Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied cAMP-dependent phosphorylation of sodium channels in rat brain neurons maintained in primary culture. In back phosphorylation studies, cells were treated with drugs to increase intracellular cAMP and sodium channels were solubilized and isolated by immunoprecipitation. Surface and intracellular pools of sodium channels were isolated separately. Purified channels were then phosphorylated with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase to incorporate 32P into available cAMP-dependent phosphorylation sites. The amount of 32P incorporated in vitro is inversely proportional to the extent of endogenous phosphorylation. Incubation of cells with forskolin (0.1-100 microM), 8-Br-cAMP (0.1-10 mM), or isobutylmethylxanthine (0.01-1.0 mM) inhibited subsequent incorporation of 32P into isolated sodium channels by 70-80%, indicating that treatment of cells with these drugs had increased endogenous phosphorylation to nearly maximum levels. The phosphopeptides phosphorylated in vivo and in vitro were identical. To examine the magnitude of basal phosphorylation and the extent of stimulated phosphorylation, the amount of 32P incorporated into sodium channels from control and stimulated cells was compared to that from matched samples which had been dephosphorylated with calcineurin. Sodium channels from control cells incorporated approximately 2-fold more 32P after dephosphorylation, indicating that cAMP-dependent sites on the channel are at least 47% phosphorylated in the basal state. Sodium channels from forskolin-treated cells incorporated 7-8-fold more 32P after dephosphorylation, indicating that cAMP-dependent phosphorylation sites are 80-90% phosphorylated after stimulation. Cell surface and intracellular pools of sodium channels were phosphorylated similarly. In cells metabolically labeled with 32P, cell surface sodium channels incorporated 2.7 mol of phosphate/mol of channel. Forskolin stimulated 32P incorporation into sodium channels 1.3-fold, consistent with the results obtained by back phosphorylation. We conclude that the rat brain sodium channel is substantially phosphorylated in both the cell surface and intracellular pools in vivo in unstimulated rat brain neurons, and the extent of phosphorylation is increased to 80-90% of maximum phosphorylation by agents that elevate intracellular cAMP.
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PMID:Cyclic-AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain neurons. 244 66

The mechanism of the cAMP involvement in regulation of cellular functions was studied here using a novel functional assay (antigen receptor-triggered exocytosis of granules) of cloned cytotoxic T lymphocytes (CTL). We suggest that cAMP-dependent protein kinase, protein kinase A, counteracts the protein kinase C and Ca2+-mediated stimulatory T-cell antigen receptor (TcR)-triggered biochemical pathway. This suggestion is supported by experimental results which satisfy criteria for protein kinase A involvement in cellular functions. Pretreatment of CTL with cholera toxin induces cAMP accumulation in CTL, partially inhibits TcR-triggered "lethal hit" delivery to the target cell, and almost completely blocks TcR-triggered exocytosis of granules from CTL. Other agents that raise the intracellular level of cAMP, including forskolin and isobutylmethylxanthine (IBMX) also inhibit TcR-triggered CTL activation. Involvement of cAMP-dependent protein kinase in an inhibitory pathway is suggested by the synergistic effects of cyclic nucleotide analogs 8-bromo-cAMP and N6-benzoyl-cAMP in inhibition of TcR-triggered exocytosis. Forskolin and IBMX inhibited TcR-triggered phosphoinositide turnover in CTL, suggesting that cAMP affected very early events in signal transduction that follow TcR cross-linking by a ligand. The ability of IBMX to inhibit CTL activation when the TcR cross-linking step was by-passed by the combination of phorbol myristate acetate and ionophore A23187 suggests that the locus of inhibitory effect of cAMP is at both the early and late stages of the TcR-triggered transmembrane signaling pathway.
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PMID:Locus of inhibitory action of cAMP-dependent protein kinase in the antigen receptor-triggered cytotoxic T lymphocyte activation pathway. 244 8

