Increased membrane affinity of the C1 domain of protein kinase Cdelta compensates for the lack of involvement of its C2 domain in membrane recruitment

Protein kinase C (PKC) family members are allosterically activated following membrane recruitment by specific membrane-targeting modules. Conventional PKC isozymes are recruited to membranes by two such modules: a C1 domain, which binds diacylglycerol (DAG), and a C2 domain, which is a Ca2+-triggered phospholipid-binding module. In contrast, novel PKC isozymes respond only to DAG, despite the presence of a C2 domain. Here, we address the molecular mechanism of membrane recruitment of the novel isozyme PKCdelta. We show that PKCdelta and a conventional isozyme, PKCbetaII, bind membranes with comparable affinities. However, dissection of the contribution of individual domains to this binding revealed that, although the C2 domain is a major determinant in driving the interaction of PKCbetaII with membranes, the C2 domain of PKCdelta does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKCdelta with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKCdelta in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKCdelta. Thus, the increased affinity of the C1 domain of PKCdelta allows this isozyme to respond to DAG alone, whereas conventional PKC isozymes require the coordinated action of Ca2+ binding to the C2 domain and DAG binding to the C1 domain for activation.     

Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells.

To determine if selective activation of individual isozymes of protein kinase C (PKC) might explain the apparently divergent effects of PKC stimulation on platelets, we purified and characterized the isozymes from both platelets and human erythroleukemia (HEL) cells, a cell line that has many features of megakaryocytes. Two peaks of platelet PKC activity were resolved by hydroxylapatite chromatography; immunoblot analysis revealed that these two peaks represented the alpha and beta isozymes of PKC. In contrast, HEL cells produced only a single peak that contained the beta isozyme. None of the other PKC isozymes were detected in these fractions. The cytosol of platelets and HEL cells, however, were both found to contain the PKC-delta isozyme. Northern hybridization analyses and mRNA amplification by the polymerase chain reaction demonstrated the presence of mRNA encoding the alpha, beta, and delta PKC isozymes in platelets, but only the beta and delta isozymes in HEL cells. Phorbol myristate acetate (PMA), thrombin, or an endoperoxide analog induced the phosphorylation of the 47-kDa substrate of PKC (pleckstrin) found in platelets and HEL cells; preincubation of either HEL cells or platelets with PMA reduced the intracellular Ca2+ rise induced by thrombin. Thus, although both HEL cells and platelets contain PKC-beta and the recently described PKC-delta isozymes, the widely distributed alpha isozyme of PKC is absent in HEL cells; however, isozymes other than PKC-alpha are sufficient for some PMA-mediated functions that are similar to those seen in stimulated platelets.     

Decoding of short-lived Ca2+ influx signals into long term substrate phosphorylation through activation of two distinct classes of protein kinase C

In electrically excitable cells, membrane depolarization opens voltage-dependent Ca(2+) channels eliciting Ca(2+) influx, which plays an important role for the activation of protein kinase C (PKC). However, we do not know whether Ca(2+) influx alone can activate PKC. The present study was conducted to investigate the Ca(2+) influx-induced activation mechanisms for two classes of PKC, conventional PKC (cPKC; PKCalpha) and novel PKC (nPKC; PKCtheta), in insulin-secreting cells. We have demonstrated simultaneous translocation of both DsRed-tagged PKCalpha to the plasma membrane and green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate to the cytosol as a dual marker of PKC activity in response to depolarization-evoked Ca(2+) influx in the DsRed-tagged PKCalpha and GFP-tagged myristoylated alanine-rich C kinase substrate co-expressing cells. The result indicates that Ca(2+) influx can generate diacylglycerol (DAG), because cPKC is activated by Ca(2+) and DAG. We showed this in three different ways by demonstrating: 1) Ca(2+) influx-induced translocation of GFP-tagged C1 domain of PKCgamma, 2) Ca(2+) influx-induced translocation of GFP-tagged pleckstrin homology domain, and 3) Ca(2+) influx-induced translocation of GFP-tagged PKCtheta, as a marker of DAG production and/or nPKC activity. Thus, Ca(2+) influx alone via voltage-dependent Ca(2+) channels can generate DAG, thereby activating cPKC and nPKC, whose activation is structurally independent of Ca(2+).     

