Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human cloned CD8+ cytotoxic T lymphocytes permeabilized with alpha-toxin of Staphylococcus aureus can be triggered by the guanosine triphosphate (GTP) analogue GTP gamma S to release the contents of their granula by exocytosis. To localize the guanosine nucleotide-binding protein (G-protein) activated by GTP gamma S in the sequence of events after T-lymphocyte triggering we have used several inhibitors of T-cell activation that inhibit early stages in T-cell triggering. The protein kinase C-inhibitor staurosporine, the immunosuppressants cyclosporin A and FK-506 and genistein, an inhibitor of tyrosine kinases, all inhibited esterase release triggered in intact cells by anti-T-cell receptor antibodies but not GTP gamma S-induced release from permeabilized cells. Cyclosporin A, FK-506 and genistein also blocked exocytosis triggered in intact cells by a combination of phorbolester and the calcium ionophore A23187. In addition, cytochalasin B, an inhibitor of actin polymerization, inhibited exocytosis in intact cells but enhanced exocytosis from permeabilized cells. These data show that the G-protein effecting exocytosis is localized distally in the cascade of events after T-cell activation.
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PMID:Functional localization of an exocytosis-triggering G-protein in human cytotoxic T lymphocytes. 138 35

Increases in the intracellular Ca2+ concentration of human platelets caused by receptor agonists, such as thrombin, 9,11-epithio-11,12-methanothromboxane A2 (STA2), platelet-activating factor (PAF) and arginine-vasopressin, were inhibited by prior addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) in time-dependent and concentration-dependent manners. The inhibitions were mostly reversed by staurosporine, and inhibitor of protein kinase C, added 1 min before TPA. Prior treatment of platelets with thrombin or STA2, the efficacious Ca2+ mobilizer, suppressed the increase in the intracellular Ca2+ concentration of the cells to other agonists, but treatment with less efficacious PAF or vasopressin did not. The heterologous receptor desensitizations were also reversed by staurosporine. The antibody, directed against the carboxy-terminal region of the alpha subunits 1 and 2 of the inhibitory guanine-nucleotide-binding proteins (Gi1 alpha and Gi2 alpha), was raised in rabbit and was used to immunoprecipitate Gi alpha in 32P-labeled platelets. The radioactivity was detected in Gi alpha after incubation of 32P-labeled platelets with TPA, thrombin or STA2, but not in the cells incubated with PAF or vasopressin. The time-dependency or concentration-dependency of TPA-induced phosphorylation of Gi alpha was similar to the dependency of its inhibitory action on agonist-induced Ca2+ mobilization. Thus, strong activation of Ca2+/phospholipid-dependent protein kinase C by phorbol ester or agonists of certain Ca(2+)-mobilizing receptors leads to phosphorylation of the alpha subunit of guanine-nucleotide-binding protein, thereby impairing the coupling of the G protein to receptors as a feedback regulatory component of the receptor-triggered intracellular Ca(2+)-mobilizing system.
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PMID:Phosphorylation of the inhibitory guanine-nucleotide-binding protein as a possible mechanism of inhibition by protein kinase C of agonist-induced Ca2+ mobilization in human platelet. 157 85

Recently, the alpha-subunit of the inhibitory guanine-nucleotide-binding protein Gi2 (alpha-Gi2) has been shown to be a substrate for phosphorylation both by protein kinase C and also by other unidentified kinase(s) which are activated as a result of elevated cyclic AMP levels in intact rat hepatocytes [Bushfield, Murphy, Lavan, Parker, Hruby, Milligan & Houslay (1990) Biochem. J. 268, 449-457]. Here we show that the incorporation of [32P]Pi into alpha-Gi2 was enhanced 3-fold by incubation of intact hepatocytes with the tumour promoter and protein phosphatase (1 and 2A) inhibitor, okadaic acid. This action was both time- and concentration-dependent and was accompanied by a loss of guanine-nucleotide-induced inhibition of adenylate cyclase. The increased labelling of alpha-Gi2 induced by okadaic acid was partially additive with that elicited by 8-bromo cyclic AMP, but not with that elicited by the protein kinase C activator phorbol 12-myristate 13-acetate. We suggest that, in the absence of hormones, the activity of alpha-Gi2 is under the control of a dynamic phosphorylation/dephosphorylation system involving protein kinase C and protein phosphatases 1 and/or 2A. This highlights the regulation of kinases and phosphatases as both providing potentially important mechanisms for causing 'cross-talk' between different signalling systems, in this instance controlling cellular responsiveness through regulation of alpha-Gi2 phosphorylation.
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PMID:Okadaic acid identifies a phosphorylation/dephosphorylation cycle controlling the inhibitory guanine-nucleotide-binding regulatory protein Gi2. 190 Sep 86

