Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A plasma membrane Ca(2+)-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several proteins were phosphorylated, but two isoforms of plasma membrane Ca(2+)-pump ATPase with molecular masses of 135- and 145-kDa were not phosphorylated. The protein that was phosphorylated by G-kinase and identified in our previous study as the 135-kDa isoform of Ca(2+)-pump ATPase, on the basis of its almost identical mobility on SDS-PAGE, was found to be another protein with a molecular mass of 138 kDa. Fractionation of the enzyme preparation after incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in the separation of all the G-kinase substrates from the two isoforms of plasma membrane Ca(2+)-pump ATPase. These results suggest that the direct phosphorylation of the Ca(2+)-pump ATPase does not occur in association with the stimulation of the plasma membrane Ca(2+)-pump ATPase by G-kinase.
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PMID:Plasma membrane Ca(2+)-pump ATPase is not a substrate for cGMP-dependent protein kinase. 132 92

We have found that a fungal strain, Talaromyces wortmannin KY12420, produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product, designated as MS-54, was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain, which was disclosed by partial tryptic digestion, was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses, preincubation studies, and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min, 0.3 microM wortmannin inhibited the activity of MLCK, while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent protein kinase II, and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore, wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl, which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor.
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PMID:Wortmannin, a microbial product inhibitor of myosin light chain kinase. 173 24

The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.
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PMID:Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation. 184 85

In the present studies we sought to determine if cicletanine, which is an antihypertensive agent of unknown mechanism, could alter cGMP metabolism via inhibition of cGMP phosphodiesterases (PDE) in vascular smooth muscle. Cicletanine was determined to be a mixed (competitive, noncompetitive) inhibitor of both calmodulin-regulated and cGMP-specific PDEs from monkey aortic smooth muscle with Ki values of 450 to 700 microM. Cicletanine also potentiated vasorelaxation by the guanylate cyclase activators sodium nitroprusside and atrial natriuretic peptide in isolated rat aortas. Potentiation was not dependent upon the contractile agonists nor was it indomethacin-sensitive. Neither potentiation nor inhibition of cGMP PDEs was stereoselective. Methylene blue attenuated a component of cicletanine-induced vasorelaxation, but did not completely obviate relaxation. Both cicletanine and the cGMP-PDE inhibitor zaprinast potentiated sodium nitroprusside-mediated cGMP formation and relaxation, although the increase in cGMP content was markedly greater with zaprinast compared to cicletanine. In further studies, cicletanine did not potentiate cGMP activation of cGMP-dependent protein kinase, but did inhibit calmodulin-activated myosin light chain kinase and protein kinase C at relatively high concentrations (approximately 1 mM). In summary, these data demonstrate that cicletanine inhibits vascular cGMP PDEs, potentiates vasorelaxation, and to a limited extent, cGMP formation by guanylate cyclase activators in vascular smooth muscle. However, these relationships for cicletanine are dissimilar from the reference cGMP PDE inhibitor, zaprinast. Thus, other mechanisms may also contribute to the vasorelaxant action of cicletanine.
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PMID:Inhibition of low Km cGMP phosphodiesterases and Ca+(+)-regulated protein kinases and relationship to vasorelaxation by cicletanine. 185 Apr 74

Cell cytosol preparations from mitotic HeLa cells exhibit a kinase activity that phosphorylates myosin light chain kinase (MLCK). This MLCK kinase activity is apparently distinct from the known MLCK kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, Ca(2+)-activated phospholipid-dependent protein kinase, or Ca(2+)-calmodulin-dependent protein kinase II, based on the following criteria. First, the MLCK kinase activity of mitotic cells does not respond to a variety of characteristic activators or inhibitors of these known kinases. Second, one- and two-dimensional peptide maps have revealed that the site of phosphorylation by the MLCK kinase of mitotic cells differs from those by these known kinases. The mitotic MLCK kinase phosphorylates MLCK at a threonine residue at a ratio of up to 1 mol of phosphate/mol of chicken gizzard MLCK. The MLCK kinase is mitosis-specific because mitotic cell extracts show much higher phosphorylation activity than nonmitotic cell extracts.
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PMID:Mitosis-specific phosphorylation of myosin light chain kinase. 193 38

Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.
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PMID:Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody. 214 59

A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.
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PMID:Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. 215 66

Effects of 8-bromo-cGMP (8-Br-cGMP) and synthetic rat atriopeptin III (APIII) on sodium absorption by isolated chicken villus enterocytes and intact chicken ileal mucosa were determined. In isolated cells, both agents significantly decreased initial rates of influx of 22Na and caused a persistent decrease in intracellular pH (pHi); effects that are not additive to those caused by amiloride (10(-3) M). The ED50 for APIII was 0.3 nM. In intact mucosa, both 8-Br-cGMP (10(-4) M) and 5-(N-methyl-N-isobutyl)amiloride (MIBA) (10(-5) M) reduced JNams and JNa.net, their effects were not additive. APIII (10(-7) M) significantly increased cellular cGMP but not cAMP. Both 8-Br-cGMP (10(-4) M) and APIII (10(-7) M) stimulated a persistent increase in cytosolic calcium (Cai), which could be prevented by pretreating the cells with the cytosolic calcium buffering agent MAPTAM or with H-8, an inhibitor of cyclic nucleotide-dependent protein kinases. Furthermore, pretreatment of cells with H-8 or the calmodulin inhibitor, calmidazolium (CM), prevented the effects of 8-Br-cGMP and APIII on pHi. However, the pHi response to subsequent addition of the calcium-ionophore ionomycin was blocked only by CM and not by H-8. These data suggest that APIII and 8-Br-cGMP inhibit amiloride-sensitive Na/H exchange by increasing Cai, an event requiring activation of cGMP-dependent protein kinase.
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PMID:Inhibition of Na/H exchange in avian intestine by atrial natriuretic factor. 216 56

Protein phosphorylation has been recognized as a major mechanism by which cellular functions are controlled by neurotransmitters and hormones. In this review, applications of molecular biological techniques to the analyses of regulatory mechanisms of protein phosphorylation by four major second messengers, cAMP, cGMP, diacylglycerol, and Ca2+, are described. 1) Complementary DNA of the regulatory subunit of the cAMP-dependent protein kinase was cloned and expressed in E. coli. Point mutations were introduced in order to analyze functional domains of the subunit. 2) The soluble isoform of guanylate cyclase was purified, and a cDNA of its 70-KD subunit was cloned. Cyclic GMP binding to purified cGMP-dependent protein kinase was characterized using a rapid filtration assay. 3) Primary structure of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A) was determined and the presence of the second isoform of the enzyme was shown by the cDNA cloning technique. 4) The regulatory domain of the protein kinase C was expressed in E. coli. Analysis using site-directed mutagenesis revealed that a "zinc finger"-like structure is responsible for the binding of phorbol esters. In these studies, the molecular biological approach has proven to be useful for clarifying the molecular mechanisms of cellular signal transduction related to second messengers and protein phosphorylation.
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PMID:[Second messengers and protein phosphorylation in cellular signal transduction]. 222 19

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
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PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35


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