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 purified bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) was rapidly phosphorylated by purified bovine lung cGMP-dependent protein kinase (cGK). Within a physiological concentration range, cGK catalyzed phosphorylation of cG-BPDE at a rate approximately 10 times greater than did equimolar concentrations of purified catalytic subunit of cAMP-dependent protein kinase (cAK). cG-BPDE was a poor substrate for either purified protein kinase C or Ca2+/calmodulin-dependent protein kinase II. Binding of cGMP to the cG-BPDE binding site was required for phosphorylation since (a) phosphorylation of cG-BPDE by the catalytic subunit of cAK was cGMP-dependent, (b) phosphorylation of cG-BPDE in the presence of a cGMP analog specific for activation of cGK was cGMP-dependent, and (c) occupation of the cG-BPDE hydrolytic site with competitive inhibitors did not produce the cGMP-dependent effect. cGMP-dependent phosphorylation of cG-BPDE by both cGK and cAK occurred at serine. Proteolytic digestion of cG-BPDE phosphorylated by either cGK or cAK revealed the same phosphopeptide pattern, suggesting that phosphorylation by the two kinases occurred at the same or adjacent site(s). Tryptic digestion of cG-BPDE phosphorylated by cGK and [gamma-32P]ATP produced a single major phosphopeptide of approximately 2 kDa with the following amino-terminal sequence: Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg- Radioactivity was released during the third cycle of Edman degradation. cG-BPDE is one of few specific in vitro cGK substrates of known function to be identified. Elevation of intracellular cGMP may cause phosphorylation of cG-BPDE by modulating the substrate site availability as well as by activating cGK. Such regulation would greatly increase the selectivity of the phosphorylation of cG-BPDE and would represent a unique mechanism of action of a cyclic nucleotide or other second messenger.
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PMID:Substrate- and kinase-directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP. 216 96

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

Relaxation of rat aorta segments with sodium nitroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) accumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiography. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachidonic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.
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PMID:Role of cyclic-GMP in relaxations of vascular smooth muscle. 240 83

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.
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PMID:Properties of a cGMP-dependent monomeric protein kinase from bovine aorta. 253 6

Treatment of cGMP-dependent protein kinase with low concentrations of trypsin generates an enzyme fragment of 65 kDa which is fully active in the absence of cGMP. The fragment has a s20,w value of 4.6 S indicating that the active fragment is a monomer of 65 kDa. Trypsin removes the first 77 amino acids which contain the aminoterminal dimerization site and the autophosphorylation sites. The Km and Vmax values of the fragment for ATP and Kemptide were essentially the same as those for the native enzyme. The fragment binds 2 mol cGMP/mol fragment with affinities close to that of the native enzyme. However, binding of cGMP to these sites was non-cooperative and shows similar characteristics to the autophosphorylated native enzyme. These results indicate that the aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.
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PMID:A catalytically active fragment of cGMP-dependent protein kinase. Occupation of its cGMP-binding sites does not affect its phosphotransferase activity. 282 99

The cGMP analogue 8-(2-carboxymethylthio)-cGMP (CMT-cGMP) was synthesized and its binding to cGMP-dependent protein kinase (cGMP kinase) was studied. CMT-cGMP bound at 4 degrees C with an over 1400-fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 microM, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 microM CMT-cGMP at 4 degrees C. The apparent site 1 selectivity was 250-fold at 30 degrees C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 microM for site 1 and 2, respectively. CMT-cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Ka values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 microM CMT-cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT-cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT-cGMP and cGMP inhibited cAMP-stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 microM and 2 microM, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself. These results indicate that CMT-cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expression of a partial enzyme activity.
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PMID:8-(2-Carboxymethylthio)-cGMP, a site-1-selective compound for cGMP-dependent protein kinase. 283 64