1. Metabolically stable analogues of GTP, e.g. guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta,gamma-imido]triphosphate (pp[NH]pG), enhance the extent of Ca2(+)-dependent secretion of beta-N-acetylglucosaminidase and beta-galactosidase from electropermeabilised human platelets in the presence of less than 5 microM Ca2+. A similar effect is observed on addition either of 1,2-dioctanoin or of GTP in in the presence or absence of thrombin. 2. In the presence of higher Ca2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca2+] of 30-40 microM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca2(+)-dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2-dioctanoin. 3. The concentration of GTP[S] or pp[NH]pG required to obtain half-maximal enhancement of lysosomal secretion is dependent on [Ca2+] for secretion of 5-hydroxytryptamine, beta-N-acetylglucosaminidase and beta-galactosidase. At two fixed [Ca2+] the median effective concentration (EC50) values for GTP[S] and pp[NH]pG which characterise enhancement of 5-hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of beta-N-acetylglucosaminidase and beta-galactosidase. 4. In the presence of a saturating concentration of GTP[S] marked 5-hydroxytryptamine and beta-N-acetylglucosaminidase secretion is observed at nanomolar [Ca2+] and these responses show little dependence on [Ca2+] over the attainable range. Secretion of beta-N-acetylglucosaminidase is also induced at nanomolar Ca2+ concentrations by addition of activators of protein kinase C. 5. Guanosine 5'-[beta-thio]diphosphate inhibits enhancement of beta-N-acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca2+ when added alone. 6. Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca2(+)-dependent platelet lysosomal secretion by activating a guanine-nucleotide-binding protein (GE) located close to the exocytotic site. However, not all the data are consistent with this postulate.
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PMID:Guanine nucleotides and Ca2(+)-dependent lysosomal secretion in electropermeabilised human platelets. 211 63

A mechanism by which protein kinase C potentiates arachidonic acid (AA) liberation in rabbit platelets was examined using [3H]AA-labeled, saponin (7 micrograms/ml)-permeabilized rabbit platelets. Pretreatment of the [3H]AA-labeled platelets with 4 beta-phorbol 12-myristate 13-acetate (PMA, 10-40 nM) or 1,2-dioctanoylglycerol (DOG, 20 microM) enhanced [3H]AA liberation induced by an addition of Ca2+ (1 mM) after cell permeabilization, whereas 4 alpha-phorbol 12,13-didecanoate (80 nM) did not exert such an effect. The potentiating effects of PMA and DOG were inhibited by staurosporine (200 nM). PMA (40 nM) also potentiated [3H]AA liberation induced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S, 100 microM), 5'-guanylyl imidodiphosphate (200 microM) or NaF (20 mM) plus AlCl3 (10 microM) in the presence of Ca2+ (100 microM). The enhancement by PMA of the GTP gamma S-induced AA liberation was also inhibited by staurosporine (200 nM). Furthermore, guanosine 5'-[beta-thio]diphosphate (GDP beta S, 0.5-2 mM) suppressed the PMA (40 nM)- and DOG (20 microM)-enhanced, Ca2+ (1 mM)-dependent [3H]AA liberation. This inhibitory effect of GDP beta S was reversed by a further addition of GTP gamma S (200 microM). However, pertussis toxin (0.2-1 micrograms/ml) had no effect on the PMA-enhanced [3H]AA liberation. These results indicate a possibility that protein kinase C may potentiate AA liberation through a guanine-nucleotide-binding protein-mediated mechanism in saponin-permeabilized rabbit platelets.
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PMID:Involvement of a guanine-nucleotide-binding protein-mediated mechanism in the enhancement of arachidonic acid liberation by phorbol 12-myristate 13-acetate and Ca2+ in saponin-permeabilized platelets. 211 77

