Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Smooth muscle preparations of human aorta or pig coronary arteries contain nearly equal amounts of cGMP-dependent protein kinase isozymes (cGMP kinase I alpha and I beta). In order to understand the roles of these isozymes in relaxing vascular smooth muscle, several new cGMP analogs were synthesized and tested for potencies in activating each enzyme and in relaxing pig coronary arteries. Analogs modified with a derivatized phenylthio group at the 8-position were as much as 72-fold more potent in activating purified cGMP kinase I alpha than cGMP kinase I beta. Electron-donating substituents, such as hydroxy, amino, and methoxy, on the phenyl ring enhanced the potencies of these analogs in activating cGMP kinase I alpha. The most potent of these cGMP analogs [8-(4-hydroxyphenylthio)-cGMP] was 17 times more potent (EC50 = 1.1 microM) as a muscle relaxant than the most efficacious analog tested previously. Among derivatives with an 8-halo group, 8-iodo-cGMP was the most potent compound (Ka = 9 nM for I alpha and 122 nM for I beta) for both I alpha and I beta. Analogs modified at the 1,N2-position or at both the 1,N2-and 8-positions of cGMP were highly potent for activating both isozymes. Within this group, 8-I-beta-phenyl-1,N2-etheno-cGMP had Ka values of 22 nM and 17 nM for cGMP kinase I alpha and I beta, respectively, whereas the Ka values of cGMP were 110 nM and 250 nM for the two isozymes. 8-I-beta-phenyl-1,N2-etheno-cGMP was the most potent muscle relaxant tested, with EC50 of 0.4 microM. For all cGMP analogs tested, there was a positive correlation between potency for activation of cGMP kinase I alpha and that for relaxation of pig coronary arteries. Assuming that the kinase assay conditions yielded a cyclic nucleotide specificity similar to that which would exist in intact cells, it was concluded that the cGMP kinase I alpha isozyme mediates the relaxation of pig coronary artery smooth muscle caused by cGMP elevation. However, an additional role for cGMP kinase I beta in the relaxation process could not be ruled out.
Mol Pharmacol 1992 Jul
PMID:Relaxation of pig coronary arteries by new and potent cGMP analogs that selectively activate type I alpha, compared with type I beta, cGMP-dependent protein kinase. 132 50

The role of cGMP-dependent protein kinase in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells was examined by studying the effects of cGMP on the phosphorylation of the Ca(2+)-ATPase regulatory protein phospholamban. Cultured rat aortic smooth muscle cells incubated with atrial natriuretic peptide II or sodium nitroprusside responded with increased phosphorylation of the 6000-Da subunit of phospholamban. The identity of phospholamban was confirmed using immunoprecipitation methods. Phosphorylation was associated with an increase in the activation of membrane-associated ATPase by Ca2+. These results indicated that at least one site of action of cGMP in smooth muscle cells is the sarcoplasmic reticulum, where phosphorylation of proteins regulating Ca2+ fluxes occurs. Studies using confocal laser scanning microscopy to define the cellular distribution of cGMP-dependent protein kinase suggested that the enzyme was localized to the same cellular region(s) as was phospholamban. Phosphorylation of proteins by cGMP in broken cell fractions from rabbit aorta was also performed. Phospholamban and other proteins were phosphorylated in the presence of cGMP but not cAMP, suggesting that only cGMP-dependent protein kinase was associated with smooth muscle membrane fractions containing phospholamban. These results suggest that one mechanism of action of cGMP in the reduction of intracellular Ca2+ is the activation of sarcoplasmic reticulum Ca(2+)-ATPase via phosphorylation of phospholamban. The data also support the concept that compartmentalization of protein kinases with substrates in the intact cell is an important factor involved in protein phosphorylation.
Mol Pharmacol 1991 Dec
PMID:Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. 183 34

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.
Mol Pharmacol 1990 Apr
PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35

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.
Mol Pharmacol 1986 Jun
PMID:Naphthalenesulfonamides as calmodulin antagonists and protein kinase inhibitors. 287 89