Ca2+-induced contraction of cat esophageal circular smooth muscle cells

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+ influx and activation of protein kinase Cepsilon (PKCepsilon). PKCepsilon, however, is known to be Ca2+ independent. To determine where Ca2+ is needed in this PKCepsilon-mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle cells permeabilized by saponin. Ca2+ (0.2-1.0 microM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCepsilon (but not by PKCbetaII or PKCgamma antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPbetas. Addition of 1 microM Ca2+ to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). Ca2+-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by AACOCF(3) (cytosolic phospholipase A(2) inhibitor). These data suggest that Ca2+ may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCepsilon, which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPgammaS augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular Ca2+ and G protein activation), producing DAG and AA, and activating PKCepsilon-dependent mechanisms to cause contraction.     

Identification of an autoinhibitory mechanism that restricts C1 domain-mediated activation of the Rac-GAP alpha2-chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.     

Sustained muscle contraction induced by agonists, growth factors, and Ca(2+) mediated by distinct PKC isozymes

The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca(2+) mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-epsilon antibody, and 3) a pseudosubstrate PKC-epsilon inhibitor. GDPbetaS abolished both initial and sustained contraction, whereas a Galpha(q/11) antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-alpha,beta,gamma antibody, and a pseudosubstrate PKC-alpha inhibitor. Ca(2+) (0.4 microM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-alpha,beta,gamma antibody. Thus initial contraction induced by Ca(2+), agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca(2+)-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.     

Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells.

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.     

C1 domains exposed: from diacylglycerol binding to protein-protein interactions

C1 domains, cysteine-rich modules originally identified in protein kinase C (PKC) isozymes, are present in multiple signaling families, including PKDs, chimaerins, RasGRPs, diacylglycerol kinases (DGKs) and others. Typical C1 domains bind the lipid second messenger diacylglycerol (DAG) and DAG-mimetics such as phorbol esters, and are critical for governing association to membranes. On the contrary, atypical C1 domains possess structural determinants that impede phorbol ester/DAG binding. C1 domains are generally expressed as twin modules (C1A and C1B) or single domains. Biochemical and cellular studies in PKC and PKD isozymes revealed that C1A and C1B domains are non-equivalent as lipid-binding motifs or translocation modules. It has been recently determined that individual C1 domains have unique patterns of ligand recognition, driven in some cases by subtle structural differences. Insights from recent 3-D studies on beta2-chimaerin and Munc13-1 revealed that their single C1 domains are sterically blocked by intramolecular interactions, suggesting that major conformational changes would be required for exposing the site of DAG interaction. Thus, it is clear that the protein context plays a major role in determining whether binding of DAG to the C1 domain would lead to enzyme activation or merely serves as an anchoring mechanism.     

A novel mechanism of FSH regulation of DNA synthesis in the granulosa cells of hamster preantral follicles: involvement of a protein kinase C-mediated MAP kinase 3/1 self-activation loop

