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)

Guanylate cyclase-A, the receptor for atrial natriuretic factor, contains a protein kinase-like domain and a catalytic domain in the intracellular region. To investigate the active site (the catalytic cavity) of guanylate cyclase-A, we amplified the catalytic domain plus three amino acids from the kinase-like domain of guanylate cyclase-A (GC-c) with polymerase chain reaction (PCR) and expressed it in Escherichia coli. During the screening of the PCR-cloned gene products with guanylate cyclase assay, a mutant that lacks enzyme activity was identified. Results of cDNA sequencing revealed that Leu 817 was replaced by an Arg residue in the mutated protein. The mutated GC-c bound to GTP-agarose as well as the wild-type protein, indicating that the binding capability of mutated GC-c to GTP is not significantly affected by the Arg substitution. Gel-filtration column chromatography showed that, like the wild-type GC-c, the mutated protein also formed a high-molecular-weight complex. Since mutation of Leu 817 to Arg abolishes the catalytic activity, Leu 817 is likely located near the active site of guanylate cyclase-A. These results demonstrate that the carboxyl fragment of guanylate cyclase-A is an ideal system for studying the active site of guanylate cyclase-A.
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PMID:Mutational inactivation of the catalytic domain of guanylate cyclase-A receptor. 772 18

The presence and physiological role of cGMP-dependent protein kinase (G-kinase) was investigated in human mononuclear phagocytes. Western blots of monocyte extracts revealed a single polypeptide band that comigrated with purified bovine lung G-kinase. G-kinase was localized by immunofluorescence microscopy in freshly isolated adherent human monocytes, monocyte-derived macrophages cultured from 4 to 14 days, and alveolar macrophages. In monocytes, G-kinase was localized in granules or vesicles in the cytoplasm, at the microtubule organizing center, on filaments, and in the nucleus. In monocyte-derived macrophages, intense staining for G-kinase was found in the vicinity of the Golgi, in vesicles throughout the cytoplasm, and diffusely in the nucleus. Dual-label confocal laser scanning microscopy demonstrated that G-kinase was colocalized with the endoplasmic reticulum. For comparison, G-kinase was localized in alveolar macrophages that were adhered from 3 to 30 min. In these cells, G-kinase was prominent within the organelle-rich area pericortical to the nucleus. However, a well-defined area of intense staining was also observed at the cell periphery at early time points during adherence and spreading. Rhodamine-labeled phalloidin showed that this peripheral area was rich in F-actin. Cytochalasin D, but not nocodazole, inhibited G-kinase targeting to the cell margin. Furthermore, the guanylate cyclase inhibitor LY83583 inhibited alveolar macrophage spreading and staining for G-kinase at the cell periphery. These data suggest that G-kinase may play an important role in cGMP-mediated regulation involved in protein processing and cell motility.
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PMID:Localization of cyclic GMP-dependent protein kinase in human mononuclear phagocytes. 772 24

Previous studies from this laboratory have shown that in cultured rat mesangial cells (MC), angiotensin II (ANG II) mediates its effects via activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PC-PLD). In addition, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating maneuvers that stimulate particulate and soluble guanylate cyclase [atrial natriuretic factor (ANF) and sodium nitroprusside (SNP), respectively] antagonize ANG II-mediated PI-PLC activation. The current study explored whether cGMP impairs ANG II-mediated PC-PLC and PLD activity. The ANG II-stimulated release of the water-soluble metabolites of PC breakdown (phosphorylcholine and choline) was blocked by ANF and SNP. ANG II-stimulated phosphatidic acid and phosphatidylethanol formation were significantly reduced by ANF and SNP, confirming that cGMP blunted PLD activity. The inhibitory effect of cGMP on PLD could be reversed by N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide, a blocker of cGMP-dependent protein kinase. In parallel experiments, ANF and SNP abrogated sustained diacylglycerol (DAG) accumulation derived from ANG II stimulation of PC hydrolysis, confirming that cGMP diminished PC-PLC activity. Inhibition of PC-derived DAG accumulation by cGMP was associated with a concomitant decrement in ANG II-mediated translocation of protein kinase C (PKC) activity from the cytosol to the membrane. In summary, in MC, cGMP antagonizes ANG II-mediated PC hydrolysis, DAG formation, and PKC activation. We propose that cGMP-mediated inhibition of phospholipid metabolism and PKC translocation plays an important role in MC vasorelaxation.
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PMID:cGMP antagonizes angiotensin-mediated phosphatidylcholine hydrolysis and C kinase activation in mesangial cells. 786 76

