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)

In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971-14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50-70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2-0.5 microM PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 microM. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.
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PMID:Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activities. 1078 99

Sulindac sulfone (exisulind), although a nonsteroidal anti-inflammatory drug derivative, induces apoptosis in tumor cells by a mechanism that does not involve cyclooxygenase inhibition. SW480 colon tumor cells contain guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) isoforms of the PDE5 and PDE2 gene families that are inhibited by exisulind and new synthetic analogues. The analogues maintain rank order of potency for PDE inhibition, apoptosis induction, and growth inhibition. A novel mechanism for exisulind to induce apoptosis is studied involving sustained increases in cGMP levels and cGMP-dependent protein kinase (PKG) induction not found with selective PDE5 or most other PDE inhibitors. Accumulated beta-catenin, shown to be a substrate for PKG, is decreased by exisulind, suggesting a mechanism to explain apoptosis induction in neoplastic cells harboring adenomatous polyposis coli gene mutations.
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PMID:Exisulind induction of apoptosis involves guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition, protein kinase G activation, and attenuated beta-catenin. 1091 34

Migration and proliferation of vascular smooth muscle cells (SMC) in response to platelet-derived growth factor (PDGF) and other mitogens play an important role in restenosis after coronary angioplasty. Elevation of both cAMP and cGMP has been shown to inhibit SMC mitogenesis. The aim of this study was to examine the antimitogenic actions of organic nitrates and sildenafil and to clarify the role of cyclic nucleotide-dependent protein kinases (PKA, PKG) in this action. Organic nitrates [glycerol trinitrate (GTN), isosorbide 5'-mononitrate (ISMN), pentaerythrityl-tetranitrate (PETN)] and the PDE5 inhibitor sildenafil reduced PDGF-induced DNA synthesis, measured by ((3)H]thymidine incorporation. GTN, ISMN, and PETN acted synergistically with sildenafil (1 microM) on inhibition of PDGF-induced DNA synthesis, increase of intracellular cyclic nucleotides, and vasodilator-stimulated phosphoprotein phosphorylation. The highly selective PKA inhibitor PKI abolished these actions of sildenafil and organic nitrates, whereas the PKG inhibitors KT5823 and (Rp)-8-pCPT-cGMPS had no effect. In addition, selective activation of PKG without inhibition of PDE3 by the cGMP analog 8-pCPT-cGMP (100 microM) had no antimitogenic effect. The data suggest that 1) organic nitrates and sildenafil exert antimitogenic actions by activation of PKA via inhibition of PDE3, but not by activation of PKG and 2) that antimitogenic effects of organic nitrates are potentiated by sildenafil at therapeutic plasma levels.
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PMID:Antimitogenic actions of organic nitrates are potentiated by sildenafil and mediated via activation of protein kinase A. 1130 86

Exisulind (Aptosyn) is a novel antineoplastic drug being developed for the prevention and treatment of precancerous and malignant diseases. In colon tumor cells, the drug induces apoptosis by a mechanism involving cyclic GMP (cGMP) phosphodiesterase inhibition, sustained elevation of cGMP, and protein kinase G activation. We studied the effect of exisulind on bladder tumorigenesis induced in rats by the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine. Exisulind at doses of 800, 1000, and 1200 mg/kg (diet) inhibited tumor multiplicity by 36, 47, and 64% and tumor incidence by 31, 38, and 61%, respectively. Experiments on the human bladder tumor cell line, HT1376, showed that exisulind inhibited growth with a GI(50) of 118 microM, suggesting that the antineoplastic activity of the drug in vivo involved a direct effect on neoplastic urothelium. Exisulind also induced apoptosis as determined by DNA fragmentation, caspase activation, and morphology. Analysis of phosphodiesterase (PDE) isozymes in HT1376 cells showed PDE5 and PDE4 isozymes that were inhibited by exisulind with IC(50)s of 112 and 116 microM, respectively. Inhibition of PDE5 appears to be pharmacologically relevant, because treatment of HT1376 cells increased cGMP and activated protein kinase G at doses that induce apoptosis, whereas cyclic AMP levels were not changed. Immunocytochemistry showed that PDE5 was localized in discrete perinuclear foci in HT1376 cells. Immunohistochemistry showed that PDE5 was overexpressed in human squamous and transitional cell carcinomas compared with normal urothelium. The data lead us to conclude that future clinical trials of exisulind for human bladder cancer treatment and/or prevention should be considered and suggest a mechanism of action involving cGMP-mediated apoptosis induction.
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PMID:Exisulind, a novel proapoptotic drug, inhibits rat urinary bladder tumorigenesis. 1135 13