The objective was to reveal whether a protein kinase C (PKC [all isozymes])-mediated self-sustaining MAPK3/1 (3/1 extracellular signal regulated kinase 2/1, also known as ERK2/1) activation loop was necessary for FSH- or epidermal growth factor (EGF)-induced DNA synthesis in the granulosa cells of intact preantral follicles. For this purpose, hamster preantral follicles were cultured with FSH or EGF in the presence of selective kinase inhibitors FSH or EGF phosphorylated RAF1, MAP2K1, and MAPK3/1. However, a relatively higher dose of EGF was necessary to sustain the MAPK3/1 activity, which was essential for cyclin-dependent kinase 4 (CDK4) activation and DNA synthesis. In intact preantral follicles, FSH or EGF stimulated DNA synthesis only in the granulosa cells. Sustained activation of MAPK3/1 beyond 3 h was independent of EGFR kinase activity but dependent on PKC activity, which appeared to form a self-sustaining MAPK3/1 activation loop by activating RAF1, MAP2K1, and PLA2G4 (phospholipase A2 [all cytosolic isozymes]). Inhibition of PKC activity as late as 4 h after the administration of FSH or EGF arrested DNA synthesis, which corresponded with attenuated phosphorylation of RAF1 and MAPK3/1, thus suggesting an essential role of PKC in MAPK3/1 activation. Collectively, these data present a novel self-sustaining mechanism comprised of MAPK3/1, PLA2G4, PKC, and RAF1 for CDK4 activation leading to DNA synthesis in granulosa cells. Either FSH or EGF can activate the loop to activate CDK4 and initiate DNA synthesis; however, consistent with our previous findings, FSH effect seems to be mediated by EGF, which initiates the event by stimulating EGFR kinase.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Spatiotemporal dynamics of lipid signaling: protein kinase C as a paradigm

The lipid second messenger diacylglycerol (DAG) controls the rate, amplitude, duration, and location of protein kinase C (PKC) activity in the cell. There are three classes of PKC isozymes and, of these, the conventional and novel isozymes are acutely controlled by DAG. The kinetics of DAG production at various intracellular membranes, the intrinsic affinity of specific isoforms for DAG-containing membranes, the coordinated use of additional membrane-binding modules, the intramolecular regulation of DAG sensitivity, and the competition from other DAG-responsive proteins together result in a unique, context-dependent activation signature for each isoform. This review focuses on the spatiotemporal dynamics of PKC activation and how it is controlled by lipid second messengers.     

Diacylglycerol kinase in the central nervous system--molecular heterogeneity and gene expression.

Diacylglycerol (DAG) is one of the important second messengers, which serves as an activator of protein kinase C (PKC). DAG kinase (DGK) phosphorylates DAG to generate phosphatidic acid, thus DGK is considered to be a regulator of PKC activity through attenuation of DAG. Recent studies have revealed molecular structures of several DGK isozymes from mammalian species, and showed that most of the isozymes are expressed in the brain in various amounts. We have cloned four DGK isozyme cDNAs from rat brain library (DGK alpha, -beta, -gamma, and -zeta) (previously also designated DGK-I, -II, -III, and -IV, respectively) and examined their mRNA expressions in rat brain by in situ hybridization histochemistry. Interestingly, it is revealed that the mRNA for each isozyme is expressed in a distinct pattern in the brain; DGK alpha is expressed in oligodendrocytes, glial cells that form myelin; DGK beta in neurons of the caudate-putamen; DGK gamma predominantly in the cerebellar Purkinje cells; and DGK zeta in the cerebellar and cerebral cortices. Molecular diversity and distinct expression patterns of DGK isozymes suggest a physiological importance for the enzyme in brain function. Furthermore, functional implications of these DGK isozymes are briefly discussed.     

Diacylglycerol (DAG)-lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate cancer cells by selective activation of PKCalpha

Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore- and receptor-guided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKCalpha and PKCdelta mediated the apoptotic effect of PMA, whereas only PKCalpha was involved in the effect of the DAG-lactone. The PKCalpha selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKCalpha and PKCdelta. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKCalpha to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKCdelta predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.     