Glyceryl trinitrate, isosorbide dinitrate, and isosorbide-5-mononitrate are organic nitrate esters commonly used in the treatment of angina pectoris, myocardial infarction, and congestive heart failure. Organic nitrate esters have a direct relaxant effect on vascular smooth muscles, and the dilation of coronary vessels improves oxygen supply to the myocardium. The dilation of peripheral veins, and in higher doses peripheral arteries, reduces preload and afterload, and thereby lowers myocardial oxygen consumption. Inhibition of platelet aggregation is another effect that is probably of therapeutic value. Effects on the central nervous system and the myocardium have been shown but not scrutinized for therapeutic importance. Both the relaxing effect on vascular smooth muscle and the effect on platelets are considered to be due to a stimulation of soluble guanylate cyclase by nitric oxide derived from the organic nitrate ester molecule through metabolization catalyzed by enzymes such as glutathione S-transferase, cytochrome P-450, and possibly esterases. The cyclic GMP produced by the guanylate cyclase acts via cGMP-dependent protein kinase. Ultimately, through various processes, the protein kinase lowers intracellular calcium; an increased uptake to and a decreased release from intracellular stores seem to be particularly important.
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PMID:Mechanisms of action of nitrates. 787 67

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family which is produced in vascular endothelial cells and may play an important paracrine role in the vasaculature. We sought to determine the regulation of CNP production by other vasoactive peptides from cultured aortic endothelial cells. The vasoconstrictors endothelin-1 and angiotensin II had little effect on the basal secretion of CNP. In contrast, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) strongly stimulated the secretion of CNP. BNP caused as much as a 400-fold enhancement above the basal accumulated secretion of CNP over 24 h at a concentration of 1 microM; this was 20 times greater than the stimulatory effect of ANP, BNP and ANP also significantly enhanced the production of new CNP protein (translation) and mRNA expressed in the BAEC. In contrast, C-ANP-4-23, a truncated form of ANP which selectively binds to the natriuretic peptide clearance receptor, did not stimulate CNP secretion. The enhanced production and secretion of CNP, caused by either ANP or BNP, was significantly prevented by LY 83583, an inhibitor of cGMP generation, and was also attenuated by KT 5823, an inhibitor of cGMP-dependent protein kinase. Our results indicate that ANP and BNP can stimulate CNP production through a guanylate cyclase receptor on endothelial cells. BNP is a much more potent stimulator of CNP secretion, compared to ANP. Our findings suggest that the vasodilatory, and anti-mitogenic effects of ANP and BNP in the vasculature could occur in part through CNP production and subsequent action if these interactions occur in vivo.
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PMID:Atrial and brain natriuretic peptides stimulate the production and secretion of C-type natriuretic peptide from bovine aortic endothelial cells. 788 64

Recent studies have implicated protein kinase-C (PKC) in the regulation of guanylate cyclase in several cell types. In view of prior experiments by our laboratory which have demonstrated that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] can activate PKC in CaCo-2 cells, it was of interest to determine whether this secosteroid influenced particulate guanylate cyclase and, if so, to determine which isoforms of PKC were involved. To address these issues, CaCo-2 cells were treated with 1 alpha,25-(OH)2D3 or other agents (see below), and crude membranes prepared from these cells were assayed for guanylate cyclase activity. In several experiments, agents were added directly to isolated membranes, and guanylate cyclase activity was then assayed. These studies demonstrated that 1) the addition of 1 alpha,25-(OH)2D3 or 12-O-tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, to intact CaCo-2 cells stimulated particulate guanylate cyclase activity in a time- and concentration-dependent manner; 2) these agents induced the translocation of PKC alpha, but not PKC zeta, from the cytosolic to the membrane fraction of these cells; 3) preincubation of cells with staurosporine (50 nM), a PKC inhibitor, or U73122 (10 microM), an inhibitor of phospholipase-C-dependent processes, significantly reduced (P < 0.05) the stimulatory effect of 1 alpha,25-(OH)2D3 (3 nM) on guanylate cyclase; 4) preincubation of isolated membranes with TPA, calcium, and Mg(2+)-ATP increased guanylate cyclase activity, an affect that was augmented by purified rat brain PKC and inhibited by the PKC inhibitor peptide, PKC-(19-36); and 5) selective down-regulation of PKC alpha by treatment of cells with TPA (200 nM) for 24 h concomitantly abolished the activation of guanylate cyclase by 1 alpha,25-(OH)2D3. Taken together, these studies demonstrate that 1 alpha,25-(OH)2D3 activates particulate guanylate cyclase at least in part via a PKC alpha-dependent mechanism.
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PMID:The role of protein kinase-C alpha in the activation of particulate guanylate cyclase by 1 alpha,25-dihydroxyvitamin D3 in CaCo-2 cells. 791 83

