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

Guanosine 5'-O-(gamma-thio)triphosphate (GTP[S]), NaF and cholecystokinin-octapeptide (CCK-8) were used to examine the participation of G proteins in agonist-induced contraction of smooth muscle cells isolated separately from circular and longitudinal muscle layer of guinea pig intestine. All three agents stimulated inositol 1,4,5-triphosphate (InsP3) production and protein kinase C activity to the same extent in permeabilized (GTP[S] and CCK-8) and nonpermeabilized (NaF and CCK-8) muscle cells. InsP3 production was 9 to 13 times higher in circular muscle cells consistent with preferential hydrolysis of phosphatidylinositol 4,5-biphosphate in this cell type. InsP3 production and protein kinase C activation in permeabilized muscle cells were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM). Maximal concentrations of GTP[S] (100 microM), CCK-8 (1 nM) and InsP3 (1 microM) elicited similar increases in [Ca++]i, net 45Ca++ efflux and contraction in permeabilized circular, but not longitudinal, muscle cells [( Ca++]i: 224 +/- 35 nM, 279 +/- 29 nM and 288 +/- 45 nM increase above basal level; 45Ca++ efflux: 35 +/- 2%, 34 +/- 3% and 37 +/- 3% decrease in cell Ca++ content; contraction: 26 +/- 2%, 24 +/- 2% and 25 +/- 2% decrease in cell length). The responses to GTP[S] and CCK-8 were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM) and heparin (10 micrograms/ml), whereas the response to InsP3 was abolished by heparin only. Maximal concentrations of NaF and CCK-8 elicited similar increases in [Ca++]i and contraction in nonpermeabilized circular and longitudinal muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Receptor-coupled G proteins mediate contraction and Ca++ mobilization in isolated intestinal muscle cells. 130 82

Recombinant forms of Gs alpha-1 and Gs alpha-4 were shown to act as substrates for a purified preparation of brain protein kinase C. Both forms of Gs alpha were thermally denatured during the incubation such that phosphorylation was virtually complete (greater than 90%) after 30 min. The quantity of phosphate incorporated into approximately equivalent starting amounts of the two forms of Gs alpha (4.8 pmol of Gs alpha-1 and 5.5 pmol of Gs alpha-4) at maximal phosphorylation were 0.23 +/- 0.08 pmol for Gs alpha-1 and 0.56 +/- 0.12 pmol for Gs alpha-4. Since both forms of Gs alpha were thermally denatured to the same extent after 30 min, the increased phosphorylation state of Gs alpha-4 provides evidence that Gs alpha-4 contains an additional phosphorylation site. Bray and co-workers [Bray, Carter, Simmons, Guo, Puckett, Kamhollz, Spiegel & Nirenberg (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8893-8897] proposed that an additional phosphorylation site may exist at the splice junction in Gs alpha-4. The guanine-nucleotide-free form of Gs alpha appears to be the preferred substrate for phosphorylation. This interpretation is based upon the following observations. (i) Guanosine 5'-[beta-thio]diphosphate at micromolar concentrations inhibits the susceptibility of Gs alpha to phosphorylation; (ii) beta gamma-subunits, which inhibit GDP release from Gs alpha-GDP at millimolar Mg2+ concentrations, also inhibit the susceptibility of Gs alpha to phosphorylation; and (iii) guanosine 5'[beta gamma-imido]triphosphate inhibits the susceptibility of Gs alpha to act as a substrate for phosphorylation. These studies suggest that there is potential for cross-talk between receptors which trigger PtdIns(4,5)P2 hydrolysis and subsequently protein kinase C activation, and receptors which stimulate adenylate cyclase via Gs.
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PMID:Phosphorylation of the spliced variant forms of the recombinant stimulatory guanine-nucleotide-binding regulatory protein (Gs alpha) by protein kinase C. 163 17

To elucidate the role of ATP in histamine release, the present study was performed using beta-escin-permeabilized rat peritoneal mast cells. Ca(2+)-induced histamine release from permeabilized cells is totally dependent upon exogenous ATP in the medium. In the presence of Ca2+, ATP caused histamine release concentration-dependently at concentrations ranging from 0.01 to 5 mmol/l. The maximum release was achieved at 3 mmol/l of ATP in the medium. When the other adenosine nucleotides (AMP, ADP), or nonhydrolyzable ATP analogues (adenylylimidodiphosphate, beta, gamma-methylene ATP) were added in place to ATP, no histamine release took place. Other ribonucleoside triphosphates (GTP, ITP, UTP and CTP) had little effect at the same concentration range. When the ribonucleoside triphosphate content of mast cells was determined by means of HPLC, ITP and CTP were not detectable. A millimolar range of the ATP content was determined in mast cells, but the amounts of other ribonucleoside triphosphates (GTP and UTP) were remarkably lower than that of ATP. These results seem to indicate that the ATP molecule plays a crucial role in histamine release from rat mast cells in association with its concurrent hydrolysis. Furthermore, 12-O-tetradecanoylphorbol-13-acetate and 1-oleoyl-2-acetylglycerol enhanced histamine release elicited in the presence of Ca2+ (0.1 mumol/l) and ATP (3 mmol/l). Calphostin C, a potent inhibitor of protein kinase C, inhibited Ca2+/ATP-dependent histamine release by approximately 60%. At the same concentration, calphostin C inhibited by 95% protein kinase C activity in the crude extract obtained from rat mast cells. It was suggested that protein kinase C activation took place in the Ca2+/ATP-dependent histamine release from permeabilized rat mast cells.
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PMID:Essential role of ATP and possibility of activation of protein kinase C in Ca(2+)-dependent histamine release from permeabilized rat peritoneal mast cells. 171 71

