Differential and regulated binding of cAMP-dependent protein kinase and protein kinase C isoenzymes to gravin in human model neurons: Evidence that gravin provides a dynamic platform for the localization for kinases during neuronal development

The membrane cortex has an important role in generating and maintaining spatially and functionally distinct domains in neurons. As a tool to functionally characterize molecules of the membrane cortex, we generated novel monoclonal antibodies against a fraction enriched for components of the neuronal membrane skeleton. We obtained two antibodies against the kinase-anchoring protein gravin. Gravin was strongly up-regulated during differentiation of human model neurons (NT2-N neurons) and was enriched at the inner peripheral cortex in close proximity to the plasma membrane where its localization primarily depended on association with membranes. In differentiated neurons, gravin colocalized in putative signaling complexes with protein kinase C (PKCbetaII) and partially with PKCalpha and cAMP-dependent protein kinase (PKA). Colocalization with PKCepsilon was not observed. PKCbetaII, PKCalpha, and PKA but not PKCepsilon coprecipitated with gravin indicating physical interaction. Binding of gravin to PKCalpha required the presence of Ca2+ and was increased after inhibition of PKC. In contrast, binding of PKCbetaII and PKA were independent of Ca2+ and PKC inhibition. Activation of PKC decreased binding of PKCalpha to gravin, decreased its association with the plasma membrane, and reduced the mean size of gravin particles. Taken together the data suggest that gravin provides a dynamic platform to localize kinases in an isoenzyme-specific and activation-dependent manner at specific sites in neurons.     

H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca(2+) handling in hydrogen sulfide (H(2)S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H(2)S donor, 10(-4) M) preconditioning (SP, 30 min). Intracellular Ca(2+) was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCalpha, PKCepsilon, and PKCdelta to membrane fraction but only translocation of PKCepsilon and PKCdelta was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca(2+)] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca(2+) removal via both accelerating uptake of Ca(2+) into sarcoplasmic reticulum and enhancing Ca(2+) extrusion through Na(+)/Ca(2+) exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca(2+) overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCalpha, PKCepsilon, and PKCdelta in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca(2+) handling.     

Glucagon-like peptide 1 activates protein kinase C through Ca2+-dependent activation of phospholipase C in insulin-secreting cells

Although the stimulatory effect of glucagon-like peptide 1 (GLP-1), a cAMP-generating agonist, on Ca(2+) signal and insulin secretion is well established, the underlying mechanisms remain to be fully elucidated. We recently discovered that Ca(2+) influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting (INS-1) cells. Building on this earlier finding, here we examined whether GLP-1-evoked Ca(2+) signaling can activate PKCalpha and PKCepsilon at a substimulatory concentration of glucose (3 mm) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKCalpha and PKCepsilon from the cytosol to the plasma membrane. Next, we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), by using MARCKS-GFP. GLP-1 translocated MARCKS-GFP to the cytosol in a Ca(2+)-dependent manner, and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors, either a broad PKC inhibitor, bisindolylmaleimide I, or a PKCepsilon inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKCepsilon-(149-164) (antp-PKCepsilon) and a conventional PKC inhibitor, G?-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122, a putative inhibitor of phospholipase C. These observations were verified in two different ways by demonstrating 1) forskolin-induced translocation of the GFP-tagged C1 domain of PKCgamma and 2) translocation of PKCalpha-DsRed and PKCepsilon-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKCalpha and PKCepsilon, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose.     