Human placenta synthesizes and secretes large amounts of CRH during the second and third trimesters. In the hypothalamus, nitric oxide (NO) has been reported to affect CRH release. We studied the effect of NO on the regulation of placental CRH secretion. The effect of the NO donor sodium nitroprusside (SNP) on basal and KCl-stimulated CRH release was examined in cultured human syncytiotrophoblasts. CRH secretion and intracellular concentrations of cGMP, calmodulin-dependent protein kinase (CaM-PK), protein kinase-G (PKG), protein kinase-C, and cAMP-dependent protein kinase holoenzyme were measured under basal conditions and after treatment with a depolarizing concentration of KCl and with SNP. The results showed that depolarization (3 h) increased CRH release 4-fold (from basal value of 5.16 +/- 0.65 to 19.31 +/- 4.46 fmol/10(6) cells); SNP (100 mumol/L) decreased both basal (0.42 +/- 0.21 fmol/10(6) cells) and KCl-stimulated CRH release (0.94 +/- 0.32 fmol/10(6) cells). KCl also increased the activity of CaM-PK in the cell membrane and both cytosolic and membrane PKG activity, whereas the activities of protein kinase-C and cAMP-dependent protein kinase holoenzyme were unchanged. SNP increased intracellular cGMP concentrations after 10, 60, and 180 min. Methylene blue (100 mumol/L), a guanylate cyclase inhibitor, blocked the inhibitory effects of SNP on CRH release. These results suggest that NO exerts inhibitory effects on both basal and KCl-stimulated CRH release from placental syncytiotrophoblasts through a cGMP-mediated pathway. In addition, as KCl-induced changes in the cell membrane were blocked by SNP, CaM-PK may be involved in KCl-stimulated CRH release. KCl may also sensitize the inhibitory pathway involved in the regulation of CRH release by increasing cellular PKG levels. The effects of KCl and SNP on CRH release are more complex than simple activation of CaM-PK and PKG activity, as other cellular signal transduction pathways are also modulated.
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PMID:Basal and KCl-stimulated corticotropin-releasing hormone release from human placental syncytiotrophoblasts is inhibited by sodium nitroprusside. 791 33

Nitric oxide (NO) down-regulates osteoclastic activity. The mechanism is unknown, although, in some cells NO acts by stimulating guanylate cyclase which activates cGMP-dependent proteins. We demonstrated cGMP-dependent protein kinase in osteoclasts by immunofluorescence microscopy. Specificity was confirmed by Western blot analysis showing a single 78 kDa band, the size of the Type I isoform, in isolated avian osteoclasts. Osteoclast function centers on HCl secretion at a specialized membrane organelle. We found that purified cGMP-dependent protein kinase inhibits ATP-dependent acid transport in reconstituted osteoclast membrane vesicles >90%, while cAMP-dependent kinase catalytic subunit, calmodulin kinase II, or cGMP alone were ineffective. This novel, direct modulation of acid transport by cGMP-dependent kinase and the occurrence of the enzyme in osteoclasts suggest that a mechanism of NO-regulation of bone turnover is via cGMP and cGMP-dependent protein kinase inhibition of HCl transport.
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PMID:Regulation of osteoclastic acid secretion by cGMP-dependent protein kinase. 798 May 15

Endothelial-derived nitric oxide (NO) is an important intercellular messenger. Although endothelial cells contain both nitric oxide synthase and soluble guanylate cyclase, the nature of receptor proteins for cGMP is uncertain. Based on previous work in vascular smooth muscle cells which indicates that the cGMP-dependent protein kinase (cGK) is partially associated with the cytoskeleton, we determined that cGK was present in non-cytosolic fractions of endothelial cells. The data reveal that cGK is found only in Triton-soluble extracts of particulate fractions from bovine aortic endothelial cells and provide the first evidence for the existence of cGK in this cell type based on immunoreactivity, immunofluorescence microscopy and phosphotransferase activity. The limited distribution of endothelial cell cGK may explain why this kinase has not been heretofore identified in endothelial cells.
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PMID:Identification and possible localization of cGMP-dependent protein kinase in bovine aortic endothelial cells. 800 83

Adrenergic stimulation of the adult pineal gland increases cAMP and cGMP production by over 100-fold. beta-Adrenergic stimulation results in Gs alpha-mediated cyclase activation; alpha 1-adrenergic activation potentiates the beta-adrenergic effects through mechanisms mediated by the intracellular Ca2+ concentration ([Ca2+]i) and Ca(2+)-phospholipid-dependent protein kinase. Development analysis of these responses has indicated that the adrenergic stimulation of cAMP is present several days after birth, but the cGMP response develops only after the second week of life. In the study presented here, the adrenergic-->cGMP response was analyzed in pineal glands from 10- and 25-day-old rats, with the intention of determining the basis of the developmental appearance of this response. Organ culture and tissue homogenate studies indicated that guanylate cyclase activity, cGMP phosphodiesterase activity, and adrenergic elevation of phospholipase-A2 were similar in pineal glands from 10- and 25-day-old rats. Norepinephrine stimulated an increase in [Ca2+]i in dispersed pinealocytes from 10-day-old rats, as has been previously demonstrated in adult pinealocytes. In contrast, several treatments that elevate [Ca2+]i had no effect on cGMP accumulation in forskolin-treated or beta-adrenergically activated glands from 10-day-old rats, but were fully effective in similarly treated glands from 25-day-old rats. However, glands from 10-day-old animals showed a 33-fold accumulation of cGMP when they were cultured together with glands from 25-day-old rats. These studies indicate that whereas many elements in the system that mediate adrenergic regulation of pineal cGMP are fully developed at 10 days of age, the developmental appearance of the cGMP response is triggered by the development of a process down-stream of the alpha 1-adrenergic stimulation of [Ca2+]i, and this process may involve a diffusible factor.
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PMID:Developmental appearance of pineal adrenergic-->guanosine 3',5'-monophosphate response is determined by a process down-stream from elevation of intracellular Ca2+: possible involvement of a diffusible factor. 809 11


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