Activation Of Conventional Protein Kinase Research Articles

VIP and muscarinic synergistic mucin secretion by salivary mucous cells is mediated by enhanced PKC activity via VIP-induced release of an intracellular Ca2+ pool

Mucin secretion by salivary mucous glands is mediated predominantly by parasympathetic acetylcholine activation of cholinergic muscarinic receptors via increased intracellular free calcium ([Ca2+]i) and activation of conventional protein kinase C isozymes (cPKC). However, the parasympathetic co-neurotransmitter, vasoactive intestinal peptide (VIP), also initiates secretion, but to a lesser extent. In the present study, cross talk between VIP- and muscarinic-induced mucin secretion was investigated using isolated rat sublingual tubuloacini. VIP-induced secretion is mediated by cAMP-activated protein kinase A (PKA), independently of increased [Ca2+]i. Synergistic secretion between VIP and the muscarinic agonist, carbachol, was demonstrated but only with submaximal carbachol. Carbachol has no effect on cAMP ± VIP. Instead, PKA activated by VIP releases Ca2+ from an intracellular pool maintained by the sarco/endoplasmic reticulum Ca2+-ATPase pump. Calcium release was independent of phospholipase C activity. The resultant sustained [Ca2+]i increase is additive to submaximal, but not maximal carbachol-induced [Ca2+]i. Synergistic mucin secretion was mimicked by VIP plus either phorbol 12-myristate 13-acetate or 0.01μM thapsigargin, and blocked by the PKC inhibitor, Gö6976. VIP-induced Ca2+ release also promoted store-operated Ca2+ entry. Synergism is therefore driven by VIP-mediated [Ca2+]i augmenting cPKC activity to enhance muscarinic mucin secretion. Additional data suggest ryanodine receptors control VIP/PKA-mediated Ca2+ release from a Ca2+ pool also responsive to maximal carbachol. A working model of muscarinic and VIP control of mucous cell exocrine secretion is presented. Results are discussed in relation to synergistic mechanisms in other secretory cells, and the physiological and therapeutic significance of VIP/muscarinic synergism controlling salivary mucous cell exocrine secretion.

PLCB3 Loss of Function Reduces Pseudomonas aeruginosa-Dependent IL-8 Release in Cystic Fibrosis.

The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-β3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca2+ release from endoplasmic reticulum and rise of Ca2+ concentration, 2) activation of conventional protein kinase C isoform β, and 3) induction of IL-8 release. These results, besides identifying S845L as a loss-of-function variant, strengthen the importance of targeting PLCB3 to mitigate the CF inflammatory response in bronchial epithelial cells without blunting the immune response.

Sibjotang Increases Atrial Natriuretic Peptide Secretion in Beating Rabbit Atria.

Sibjotang (Shizaotang), traditional herbal medicine formula, which was first documented in the Shanghanlun, has long been prescribed for the treatment of impairment of the body fluid homeostasis. The purpose of the present study was to identify the effects of Sibjotang on the secretion of a cardiac hormone, atrial natriuretic peptide (ANP), one of the main hormones involved in the regulation of the body fluid and blood pressure homeostasis. Water extract of Sibjotang increased ANP secretion concomitantly with an increase in atrial dynamics in a concentration-dependent manner. Sibjotang-induced increase in ANP secretion and positive inotropic effect were attenuated by GO6976 and LY333531, selective inhibitors of conventional protein kinase C, but not Rottlerin, an inhibitor of novel PKCδ. Similarly to the effect of Sibjotang, extracts of components of Sibjotang, Euphorbia kansui, and Daphne genkwa, but not Euphorbia pekinensis and Ziziphus jujuba, increased ANP secretion and atrial dynamics. Ingredients of Sibjotang, apigenin, rosmarinic acid, and salvianolic acid B decreased ANP secretion and atrial dynamics. These findings suggest that Sibjotang increases ANP secretion and atrial dynamics via activation of conventional protein kinase C signaling. This finding provides experimental evidence for the rationale in the use of Sibjotang in the treatment of impairment of the regulation of body fluid and blood pressure homeostasis.

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca(2+). Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca(2+)-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCα not only reduces the Ca(2+)-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCα to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.

Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps

BackgroundActivation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation.MethodsPharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood.ResultsPan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001).ConclusionsConventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation.