Chronic opioid treatment of neuroblastoma x glioma NG108-15 cells induces desensitization of the opioid receptor and this may involve a change in membrane protein phosphorylation. In an attempt to mimic this possible mechanism, we studied effects of phorbol ester activation of protein kinase C on opioid receptor activity. Incubation of NG108-15 hybrid cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) abolished up to 45% of opioid inhibition of cyclic AMP accumulation in intact cells, while basal accumulation and prostaglandin E1-stimulated cyclic AMP accumulation were unaltered. This decrease of opioid inhibition was dose- and time-dependent and the potency order of phorbol esters and apparent K activation (90 nM) for TPA were consistent with phorbol esters acting through the stimulation of protein kinase C. TPA also decreased the inhibition of cyclic AMP accumulation mediated through muscarinic and alpha-2 adrenergic receptors. These effects of TPA were best explained by a TPA-induced alteration of the inhibitory nucleotide-binding protein (Gi), the common transducer protein of these receptors. Impairment of Gi by TPA treatment was evidenced by a reduction in agonist-stimulated GTP hydrolysis and activation by GTP. Quantification of Gi by pertussis toxin-catalyzed ADP-ribosylation revealed that TPA decreased maximal labeling. In summary, phorbol esters appeared to attenuate opioid receptor activity by altering the activity of the transducer protein Gi.
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PMID:Attenuation of opioid receptor activity by phorbol esters in neuroblastoma x glioma NG108-15 hybrid cells. 215 50

The potentiation of corticotropin-releasing factor (CRF)-stimulated cAMP production by vasopressin (VP) in the pituitary cell was investigated by studies on the interaction of CRF, VP, and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) on cAMP, adenylate cyclase and phosphodiesterase. Addition of VP or PMA (0.01-100 nM) alone did not alter cellular cAMP content, but markedly increased the effect of 10 nM CRF with ED50 of about 1 nM. Treatment of the cells with 200 ng/ml pertussis toxin for 4 h increased CRF-stimulated cAMP accumulation by 3.2-fold, an effect that was not additive to those of VP and PMA. Incubation of pituitary cells with 2 mM 1-methyl-3-isobutylxanthine increased CRF-stimulated cAMP accumulation and decreased the relative effect of VP and PMA, suggesting that the actions of VP and PMA are partially due to inhibition of phosphodiesterase. This was confirmed by the demonstration of a 30% inhibition of the low-affinity phosphodiesterase activity in cytosol and membranes prepared from cells preincubated with VP or PMA. In intact cells, following [3H]adenine prelabeling of endogenous ATP pools, measurement of adenylate cyclase in the presence of 1-methyl-3-isobutylxanthine showed no effect of VP and PMA alone, but did show a 2-fold potentiation of the effect of CRF. Measurement of adenylate cyclase in pituitary homogenates by conversion of [alpha-32P]ATP to [32P]cAMP showed a paradoxical GTP-dependent inhibition by VP of basal and CRF-stimulated adenylate cyclase activity, suggesting that the VP receptor is coupled to an inhibitory guanyl nucleotide-binding protein. Pertussis toxin pretreatment of the cells prevented the VP inhibition of adenylate cyclase activity observed in pituitary cell homogenates. These findings indicate that besides inhibition of phosphodiesterase, VP has a dual interaction with the pituitary adenylate cyclase system; a direct inhibitory effect, manifested only in broken cells, that is mediated by a receptor-coupled guanyl nucleotide-binding protein, and a physiologically predominant indirect stimulatory effect in the intact cell, mediated by protein kinase C phosphorylation of one of the components of the CRF-activated adenylate cyclase system.
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PMID:Phorbol 12-myristate 13-acetate and vasopressin potentiate the effect of corticotropin-releasing factor on cyclic AMP production in rat anterior pituitary cells. Mechanisms of action. 243 73