Our previous work demonstrated that 8-bromo-cAMP promotes the secretion of both hCG and progesterone by cultured cytotrophoblasts. This study was conducted to characterize the adenylate cyclase of cytotrophoblasts and to examine the effects of agents that stimulate adenylate cyclase on hCG secretion. Adenylate cyclase activity was detected in purified cytotrophoblasts, as were membrane-bound stimulatory and inhibitory guanine nucleotide regulatory proteins, Gs and Gi. Adenylate cyclase was stimulated by MnCl2 and MgCl2, and the effects of MgCl2 were amplified by the GTP analog guanylylimidodiphosphate. Cholera toxin stimulated both cAMP and hCG production by cultured cytotrophoblasts, confirming the coupling of Gs to the adenylate cyclase. Forskolin also stimulated adenylate cyclase, cAMP synthesis, and hCG secretion. Pertussis toxin did not affect hCG secretion in either the absence or presence of forskolin. 8-Bromo-cAMP stimulated cytotrophoblast protein kinase activity, resulting in the increased phosphorylation of a protein with a mol wt of about 70,000, and produced a marked stimulation of hCG secretion. Our findings suggest that the level of expression of adenylate cyclase activity is one determinant of the endocrine function of the differentiating trophoblast.
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PMID:Adenylate cyclase in human cytotrophoblasts: characterization and its role in modulating human chorionic gonadotropin secretion. 244 28

1. The reduction of cytoplasmic free calcium, [Ca2+]i following stimulation, has been investigated in fura-2-loaded human platelets in the presence of low extracellular calcium concentration. Thrombin produced a rapid rise in [Ca2+]i which then fell back to the basal level within 2 min. 2. Ionomycin produced a rapid elevation in [Ca2+]i which then declined to a plateau well above the basal calcium level. The addition of thrombin after ionomycin accelerated the decline in [Ca2+]i back towards basal levels, an action mimicked by phorbol myristate acetate (PMA). 3. Thrombin promoted the efflux of 45Ca2+ from cells co-loaded with fura-2 and the isotope. Ionomycin also promoted an efflux of 45Ca2+ which was increased by the subsequent addition of thrombin or PMA. These results confirm the ability of thrombin and PMA to stimulate Ca2+ removal from the cells. 4. The complete substitution of extracellular Na+ with N-methyl-D-glucamine (NMDG) did not alter the time course of the return of [Ca2+]i to basal following stimulation by thrombin, nor the ability of thrombin or PMA to promote Ca2+ efflux after elevation of [Ca2+]i by ionomycin. 5. The insensitivity to external Na+ suggests that the stimulated Ca2+ efflux is mediated by a Ca2+-ATPase rather than Na+-Ca2+ exchange. This pump does not appear to be activated by Ca2+-calmodulin since [Ca2+]i remains high when elevated by ionomycin. The ability of PMA to stimulate removal suggests that its known target, protein kinase C, can stimulate the Ca2+ pump. Forskolin, which stimulates adenylate cyclase, did not stimulate a fall in [Ca2+]i in the presence of ionomycin, indicating that cyclic AMP-dependent protein kinase does not stimulate Ca2+ extrusion.
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PMID:Stimulated calcium efflux from fura-2-loaded human platelets. 245 43

Incubating skeletal muscle fibers with forskolin, an activator of adenylate cyclase, increases the rate at which nicotinic acetylcholine receptors (AChRs) desensitize when exposed to ACh. Several reports indicate that this is due to the phosphorylation of AChRs by cAMP-dependent protein kinase, but other studies suggest that forskolin interacts with AChRs directly and that second-messenger systems are not required. To help clarify this issue, we studied the effects of forskolin and several other drugs on AChR function in embryonic rat myotubes. AChR function was studied by recording ACh-induced membrane depolarizations and ACh-induced single-channel currents. Our results indicate that forskolin at low concentrations enhances AChR desensitization through the action of a second messenger, most likely cAMP. An analog of forskolin that is much less effective in activating adenylate cyclase (1,9-dideoxyforskolin) is also much less potent in enhancing desensitization. Forskolin at low concentrations does not alter single-channel conductance or mean channel open time. However, when used at concentrations above 20 microM, forskolin may also exert direct drug effects on AChRs.
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PMID:Desensitization of acetylcholine receptors in rat myotubes is enhanced by agents that elevate intracellular cAMP. 245 25