Unloading of intercellular tension induces the directional translocation of PKCα

The migration of endothelial cells (ECs) is closely associated with a Ca²⁺-dependent protein, protein kinase Cα (PKCα). The disruption of intercellular adhesion by single-cell wounding has been shown to induce the directional translocation of PKCα. We hypothesized that this translocation of PKCα is induced by mechanical stress, such as unloading of intercellular tension, or by intercellular communication, such as gap junction-mediated and paracrine signaling. In the current study, we found that the disruption of intercellular adhesion induced the directional translocation of PKCα even when gap junction-mediated and paracrine signaling were inhibited. Conversely, it did not occur when the mechanosensitive channel was inhibited. In addition, the strain field of substrate attributable to the disruption of intercellular adhesion tended to be larger at the areas corresponding with PKCα translocation. Recently, we found that a direct mechanical stimulus induced the accumulation of PKCα at the stimulus area, involving Ca ²⁺ influx from extracellular space. These results indicated that the unloading of intercellular tension induced directional translocation of PKCα, which required Ca ²⁺ influx from extracellular space. The results of this study indicate the involvement of PKCα in the Ca ²⁺ signaling pathway in response to mechanical stress in ECs.     

Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.     

Molecular conformation dictates signaling module formation: example of PKCepsilon and Src tyrosine kinase

Our laboratory has conducted multiple functional proteomic analyses to characterize the components of protein kinase C (PKC)epsilon cardioprotective signaling complexes and found that activation of PKCepsilon induces dynamic modulation of these complexes. In addition, it is known that signal transduction within a complex involves the formation of modules, one of which has been shown to include PKCepsilon and Src tyrosine kinase in the rabbit heart. However, the cellular mechanisms that define the assembly of PKCepsilon modules remain largely unknown. To address this issue, the interactions between PKCepsilon and Src were studied. We used recombinant proteins of wild-type PKCepsilon (PKCepsilon-WT) and open conformation mutants of the kinase (PKCepsilon-AE5 and PKCepsilon-AN59), the regulatory and catalytic domains of PKCepsilon, along with glutathione-S-transferase (GST) fusion proteins of Src (GST-Src) and two domains of Src (GST-SH2 and GST-SH3). GST pulldown assays demonstrated that Src and PKCepsilon are binding partners and that the interaction between PKCepsilon and Src appears to involve multiple sites. This finding was supported for endogenous PKCepsilon and Src in the murine heart using immunofluorescence-based confocal microscopy and coimmunoprecipitation. Furthermore, PKCepsilon-WT and GST-Src interactions were significantly enhanced in the presence of phosphatidyl-L-serine, an activator of PKC, indicating that Src favors interaction with activated PKCepsilon. This finding was confirmed when the PKCepsilon-WT was replaced with PKCepsilon-AE5 or PKCepsilon-AN59, demonstrating that the conformation of PKCepsilon is a critical determinant of its interactions with Src. Together, these results illustrate that formation of a signaling module between PKCepsilon and Src involves specific domains within the two molecules and is governed by the molecular conformation of PKCepsilon.     

Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.     

Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.     

Protein kinase C isoform expression and activity in the mouse heart

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.     

Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver

Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors G?6850 (1 micromol/liter) and G?6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells. However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. G?6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.     

The very C-terminus of protein kinase Cepsilon is critical for the full catalytic competence but its hydrophobic motif is dispensable for the interaction with 3-phosphoinositide-dependent kinase-1

In this article, we explore the role of the C-terminus (V5 domain) of PKCepsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCepsilon resulted in a PKCepsilon-Delta731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCepsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCepsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCepsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCepsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.     

Control of astrocyte Ca(2+) oscillations and waves by oscillating translocation and activation of protein kinase C

BACKGROUND: Glutamate-induced Ca2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Here, we use total internal reflection fluorescence (TIRF) microscopy to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca2+ oscillations and waves. RESULTS: Glutamate stimulation of astrocytes triggered highly localized GFP-PKCgamma plasma membrane translocation events, induced rapid oscillations in GFP-PKCgamma translocation, and generated GFP-PKCgamma translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca2+-sensitive C2 domains of PKCgamma and were driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca(2+) waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCgamma or PKCdelta that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca(2+) and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, led to a marked prolongation of Ca(2+) spikes, suggesting that PKC activation is involved in terminating individual Ca(2+) spikes and waves and in defining the time period between Ca(2+) spikes. CONCLUSIONS: Our study suggests that cPKCs have a negative feedback role on Ca(2+) oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca(2+) concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca(2+) spikes and waves and can define the Ca(2+) spike and wave frequencies.     