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates. 283 78

The purified catalytic subunit (C) of cAMP-dependent protein kinase produced a 2-fold activation of the low Km phosphodiesterase in crude microsomes (P-2 pellet) of rat adipocytes. This activation was C subunit concentration-dependent, ATP-dependent, blocked by a specific peptide inhibitor, and lost if the C subunit was first heat denatured. The concentration of ATP necessary for half-maximal activation of the low Km phosphodiesterase was 4.50 +/- 1.1 microM, which was nearly the same as the known Km of C subunit for ATP (3.1 microM) using other substrates. The concentration of C subunit producing half-maximal activation of phosphodiesterase was 0.22 +/- 0.04 microM, slightly less than the measured concentration of total C subunit in adipocytes (0.45 microM). The activation of the low Km phosphodiesterase by C subunit was specific, since on an equimolar basis, myosin light chain kinase, cGMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II did not activate the enzyme. The percent stimulation of phosphodiesterase by C subunit was about the same as that produced by incubation of adipocytes with a cAMP analog, and the enzyme first activated in vivo with the analog was not activated to the same extent (on a percentage basis) by in vitro treatment with C subunit. Treatment of the crude microsomes with trypsin resulted in transfer of phosphodiesterase catalytic activity from the particulate to the supernatant fraction, but the enzyme in the supernatant was minimally activated by C subunit, suggesting either loss or dislocation of the regulatory component. The C subunit-mediated activation of phosphodiesterase was preserved after either transfer of phosphodiesterase activity to the supernatant fraction by nonionic detergents or partial purification of the transferred enzyme. The present findings are consistent with the suggestion that protein kinase regulates the concentration of cAMP through phosphodiesterase activation and provide direct evidence that the mechanism of activation involves phosphorylation.
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PMID:Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase. 283 86

N-(6-Aminoethyl)-5-chloro-1-naphthalenesulfonamide (A-3), which is a shorter alkyl chain derivative of the calmodulin (CaM) antagonist, W-7, was found to inhibit smooth muscle myosin light chain kinase (MLC-kinase) through a mechanism different from that related to W-7. Both the holoenzyme and the catalytic fragment, which is active without CaM, were susceptible to A-3 with a similar concentration dependency, thereby indicating that the inhibitory effect is due to the direct interaction of the compound with the enzyme molecule and not with the enzyme activator. Naphthalenesulfonamides are both CaM antagonists and direct inhibitors of MLC-kinase, and these actions depend on the length of the alkyl chain (C2-C6). Although the potencies in inhibiting CaM functions increased, the direct effects on MLC-kinase decreased with extension of the carbon chain of the derivatives. Kinetic studies indicated that A-3 inhibited MLC-kinase competitively with respect to ATP and that the Ki value was 7.4 microM. A-3 was also a competitive inhibitor of cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, casein kinase I, and casein kinase II, with respect to ATP. The Ki values of naphthalenesulfonamides for these enzymes also increased with extension of the carbon chain of the derivatives. These results suggest that naphthalenesulfonamides inhibit protein phosphorylation not only by inhibition of the enzyme-activating process but also by inhibition of the catalytic process. The mode of interaction between the derivatives and protein kinases differs from the interaction between the derivatives and CaM.
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PMID:Naphthalenesulfonamides as calmodulin antagonists and protein kinase inhibitors. 287 89

The cytosolic fraction of insulin-treated adipocytes exhibits a 2-fold increase in protein kinase activity when Kemptide is used as a substrate. The detection of insulin-stimulated kinase activity is critically dependent on the presence of phosphatase inhibitors such as fluoride and vanadate in the cell homogenization buffer. The cytosolic protein kinase activity exhibits high sensitivity (ED50 = 2 X 10(-10) M) and a rapid response (maximal after 2 min) to insulin. Kinetic analyses of the cytosolic kinase indicate that insulin increases the Vmax of Kemptide phosphorylation and ATP utilization without affecting the affinities of this enzyme toward the substrate or nucleotide. Upon chromatography on anion-exchange and gel filtration columns, the insulin-stimulated cytosolic kinase activity is resolved from the cAMP-dependent protein kinase and migrates as a single peak with an apparent Mr = 50,000-60,000. The partially purified kinase preferentially utilizes histones, Kemptide, multifunctional calmodulin-dependent protein kinase substrate peptide, ATP citrate-lyase, and acetyl-coenzyme A carboxylase as substrates but does not catalyze phosphorylation of ribosomal protein S6, casein, phosvitin, phosphorylase b, glycogen synthase, inhibitor II, and substrate peptides for casein kinase II, protein kinase C, and cGMP-dependent protein kinase. Phosphoamino acid analyses of the 32P-labeled substrates reveal that the insulin-stimulated cytosolic kinase is primarily serine-specific. The insulin-activated cytosolic kinase prefers Mn2+ to Mg2+ and is independent of Ca2+. Unlike ribosomal protein S6 kinase and protease-activated kinase II, the insulin-sensitive cytosolic kinase is fluoride-insensitive. Taken together, these results indicate that a novel cytosolic protein kinase activity is activated by insulin.
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PMID:Insulin stimulates a novel Mn2+-dependent cytosolic serine kinase in rat adipocytes. 296 Jun 79


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