin-induced Ca2+ binding and/or the direct binding of regucalcin to protein kinase C.     

Cross-talk between protein kinase C and multifunctional Ca2+/calmodulin-dependent protein kinase.

Protein kinase C (PKC) exhibits both negative and positive cross-talk with multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in PC12 cells. PKC effects negative cross-talk by inhibiting the mobilization of intracellular Ca2+ stores and by inhibiting Ca2+ influx through voltage-sensitive Ca2+ channels. In the absence of cross-talk, Ca2+ influx induced by depolarization with 56 mM K+ stimulates CaM kinase and its autophosphorylation and converts up to 50% of the enzyme to a Ca(2+)-independent or autonomous species. Acute treatment with phorbol myristate acetate (PMA) elicits a parallel reduction in depolarization-induced Ca2+ influx and in generation of autonomous CaM kinase. Negative cross-talk also occurs during stimulation of the phosphatidylinositol signaling system with bradykinin, which activates both PKC and CaM kinase. The extent of CaM kinase activation is attenuated by the simultaneous activation of PKC; it is enhanced by prior down-regulation of PKC. PKC also exhibits positive cross-talk with CaM kinase. Submaximal activation of CaM kinase by ionomycin is potentiated by concurrent activation of PKC with PMA. Such PMA treatment is found to increase the level of cytosolic calmodulin. Enhanced activation of CaM kinase by PKC may result from PKC-mediated phosphorylation of calmodulin-binding proteins, such as neuromodulin and MARCKS, and the subsequent increase in the availability of previously bound calmodulin for activation of CaM kinase.     

Regulation of protein kinase C by Zn(2+)-dependent interaction with actin.

Zn2+ influences diverse cellular processes by poorly understood mechanisms. Some of these effects may be mediated by the protein kinase C (PKC) family of enzymes, since an influx of Zn2+ greatly increases their binding of regulatory ligand phorbol ester and induces their translocation from cytosol to the cytoskeleton. Using a model with purified components, we now show that Zn2+ acts by forming a phospholipid-dependent complex of PKC with filamentous actin, which results in expression of new binding sites for phorbol ester and phosphorylation of actin. These results provide a basis for the observed localization of PKC at actin-membrane junctions, in-vivo.