To date, relative cellular levels of cGMP and cGMP-binding proteins have not been considered important in the regulation of smooth muscle or any other tissue. In rabbit penile corpus cavernosum, intracellular cGMP was determined to be 18 +/- 4 nM, whereas the cGMP-binding sites of types Ialpha and Ibeta cGMP-dependent protein kinase (PKG) and cGMP-binding cGMP-specific phosphodiesterase (PDE5) were 58 +/- 14 nM and 188 +/- 6 nM, respectively, as estimated by two different methods for each protein. Thus, total cGMP-binding sites (246 nM) greatly exceed total cGMP. Given this excess of cGMP-binding sites and the high affinities of PKG and PDE5 for cGMP, it is likely that a large portion of intracellular cGMP is associated with these proteins, which could provide a dynamic reservoir for cGMP. Phosphorylation of PDE5 by PKG is known to increase the affinity of PDE5 allosteric sites for cGMP, suggesting the potential for regulation of a reservoir of cGMP bound to this protein. Enhanced binding of cGMP by phosphorylated PDE5 could reduce the amount of cGMP available for activation of PKG, contributing to feedback inhibition of smooth muscle relaxation or other processes. This introduces a new concept for cyclic nucleotide signaling.
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PMID:Allosteric sites of phosphodiesterase-5 (PDE5). A potential role in negative feedback regulation of cGMP signaling in corpus cavernosum. 1138 33

The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole 3',5'-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC.
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PMID:Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle. 1169 8

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.
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PMID:Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. 1172 16

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.
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PMID:Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase. 1174 88

Several transmembrane transporters of organic compounds are regulated by phosphorylation/dephosphorylation mechanisms. The aim of this study was to investigate the possible regulation of the human extraneuronal monoamine transporter, hEMT, by these mechanisms. The experiments were performed using HEK293 cells stably transfected with pcDNA3hEMT (293hEMT). The characteristics of hEMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) were studied by incubating the cells at 37 degrees C for 1 min with 200 nM [3H]MPP+. Uptake of [3H]MPP+ by 293hEMT cells was not affected or only slightly reduced by modulators of protein kinase A, protein kinase C, or protein kinase G. It was not affected by an inhibitor of protein tyrosine kinase and was reduced by mitogen-activated protein kinase inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was independent of extracellular Ca2+ and strongly reduced by Ca2+/calmodulin pathway inhibitors. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced in the presence of non-selective phosphodiesterase inhibitors (IBMX, caffeine, theophylline). The effect of IBMX was independent of extracellular Ca2+ its IC50 was found to be 82.0 microM (66.2-101.6 microM; n=4), and its inhibitory effect resulted from a significant decrease in the maximal velocity of [3H]MPP+ uptake, with no change in the Michaelis-Menten constant. [3H]MPP+ uptake was reduced by 8-methoxy-methyl-IBMX, a selective inhibitor of the Ca2+/calmodulin-dependent phosphodiesterase (PDE1), but not by zaprinast, a selective inhibitor of PDE5. Uptake of [3H]MPP+ by 293hEMT cells was strongly reduced by protein tyrosine phosphatase inhibitors, by an alkaline phosphatase inhibitor and, by contrast. showed an increase in the presence of exogenous alkaline phosphatase. In conclusion, these results suggest that hEMT is regulated by phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state.
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PMID:Regulation of human extraneuronal monoamine transporter (hEMT) expressed in HEK293 cells by intracellular second messenger systems. 1177 2

Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively). [(3)H]cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.
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PMID:Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity. 1235 32


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