Receptor-linked activation of phospholipase D has been demonstrated recently in a variety of intact cell types including granulocytes, but little is known about the enzyme, its cofactor requirements, and regulation. Using [3H]alkyllysophosphatidylcholine to prelable an endogenous phosphatidylcholine substrate pool in conjunction with transphosphatidylation using ethanol to generate labeled phosphatidylethanol, we demonstrated a novel phospholipase D activity in neutrophil subcellular fractions. Guanosine 5'-O-3-(thiotriphosphate) (GTP gamma S) and phorbol 12-myristate 13-acetate (PMA) activated both phosphatidic acid generation and transphosphatidylation. Activity using both activators required the presence of not only plasma membrane but also cytosol, and proteolytic and thermal inactivation demonstrated the requirement for protein factors in both fractions. Using both stimuli, activity increased with increasing cytosol concentration. Product formation was approximately linear for about 10 min with PMA and 30 min with GTP gamma S, and both activators resulted in the total hydrolysis of up to 10% of the labeled phosphatidylcholine. The activity using both activators showed similar broad neutral pH optima, and both required the presence of micromolar levels of calcium, which by itself failed to activate at concentrations up to 1 mM. At low micromolar concentrations of nucleotides, activation was specific for guanine nucleotides and showed a specificity of GTP gamma S greater than guanyl-5'-yl imidodiphosphate greater than GTP, with no effect of GDP and GMP or adenine nucleotides, consistent with the participation of a guanine nucleotide regulatory protein. PMA activation was dependent on the presence of ATP, in particular when dialyzed cytosol was used, and was inhibited by about 50% by staurosporine, supporting a role for protein kinase C. However, purified protein kinase C failed to substitute for cytosol, implicating an additional cytosolic factor(s) in this response. These results indicate that the granulocytic phospholipase D pathway is a complex system that is regulated by at least two activation pathways, each comprised of components in two subcellular compartments.
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PMID:Phospholipase D activation in a cell-free system from human neutrophils by phorbol 12-myristate 13-acetate and guanosine 5'-O-(3-thiotriphosphate). Activation is calcium dependent and requires protein factors in both the plasma membrane and cytosol. 189 16

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

The effect of the hydrolysis-resistant GTP analogs, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin-permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca2+ (10 microM) was not affected by GTP gamma S or GMPPNP, secretion in the absence of Ca2+ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTP gamma S or GMPPNP was approximately 80% of that induced by 10 microM Ca2+. Half-maximum stimulation was induced by 30 microM GTP gamma S or GMPPNP. Both Ca2(+)-stimulated and GTP gamma S-stimulated secretion were ATP dependent and inhibited by N-ethylmaleimide. The GTP gamma S-stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca2+ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12-O-tetradecanoylphorbol 13-acetate caused a 50% increase in both Ca2(+)-stimulated and GTP gamma S-stimulated secretion. Cholera and pertussis toxins did not affect Ca2(+)-stimulated or GTP gamma S-stimulated NE secretion. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and GTP inhibited GTP gamma S-stimulated secretion but not Ca2(+)-stimulated secretion. The inability of GDP beta S to inhibit Ca2(+)-stimulated secretion indicates that the process affected by GTP gamma S is not an essential step in the Ca2(+)-stimulated pathway.
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PMID:Hydrolysis-resistant GTP analogs stimulate catecholamine release from digitonin-permeabilized PC12 cells. 211 52

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorbol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+free] of 1 microM, PLD activity was detected in control membranes, but was 5.4 +/- 0.8-fold (mean +/- S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated PLD by 18 +/- 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 +/- 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 microM-Ca2+ (3.5 +/- 0.2-fold and 1.8 +/- 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[beta-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.
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PMID:Phorbol ester treatment of intact rabbit platelets greatly enhances both the basal and guanosine 5'-[gamma-thio]triphosphate-stimulated phospholipase D activities of isolated platelet membranes. Physiological activation of phospholipase D may be secondary to activation of phospholipase C. 212 96