IL-1beta enhances beta2-adrenergic receptor expression in human airway epithelial cells by activating PKC

Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1beta upregulates beta2-adrenergic receptor (beta2-AR) density and beta2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1beta-induced beta-AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta-AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on beta-AR density, and 3) IL-1beta treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1beta (0.2 nM for 18 h) increased beta-AR density to 213% of control values (P < 0.001). PMA (1 microM for 18 h) increased beta-AR density to 225% of control values (P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1beta-induced upregulation of beta-AR in dose-dependent fashion. PKC isozymes detected by Western blotting included alpha, betaII, epsilon, mu, zeta, and lambda/iota. IL-1beta increased PKC-mu immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1beta-induced beta-AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-mu isozyme. We conclude that signal transduction pathways activated by PKC-mu upregulate beta2-AR expression in human airway epithelial cells.     

Signalling events mediating the activation of protein kinase C by interleukin-2 in cytotoxic T cells.

Interleukin-2 (IL-2) plays a vital role in the generation and regulation of the immune response, including important aspects of T cell survival. IL-2-mediated survival of T cells appears to be dependent on the activation of a pool of membrane-associated protein kinase C (PKC) that occurs in the absence of detectable translocation of the enzyme from the cytosol to membranes. In this report we investigate the mechanism(s) responsible for this PKC activation after IL-2 stimulation in the cytotoxic T cell line, CTLL-2. Tyrosine kinase activity, activated after IL-2 stimulation, was found not to be linked to the activation of PKC by the cytokine. On the other hand, a pertussis toxin (PTX)-sensitive G protein did appear coupled to PKC activation since PTX effectively blocked IL-2 stimulated PKC activity. Diacylglycerols (DAG), but not inositol 1,3,5-triphosphate (IP3) and intracellular Ca2+, increased after IL-2 stimulation suggesting that DAGs were generated via the phosphatidylcholine-phospholipase C (PC-PLC) or phosphatidylcholine-phospholipase D (PC-PLD) pathways. The increase in DAG by IL-2 was probably necessary for activation of membrane-resident PKC since exogenously applied DAG stimulated this PKC pool in both intact cells and in isolated membranes. IL-2 also increased arachidonic acid (AA) production in CTLL-2 cells, probably via phospholipase A2 (PLA2) since the PLA2 inhibitors oleoyloxyethyl phosphocholine and AACOCF3 (AACF) effectively blocked IL-2 stimulated PKC activation. Exogenous AA also increased PKC activity in intact cells and isolated membranes, suggesting that AA produced by IL-2 receptor stimulation was probably linked to PKC activation. These results suggest that the activation of membrane-resident PKC by IL-2 involves multiple second messengers, including G proteins, DAG and AA.     

Activation and inhibition of protein kinase C isozymes alpha and beta by Gd3+.

Gd3+ was evaluated as a probe for Ca2+ sites on protein kinase C (PKC) by studying its ability to replace Ca2+ in activation of PKC isozymes II (beta) and III (alpha) in the lipid systems phosphatidylserine/1,2-dioleoyl-sn-glycerol (PS/DO) and diheptanoylphosphatidylcholine (PC7)/DO. PKC beta was stimulated by Ca2+ or Gd3+ in PS/DO whereas activity in PC7/DO was independent of these metals. Thus, it is suggested that Gd3+ replaces Ca2+ at a site involving metal-lipid interactions. High concentrations of Ca2+ or Gd3+ inhibited activity in both lipid systems. Analysis of the Gd3+ inhibition in the PC7/DO system suggests that it is due to formation of GdATP, which competes at the MgATP site. Activity of PKC alpha was dependent on low concentrations of Ca2+ in both lipid systems. The ability of Gd3+ to substitute for Ca2+ could not be evaluated in the PS system due to the inability to completely remove contaminating Ca2+ without chelating buffers. Successful reduction of contaminating Ca2+ was achieved in the PC7 system but Gd3+ failed to substitute for Ca2+ in activating PKC alpha and only caused inhibition. This is consistent with binding of Gd3+ to a Ca2+ site at or near the active site of the enzyme rather than to a site on the lipid. These results indicate that interactions between PKC and Gd3+ are complex, involving occupation of more than one class of sites. Conditions for separately evaluating the individual sites can be manipulated by selection of isozyme and lipid system.