Rediocide A, an Insecticide, Induces G-Protein-Coupled Receptor Desensitization via Activation of Conventional Protein Kinase C

In order to identify small-molecule antagonists of Methuselah (Mth), a Drosophila G-protein-coupled receptor (GPCR) involved in life-span control, a library of natural compounds was screened, and it was found that rediocide A (1), a daphnane ester from the roots of Trigonostemon reidioides and used currently for flea control, potently inhibited calcium mobilization mediated by this receptor. Compound 1 inhibited calcium mobilization in GPCRs other than Mth, indicating that the inhibitory effect was not due to receptor antagonism but rather to a more general mechanism. It was found that 1 can induce GPCR desensitization and internalization, and such effects were mediated by the activation of conventional protein kinase C.

Bryostatin-5 Blocks Stromal Cell–Derived Factor-1 Induced Chemotaxis via Desensitization and Down-regulation of Cell Surface CXCR4 Receptors

The chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), play important roles in hematopoiesis regulation, lymphocyte activation, and trafficking, as well as in developmental processes, including organogenesis, vascularization, and embryogenesis. The receptor is also involved in HIV infection and tumor growth and metastasis. Antagonists of CXCR4 have been widely evaluated for drugs against HIV and tumors. In an effort to identify novel CXCR4 antagonists, we screened a small library of compounds derived from marine organisms and found bryostatin-5, which potently inhibits chemotaxis induced by SDF-1 in Jurkat cells. Bryostatin-5 is a member of the macrolactones, and its analogue bryostatin-1 is currently being evaluated in clinical trials for its chemotherapeutic potential. The involvement of bryostatins in the SDF-1/CXCR4 signaling process has never been reported. In this study, we found that bryostatin-5 potently inhibits SDF-1-induced chemotaxis but does not affect serum-induced chemotaxis. Further studies indicate that this inhibitory effect is not due to receptor antagonism but rather to bryostatin-5-induced receptor desensitization and down-regulation of cell surface CXCR4. We also show that these effects are mediated by the activation of conventional protein kinase C.

Upregulation of AT1 receptor gene on activation of protein kinase Cβ/nicotinamide adenine dinucleotide diphosphate oxidase/ERK1/2/c-fos signaling cascade mediates long-term pressor effect of angiotensin II in rostral ventrolateral medulla

Angiotensin II induces the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2 via the activation of nicotinamide adenine dinucleotide diphosphate (NADPH) oxidase on stimulation of the angiotensin subtype 1 receptor (AT1R) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for the maintenance of vasomotor tone and blood pressure are located. Angiotensin II-activated p38 MAPK in RVLM promotes a short-term pressor effect via augmented glutamatergic neurotransmission. We tested the hypothesis that the NADPH oxidase-dependent phosphorylation of ERK1/2 after the activation of conventional protein kinase C (PKC) mediates the AT1R-dependent long-term pressor effects of angiotensin II via transcriptional induction of the proto-oncogene c-fos gene in RVLM. In Sprague-Dawley rats, a microinjection of angiotensin II bilaterally into the RVLM induced membrane-bound translocation of the conventional PKCalpha, PKCbeta or PKCgamma isoform, phosphorylation of the p47 subunit of NADPH oxidase and ERK1/2, followed by phosphorylation of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB), and c-fos induction. The PKC inhibitor antagonized angiotensin II-induced p47 phosphorylation, and an antisense oligonucleotide (ASON) complementary to PKCbeta messenger RNA suppressed angiotensin II-induced ERK1/2 activation, phosphorylation or DNA binding activity of CREB, and upregulation of c-fos mRNA expression in the ventrolateral medulla. Furthermore, a microinjection of ERK1/2, CREB or c-fos ASON into the RVLM significantly reduced the long-term pressor effect and augmented AT1R expression in the ventrolateral medulla induced by intracerebroventricular infusion of angiotensin II. We concluded that the PKCbeta/NADPH oxidase/ERK1/2/CREB/c-fos cascade represents a novel signaling cascade that mediates the long-term pressor effect induced by angiotensin II in the RVLM.