The following studies were conducted with the goal of understanding some of the molecular mechanisms by which cAMP alters granulosa cell function. In this regard, we characterized the expression of messages for the cholesterol side-chain cleavage cytochrome P450 (P450scc) enzyme and a type II cAMP-dependent protein kinase subunit (RII beta) in granulosa cells isolated from small antral follicles and cultured in 1% fetal bovine serum-containing medium. Forskolin (FSK) stimulated cAMP production followed by accumulation of RII beta mRNAs, induction of P450scc mRNA, and, finally, progesterone biosynthesis. The regulation of each mRNA displayed a different sensitivity to actinomycin-D treatment. To determine if the modulation of RII beta or the induction of P450scc could be mediated by the enhancer activity of a cAMP-responsive DNA element (CRE), plasmid DNA containing the CRE and TATA box of the human glycoprotein alpha-subunit (alpha G) gene fused to the chloramphenicol acetyl transferase (CAT) gene was introduced into cultured granulosa or interstitial cells, and the ability of FSK to stimulate the expression of the CAT enzyme was measured. When granulosa cells were isolated from preantral/small antral follicles and maintained for at least 5 days in culture, 5 or 10 microM FSK reversibly stimulated expression of the CAT enzyme within 18 h posttransfection. Under similar conditions, interstitial ovarian cells (prepared from the residual ovarian tissue remaining after granulosa cell isolation) were unable to express the template unless the cells were pretreated for 24 h with FSK before transfection. Thereafter, CAT gene expression by interstitial cells was maintained in a FSK-insensitive manner. To determine if cAMP-dependent transcription of the reporter gene required the same sequences that had been characterized for placenta-derived cells, a truncated plasmid lacking the CRE of the alpha G gene was transfected. Under no condition was expression of the CAT gene observed from a CRE-deficient template. The acute transcriptional activation of a CRE/TATA box-containing gene by cAMP in granulosa cells indicated that transcription-activating proteins interacting with the CRE were either present in these cells or were rapidly synthesized after stimulation. To distinguish between these two possibilities, protein synthesis was transiently inhibited after transfection, before the addition of FSK, by incubating the cells for 2 h with 25 micrograms/ml cycloheximide. Cycloheximide treatment alone did not stimulate transcription from the CRE-containing molecule. Incubation with cycloheximide, followed by treatment with FSK, increased CAT activity 2-fold compared to t
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PMID:An adenosine 3'5'-monophosphate-responsive deoxyribonucleic acid element confers forskolin sensitivity on gene expression by primary rat granulosa cells. 247 38

The possible involvement of three different second-messenger systems, namely cyclic AMP/protein kinase (PK)-A, cyclic GMP/PK-G, and diacylglycerol (DG)/PK-C systems, in the perivascular nerve terminals of guinea pig mesenteric artery was examined by intracellular microelectrode recording. Excitatory junction potentials (EJPs) were evoked by perivascular nerve stimulation. Isoproterenol (0.1 microM) enhanced the EJP amplitude without modifying the passive membrane properties of the vascular smooth muscle (VSM) cells. The facilitatory effect of isoproterenol on EJP amplitude was completely abolished by beta-adrenergic blockade (0.3 microM propranolol). Forskolin (activator of adenylate cyclase) also augmented the EJP amplitude in a concentration-dependent manner (EC50 congruent to 10 microM), without affecting the passive membrane properties of the VSM cells. In addition, forskolin (1-10 mM) markedly potentiated the isoproterenol-induced stimulation of EJP amplitude (EC50 congruent to 2 microM). A permeant analogue of cyclic AMP, 8-bromo-cyclic AMP (0.1 and 1 mM), enhanced the EJP amplitude, thus mimicking the effects of isoproterenol and forskolin. 8-Bromo-cyclic AMP had no effect on the resting potential or current-voltage relationship of the VSM cells, thus suggesting that the membrane properties of the VSM cells were not altered. 8-Bromo-cyclic GMP (1 mM) also augmented the EJP amplitude, but its facilitatory effect was weaker than that of 8-bromo-cyclic AMP. 8-Bromo-cyclic GMP hyperpolarized the VSM membrane by 4 mV and decreased the input resistance, presumably due to an increase in K+ conductance. Phorbol-12-myristate-13-acetate (PMA, 30-300 nM), a direct activator of PK-C, significantly enhanced the EJP amplitude after 40 min in a concentration-dependent manner, without affecting the resting potential of the VSM cells. From these results, we suggest that cyclic AMP/PK-A, cyclic GMP/PK-G, and DG/PK-C systems might be involved in regulation of the release of neurotransmitter in the perivascular nerve terminals. However, the possibility of some action on the postsynaptic VSM cell cannot be excluded.
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PMID:Cyclic nucleotide regulation of neurotransmission in guinea pig mesenteric artery. 248 77