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca2+]i. Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature (London) 316, 440-442]. Treatment with acetylcholine resulted in the rapid (within seconds), selective, and reversible inhibition of the rapidly inactivated, N-type current, whereas the long-lasting L-type current remained unaffected. The high sensitivity to blocker drugs (atropine, pirenzepine) indicated that this effect of acetylcholine was due to a muscarinic M1 receptor. Intracellular perfusion with nonhydrolyzable guanine nucleotide analogs or pretreatment of the neurons with pertussis toxin had profound effects on the Ca2+ current modulation. Guanosine 5'-[gamma-thio]triphosphate caused the disappearance of the N-type current (an effect akin to that of acetylcholine, but irreversible), whereas guanosine 5'-[beta-thio]diphosphate and pertussis toxin pretreatment prevented the acetylcholine-induced inhibition. In contrast, cAMP, applied intracellularly together with 3-isobutyl-1-methylxanthine, as well as activators and inhibitors of protein kinase C, were without effect. Acetylcholine caused shortening of action potentials in neurons treated with tetraethylammonium to partially block K+ channels. Moreover, when applied to neurons loaded with the fluorescent indicator fura-2, acetylcholine failed to appreciably modify [Ca2+]i at rest but caused a partial blunting of the initial [Ca2+]i peak induced by depolarization with high K+. This effect was blocked by muscarinic antagonists and pertussis toxin and was unaffected by protein kinase activators. Thus, muscarinic modulation of the N-type Ca2+ channels appears to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein and independent of both cAMP-dependent protein kinase and protein kinase C.
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PMID:Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+ current in rat sympathetic neurons. 243 97

Pretreatment with pertussis toxin inhibits angiotensin II-induced activation of polyphosphoinositide phosphodiesterase in rat renal mesangial cells [Pfeilschifter & Bauer (1986) Biochem. J. 236, 289-294]. Furthermore, activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and by 1-oleoyl-2-acetylglycerol (OAG) abolishes angiotensin II-induced formation of inositol trisphosphate (IP3) in mesangial cells [Pfeilschifter (1986) FEBS Lett. 203, 262-266]. Using membrane preparations of [3H]inositol-labelled mesangial cells we tried to obtain further insight as to the step at which protein kinase C might interfere with the signal transduction mechanism in mesangial cells. Angiotensin II (100 nM) stimulates IP3 formation from membrane preparations of [3H]inositol-labelled mesangial cells with a half-maximal potency of 1.1 nM. The angiotensin II-induced formation of IP3 is enhanced by GTP. This effect of angiotensin II is completely blocked by the competitive antagonist [Sar1,Ala8]angiotensin II. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p), non-hydrolysable analogues of GTP, stimulate IP3 production in the absence of angiotensin II with Kd values of 0.19 microM and 2.4 microM, respectively. Angiotensin II augments the increase in IP3 formation induced by GTP gamma S. However, when mesangial cells were pretreated with TPA there was a dose-dependent inhibition of the synergistic action of angiotensin II on GTP gamma S-induced IP3 production. Comparable results are obtained with OAG, while the non-tumour-promoting phorbol ester 4 alpha-phorbol 12,13-didecanoate is without effect. These results suggest that activation of protein kinase C in mesangial cells does not impair phosphoinositide hydrolysis by stable GTP analogues but somehow seems to interfere with the stimulatory interaction of the occupied angiotensin II receptor with the transducing G-protein.
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PMID:Different effects of phorbol ester on angiotensin II- and stable GTP analogue-induced activation of polyphosphoinositide phosphodiesterase in membranes isolated from rat renal mesangial cells. 282 20

Activation of the NADPH oxidase was examined in electrically permeabilized human neutrophils exposed to non-hydrolysable guanine nucleotides. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced a marked increase in the rate of O2 consumption, which was partially resistant to staurosporine, an inhibitor of protein kinase C, under conditions where the response to diacylglycerol was virtually abolished. The respiratory burst elicited by GTP[S] was dependent on the presence of ATP and Mg2+, suggesting involvement of phosphorylation reactions. Accordingly, phosphoprotein formation was greatly stimulated by the guanine nucleotide. The polypeptide phosphorylation pattern induced by GTP[S] was similar to, but not identical with, that observed with diacylglycerol, indicating the activation of kinases other than protein kinase C by the guanine nucleotide. The possible involvement of tyrosine kinases was assessed by immunoblotting using anti-phosphotyrosine antibodies. Treatment of electroporated cells with GTP[S] stimulated the accumulation of tyrosine-phosphorylated proteins. This effect was not induced by diacylglycerol, indicating that tyrosine phosphorylation is not secondary to stimulation of protein kinase C. The results indicate that, in neutrophils, activated G-proteins can stimulate tyrosine kinase and/or inhibit tyrosine phosphatase activity. Changes in the amounts of tyrosine-phosphorylated proteins may signal activation of the respiratory burst.
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PMID:Guanine nucleotides induce tyrosine phosphorylation and activation of the respiratory burst in neutrophils. 293 Apr 92


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