Bradykinin augments EGF-induced airway smooth muscle proliferation by activation of conventional protein kinase C isoenzymes

This study aims to investigate the effects of bradykinin, alone and in combination with growth factors on proliferation of cultured bovine tracheal smooth muscle cells. Bradykinin did not induce mitogenic responses by itself, but concentration-dependently augmented growth factor-induced [ 3H]thymidine incorporation and cell proliferation. The bradykinin effect was mediated by bradykinin B 2 receptors, and not dependent on cyclo-oxygenase. Bradykinin-induced synergism with epidermal growth factor (EGF) could be suppressed by the protein kinase C (PKC) inhibitors GF 109203X (Bisindolylmaleimide I; specific for conventional and novel PKCs) and Gö 6976 (12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; specific for conventional PKCs). In addition, sole activation of PKC using Phorbol 12-myristate 13-acetate (PMA) was sufficient for a synergistic interaction with EGF. In contrast to bradykinin however, PMA was mitogenic by itself which was not at all affected by Gö 6976, but abolished by GF 109203X. Bradykinin transiently activated the p42/p44 MAP kinase pathway, whereas PMA-induced activation of p42/p44 mitogen activated protein (MAP) kinase was sustained. Neither the combination of bradykinin and EGF nor that of PMA and EGF induced synergistic activation of p42/p44 MAP kinase, however. These results show that bradykinin B 2 receptor-stimulation augments growth factor-induced mitogenic responses of airway smooth muscle cells through activation of conventional PKC isozymes. In addition, the results show that PKC isozyme-specificity underlies stimulus-specific differences in mitogenic capacity for bradykinin and PMA.

Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains.

Activation of conventional protein kinase C by phorbol ester triggers the Src-dependent remodelling of the actin cytoskeleton and the formation of podosomes in vascular smooth muscle cells. Rearrangement of actin cytoskeleton in response to phorbol-12,13-dibutyrate is characterised by the simultaneous disassembly of peripheral actin stress fibres and focal adhesions, focal de novo actin polymerisation and actomyosin contraction in the cell center, indicating a spatially and temporally segregated, differential modulation of actin-cytoskeleton stability and turnover. Taking advantage of the prominent actin cytoskeleton in A7r5 cells we show here, that the molecular basis for the local inhibition of contractility is the specific recruitment of p190RhoGAP to specialised microdomains at the focal adhesion/stress fibre interface, which are constitutively enriched in cortactin. The microdomains contain structurally altered actin filaments inaccessible to phalloidin. However, the filaments remain decorated with high molecular weight tropomyosins. Clustering of cortactin during podosome formation causes the rapid, local dispersion of myosin and tropomyosin, and interferes with the F-actin binding of h1calponin, consistent with a RhoGAP-mediated reduction of contractility. Phorbol ester-induced podosome formation is efficiently blocked by expression of constitutively active Dia1, which leads to the dispersion of cortactin. The results provide direct evidence for the spatially restricted inhibition of contractility via the recruitment and accumulation of cortactin and p190RhoGAP.

Effect of phospholipase Cβ4 lacking in thalamic neurons on electroencephalogram

Activity of thalamic neurons has been shown to be modulated via type-1 metabotropic glutamate receptor (mGluR1) activation, which initiates an intracellular Ca signaling cascade involving phospholipase Cβ4 (PLCβ4) and leading to the activation of conventional protein kinase C (cPKC). In the present study, we investigated the role of PLCβ4 in thalamic neuron. PLCβ4-deficient mutant mice were found to exhibit three phenotypic characteristics: (1) a 2-Hz increase in the peak frequency of electroencephalogram (EEG) of rapid eye movement (REM) sleep, (2) an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) recorded in thalamus, and (3) waveform distortion of EEG. We postulate here that changes in protein phosphorylation due to reduced cPKC activity by PLCβ4 deletion in thalamic neurons may give rise to these phenotypic characteristics. Taken together, these results indicate that reduced PLCβ4 activity in thalamic neurons may underlie high-cortical oscillation frequency.

Control of astrocyte Ca2+ 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-PKCγ plasma membrane translocation events, induced rapid oscillations in GFP-PKCγ translocation, and generated GFP-PKCγ translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca2+-sensitive C2 domains of PKCγ and were driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca2+ waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCγ or PKCδ 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 Ca2+ 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 Ca2+ spikes, suggesting that PKC activation is involved in terminating individual Ca2+ spikes and waves and in defining the time period between Ca2+ spikes.Conclusions: Our study suggests that cPKCs have a negative feedback role on Ca2+ oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca2+ concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca2+ spikes and waves and can define the Ca2+ spike and wave frequencies.