Muscarinic cholinergic agonists such as acetylcholine attenuate phosphorylation of phospholamban induced by agents that activate cAMP-dependent protein kinase. However, cAMP accumulation is variably affected or only slightly reduced; thus, the choline ester might produce effects in addition to inhibition of adenylate cyclase. We hypothesized that acetylcholine might regulate a phosphatase in mammalina myocardium. Exposure of Langendoff-perfused guinea pig ventricles to isoproterenol (10 nM) for 45 s increased phosphatase inhibitor-1 activity 2-fold. Co-administration of acetylcholine (100 nM) antagonized the effect of isoproterenol, and atropine (1 microM) blocked the effect of acetylcholine. Forskolin (1 microM) caused a 3-fold increase in inhibitor-1 activity, and acetylcholine markedly attenuated the effect of forskolin. However, acetylcholine did not lower cAMP levels in the same tissues. Both isoproterenol and forskolin reduced the type 1 phosphatase activity intrinsic to sarcoplasmic reticulum by 25-50%, using [32P]phosphorylase a or 32P-labeled membrane vesicles as a substrate for the phosphatase. Co-administration of acetylcholine markedly attenuated these effects of isoproterenol and forskolin. Acetylcholine alone caused a 50% increase in type 1 phosphatase activity. We concluded that inhibitor-1 and type 1 phosphatase can be regulated in intact cardiac muscle by agents that increase intracellular cAMP and by acetylcholine.
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PMID:Autonomic regulation of type 1 protein phosphatase in cardiac muscle. 253 94

Here we show the activation of G-protein by inositol trisphosphate (IP3) or caffeine in sarcoplasmic reticulum (SR) of skeletal muscle and the consequent existence of a common mechanism of Ca2+ release from SR induced by caffeine and by IP3. (i) Indomethacin inhibits Ca2+ release induced by IP3 or caffeine. (ii) PGE1 does not induce Ca2+ release itself, but does stimulate Ca2+ release induced by IP3, or caffeine, from SR. (iii) Forskolin stimulates both types of Ca2+ release. The inhibitory effect of indomethacin on both forms of Ca2+ release, and the stimulatory effect of PGE1 and forskolin on either Ca2+ release suggest that there exists a common mechanism between IP3- and caffeine-induced Ca2+ release. (iv) Caffeine or IP3 activates G-protein via inhibition of a GTPase activity. (v) Indomethacin itself inactivates this G-protein by stimulation of a GTPase activity and reverses the activation of G-protein induced by IP3 or caffeine. (vi) PGE1 competes with the inhibitory effect of indomethacin on GTPase activity and PGE1 itself activates G-protein through inhibition of GTPase activity. From these results, it could be suggested that caffeine or IP3 induces Ca2+ release from the SR via activation of G-protein, which affects the Ca2+ channel and cAMP which seems to affect G-protein via A-kinase.
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PMID:A G-protein of sarcoplasmic reticulum of skeletal muscle is activated by caffeine or inositol trisphosphate. 253 55

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein phosphorylation induced by phorbol esters and cyclic AMP in anterior pituitary cells: possible role in adrenocorticotropin release and synthesis. 253 66


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