Injection of small volumes of N-methyl-D-aspartate (NMDA) or Sin-1 molsidomine (a nitric oxide releasing agent) onto the dendrites of granule cells in the hippocampal dentate gyrus leads to changes in the level of expression of a number of genes. There is a fall in prodynorphin mRNA levels with a corresponding increase in proenkephalin mRNA levels. Similar changes in opioid gene expression occur following the induction of long-term potentiation (LTP). We report here that at short time periods (1-6 h) after injections of NMDA or sin-1 molsidomine, there is an increase in the levels of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII alpha), consistent with a report of elevated CaMKII alpha mRNA in postsynaptic neurons in the CA1 region of the hippocampus following LTP induction [54]. However, we also report that 24 h after injection of NMDA or sin-1, there is a dramatic decrease in CaMKII alpha mRNA levels in the vicinity of the injection. This effect is specific for CaMKII alpha mRNA, in that many other mRNA species are not affected, and occurs in the dendritic population of CaMKII alpha mRNA as well as in the pool of mRNA in the granule cell bodies. The effect is blocked by an inhibitor of cGMP-dependent protein kinase. The biphasic regulation of CaMKII alpha mRNA may be of considerable functional importance for the long-term response of granule cells to local stimulation of NMDA receptors or NO release.
Brain Res Mol Brain Res 1995 Jul
PMID:N-methyl-D-aspartate and nitric oxide regulate the expression of calcium/calmodulin-dependent kinase II in the hippocampal dentate gyrus. 747 22

Nitric oxide is a signaling molecule involved in events crucial to neuronal cell function, such as neurotransmitter release, gene transcription, and neurotoxicity, i.e., a number of processes in which a key role appears to be played by increases in intracellular Ca2+ concentration. In the neurosecretory/neuronal cell line PC-12, we have investigated the role of nitric oxide in the modulation of Ca2+ release from intracellular stores elicited by activation of three different receptors coupled to phosphatidyl-inositol-4,5-bisphosphate hydrolysis, i.e., the purinergic P2U, muscarinic M3, and bradykinin B2 receptors. The results obtained show that nitric oxide donors have an inhibitory effect on agonist-evoked Ca2+ release. This effect is not due to nitric oxide-induced modifications of Ca2+ storage, because the total releasable Ca2+ pool, measured as the radioactivity released by thapsigargin and ionomycin in cells loaded at equilibrium with 45Ca2+, was unchanged. In contrast, nitric oxide donors decreased agonist-evoked inositol-1,4,5-trisphosphate generation and total inositol phosphate accumulation. Similarly, nitric oxide inhibited total inositol phosphate accumulation stimulated by either aluminium fluoride or Ca2+. All of these effects were mimicked by the cGMP analogue 8-bromo-cGMP. When cells were incubated with nitric oxide synthase inhibitors, the results observed were opposite those produced by nitric oxide donors. All of the effects of nitric oxide were abolished when cells were treated with the cGMP-dependent protein kinase I inhibitor KT5823. Furthermore, KT5823 mimicked the effects of nitric oxide synthase inhibitors. We conclude that nitric oxide and Ca2+ signaling pathways are interconnected in PC-12 cells. Modulation of inositol phosphate generation and Ca2+ release by nitric oxide appears to be exerted primarily at the level of phospholipase C functioning and to be mediated by the activation of cGMP-dependent protein kinase I.
Mol Pharmacol 1995 Mar
PMID:Nitric oxide modulation of agonist-evoked intracellular Ca2+ release in neurosecretory PC-12 cells: inhibition of phospholipase C activity via cyclic GMP-dependent protein kinase I. 753 79

To help define essential interactions of cGMP with the catalytic site, we tested a series of cGMP analogs as competitive inhibitors of each cyclic nucleotide phosphodiesterase (PDE) family known to hydrolyze cGMP (PDE1, PDE2, PDE3, PDE5, and PDE6). IC50 values, relative to cGMP, were used to predict which functional groups of cGMP contribute to binding by the catalytic sites of each isozyme. The results indicate that the N1-nitrogen of cGMP contributes to binding at the catalytic site of all PDEs, probably as a hydrogen donor. All PDEs tested, with the exception of PDE2, also use the 6-oxo group, probably as a hydrogen acceptor. In contrast to other cGMP-binding enzymes, the 2-amino and 2'-hydroxyl groups of cGMP are not major requirements for binding to any PDE. The 8-bromo- and 8-p-chlorophenylthio-substituted analogs inhibit PDE1, PDE2, and PDE6 activity with high relative affinities, suggesting that these PDEs are not sterically hindered with bulky 8-position substitutions and that they do not preferentially bind the anti-conformation of cGMP. PDE3 and PDE5 have reduced apparent affinity for these analogs and therefore either are sterically hindered with these substitutions or bind cGMP in the anti-conformation. Overall, the data show substantial differences in structural requirements for cGMP binding to the catalytic sites of the different PDE families. Comparisons with published data show different structural requirements for binding to the catalytic, compared with noncatalytic, binding domains of PDEs. Even larger differences are seen between the requirements for binding to PDE catalytic sites and those for the cGMP-dependent protein kinase and the cGMP-gated cation channel.
Mol Pharmacol 1995 Feb
PMID:Characterization of cyclic nucleotide phosphodiesterases with cyclic GMP analogs: topology of the catalytic domains. 787 41