Single cell analysis and temporal profiling of agonist-mediated inositol 1,4,5-trisphosphate, Ca2+, diacylglycerol, and protein kinase C signaling using fluorescent biosensors

The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M(3) muscarinic acetylcholine and alpha(1B) -adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), using the pleckstrin homology domain of PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca(2+) release via Ins(1,4,5)P(3) receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.     

Cross-desensitization among CXCR1, CXCR2, and CCR5: role of protein kinase C-epsilon

The IL-8 (or CXCL8) chemokine receptors, CXCR1 and CXCR2, activate protein kinase C (PKC) to mediate leukocyte functions. To investigate the roles of different PKC isoforms in CXCL8 receptor activation and regulation, human mononuclear phagocytes were treated with CXCL8 or CXCL1 (melanoma growth-stimulating activity), which is specific for CXCR2. Plasma membrane association was used as a measure of PKC activation. Both receptors induced time-dependent association of PKCalpha, -beta1, and -beta2 to the membrane, but only CXCR1 activated PKCepsilon. CXCL8 also failed to activate PKCepsilon in RBL-2H3 cells stably expressing CXCR2. DeltaCXCR2, a cytoplasmic tail deletion mutant of CXCR2 that is resistant to internalization, activated PKCepsilon as well as CXCR1. Expression of the PKCepsilon inhibitor peptide epsilonV1 in RBL-2H3 cells blocked PKCepsilon translocation and inhibited receptor-mediated exocytosis, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization. epsilonV1 also inhibited CXCR1-, CCR5-, and DeltaCXCR2-mediated cross-regulatory signals for GTPase activity, Ca(2+) mobilization, and internalization. Peritoneal macrophages from PKCepsilon-deficient mice (PKCepsilon(-/-)) also showed decreased CCR5-mediated cross-desensitization of G protein activation and Ca(2+) mobilization. Taken together, the results indicate that CXCR1 and CCR5 activate PKCepsilon to mediate cross-inhibitory signals. Inhibition or deletion of PKCepsilon decreases receptor-induced exocytosis and cross-regulatory signals, but not phosphoinositide hydrolysis or peak intracellular Ca(2+) mobilization, suggesting that cross-regulation is a Ca(2+)-independent process. Because DeltaCXCR2, but not CXCR2, activates PKCepsilon and cross-desensitizes CCR5, the data further suggest that signal duration leading to activation of novel PKC may modulate receptor-mediated cross-inhibitory signals.     

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.     

A novel diacylglycerol-lactone shows marked selectivity in vitro among C1 domains of protein kinase C (PKC) isoforms alpha and delta as well as selectivity for RasGRP compared with PKCalpha

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms alpha, beta, gamma, delta, and epsilon, with apparent Ki values ranging from 340 nm for PKCalpha to 29 nm for PKCepsilon. When binding to isolated C1 domains of PKCalpha and -delta, 130C037 showed good affinity (Ki= 1.78 nm) only for deltaC1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of deltaC1b. 130C037 bound intact RasGRP1 and RasGRP3 with Ki values of 3.5 and 3.8 nm, respectively, reflecting 8- and 90-fold selectivity relative to PKCepsilon and PKCalpha. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED50 = 286 nm), partial reduction of PKCepsilon (ED50 > 10 microm), and no effect on PKCalpha. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKCepsilon, and no translocation of PKCalpha. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets.     

ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain

The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.     

Assembly of the tight junction: the role of diacylglycerol

Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the TJ-associated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin filaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca2+ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca2+. diC8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherin-mediated cell-cell adhesion. In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca2+, suggesting that the addition of Ca2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.