In rabbit platelets, collagen (50 micrograms/ml)- or thrombin (0.5 U/ml)-induced diacylglycerol formation was dose-dependently prevented by phorbol 12-myristate 13-acetate (PMA, 2-50 nM). However, collagen-induced arachidonic acid liberation and lysophosphatidylcholine formation were rather enhanced by PMA, while the thrombin-induced liberation was not. We also demonstrated with saponin-permeabilized platelets that collagen (100 micrograms/ml)-induced arachidonic acid liberation was enhanced by GTP gamma S and inhibited by GDP beta S, both dose-dependently. Since these results lead us to consider that protein kinase C affects a guanine-nucleotide-binding protein (G-protein) to modulate phospholipase A2 and C, we investigated this dual effect of PMA on arachidonic acid liberation and diacylglycerol formation induced by G-protein activator. Addition of GTP gamma S (100 microM) to saponin-permeabilized platelets significantly induced these responses, and PMA (2-10 nM)-pretreatment before the cell permeabilization inhibited diacylglycerol formation and enhanced arachidonic acid liberation and lysophosphatidylcholine formation, dose-dependently. Likewise, PMA (20 nM) had differential effects on the similar NaF (20 mM)-induced responses in intact platelets. Contrarily, 10 nM PMA had no effect on diacylglycerol formation caused by an addition of high concentration of Ca2+ (1 mM) alone after the cell permeabilization, while it still had a potentiating effect on arachidonic acid liberation under the condition. These results suggest that protein kinase C may have a dual regulatory effect on the activation of phospholipase A2 (positive feedback) and phospholipase C (negative feedback), probably through influences on two distinct G-proteins associated separately with these two enzymes.
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PMID:Differential effects of phorbol 12-myristate 13-acetate on GTP gamma S-induced diacylglycerol formation and arachidonic acid liberation in saponin-permeabilized rabbit platelets. 249 43

The addition of the protein kinase C activator phorbol esters to cell suspension a few minutes prior to stimulation inhibits the agonists-induced biochemical changes and cell responses. This inhibition is prevented by protein kinase C inhibitors. Activation of protein kinase C down regulates the stimulated responses by affecting one or more of the steps in the exitation-response coupling. This includes the receptors, the quanine-nucleotide-binding protein, the activity or distribution of phospholipase C, and other steps.
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PMID:Inhibition of stimulated cell responses by phorbol esters and other activators of protein kinase C: sites of action. 285 61

Development of an enriched cultured cell system allowed us to investigate the mechanism of cholinergic inhibition of somatostatin release stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) and Ca2+-protein kinase C-dependent pathways of cell activation. After a 24-h culture on rat tail collagen, D-cells, quantified by immunohistochemistry, were 18-fold enriched compared with unelutriated dispersed cells. Somatostatin release from cultured cells was expressed as a percent of the somatostatin released by a specific stimulus in control cells. Under basal conditions release of somatostatin was 2.3 +/- 0.6% of the total cell content. Epinephrine (1 microM) and cholecystokinin octapeptide (10 nM) increased somatostatin release to 6.98 +/- 1.25 and 10.72 +/- 1.64%, respectively. Carbachol (1 microM) completely inhibited somatostatin release stimulated by epinephrine and reduced cholecystokinin octapeptide-stimulated release to 75% of control levels. Carbachol inhibition of the response to both epinephrine and cholecystokinin octapeptide was totally prevented by 5 h of treatment of the cells with pertussis toxin (300 ng/ml). Somatostatin release in response to the diterpene forskolin (10 microM), dibutyryl cAMP (300 microM), the phorbol ester beta-phorbol 12-myristate 13-acetate (0.1 microM), and the calcium ionophore A23187 (1 microM) was also inhibited by carbachol and prevented by pertussis toxin pretreatment. The ADP-ribosylase inhibitor isonicotinamide (1 mM) selectively blocked the effect of pertussis toxin without altering other stimulatory or inhibitory responses. These data are consistent with the view that carbachol inhibits somatostatin release at guanyl nucleotide-binding protein and/or another pertussis toxin-sensitive site.
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PMID:Pertussis toxin-sensitive cholinergic inhibition of somatostatin release from canine D-cells. 290 2


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