The role of the cGMP-dependent protein kinase (cGK) and one of its major substrates, the vasodilator-stimulated phosphoprotein (VASP), in the regulation of a receptor-evoked calcium response was investigated. The human type I beta cGK was stably transfected in human embryonic kidney 293 cells and Swiss mouse 3T6 fibroblasts, which contained significant or no detectable levels of the focal adhesion protein VASP, respectively. Western blot analysis and protein kinase activity measurements demonstrated an 8-fold overexpression of cGK-I beta in 293 cells (7-fold in 3T6 cells), representing an intracellular cGK concentration of 0.33 microM. In experiments with intact 293 cells expressing cGK-I beta, beta-phenyl-1,N2-etheno-cGMP and 8-(p-chlorophenylthio)-cGMP were capable of converting up to 30-40% of the 46-kDa VASP to its 50-kDa phospho- form, equivalent to results observed with cGMP analogs that cause a marked inhibition of the stimulated Ca2+ transient in intact human platelets. In contrast to platelets, preincubation of fura-2-loaded 293 and 3T6 cells with 8-(p-chlorophenylthio)-cGMP did not significantly inhibit thrombin-evoked calcium transients, although sufficient cGK-mediated VASP phosphorylation was clearly detectable under these conditions in cGK-I beta-expressing 293 cells. These results demonstrate that cGK inhibition of agonist-evoked calcium mobilization is not a mechanism common to all cell types and that VASP phosphorylation may not be an essential or sufficient component of the cGK effect on calcium levels. In contrast, the observed VASP phosphorylation mediated by recombinant human cGK-I beta in intact 293 cells does support the hypothesis that focal adhesions and their associated proteins are important cellular sites of cGK action.
Mol Pharmacol 1994 Aug
PMID:Human cyclic GMP-dependent protein kinase I beta overexpression increases phosphorylation of an endogenous focal contact-associated vasodilator-stimulated phosphoprotein without altering the thrombin-evoked calcium response. 807 90

We have investigated the role played by cyclic nucleotide phosphodiesterases (EC 3.1.4.17) in the control of T-lymphocyte response to mitogenic agents by their ability to influence the cellular level of cAMP. The importance of this messenger as a negative regulator in this cell type is well established. Multiple isoenzymes of phosphodiesterase were fractionated from the cytosol of rat thymic lymphocytes by high performance liquid chromatography on an anion exchange column. In addition to the type II, III, IV isoforms that we have already described [Valette et al., Biochem. Biophys. Res. Commun. 169:864-872 (1990)], a phosphodiesterase fraction sharing several of the characteristics of type V, cGMP-binding phosphodiesterase, was detected. Non-isoform-selective inhibitors of phosphodiesterase such as dipyridamole, papaverine, and methyl-isobutylxanthine were able to totally prevent the proliferative response of thymocytes to stimulation by the mitogenic lectin concanavalin A. In contrast, the selective inhibitor of type IV phosphodiesterases rolipram induced a rather moderate inhibition of proliferation, not exceeding 60%; and the selective inhibitors of type III and type V phosphodiesterases, milrinone and M&B 22,948, respectively, displayed only marginal inhibitory effects. The association of the type III and IV phosphodiesterase inhibitors produced synergistic inhibition of proliferation, which could then be almost totally suppressed. These inhibitory effects on cell multiplication were reflected at the level of the cell cAMP content; only rolipram was able to induce a significant (approximately 50%) increase in cAMP, and this increase was potentiated by the presence of milrinone, reaching almost 100%. The type V phosphodiesterase selective inhibitor M&B 22,948 displayed similar properties to those of milrinone, which suggests that it indirectly inhibited the type III, cGMP-inhibitable isoenzyme, by inducing a cGMP rise. This hypothesis was supported by evidence of a significant raising effect of M&B 22,948 on cGMP level, and by the ability of a cGMP-elevating agent, sodium nitroprusside, to mimic the synergistic effects of milrinone associated with rolipram. Furthermore, 8-bromo-cGMP, a potent activator of cGMP-dependent protein kinase, which showed only weak inhibitory effects on thymic type III phosphodiesterase, failed to alter the effects of rolipram on the cell proliferation. These results allow us to delineate a role for types III, IV, and V phosphodiesterase in the control of cAMP level during the proliferative response of thymic lymphocytes. They also suggest that endogenously formed cGMP might participate in the regulation of cAMP level in the cells by means of the inhibition of the type III phosphodiesterase.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1993 Nov
PMID:Modulation of rat thymocyte proliferative response through the inhibition of different cyclic nucleotide phosphodiesterase isoforms by means of selective inhibitors and cGMP-elevating agents. 824 5

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.
Mol Biol Cell 1995 Dec
PMID:Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions. 859 Aug


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