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:2.7.11.12 (PKG)
2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The heat-stable protein (protein kinase modulator), partially purified from fresh bovine heart, possessed the ability to inhibit and stimulate adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase and guanosine 3':5'-monophosphate (cGMP)-dependent protein kinase activities, respectively. The inhibitory activity of protein kinase modulator on cAMP-dependent protein kinase was abolished almost completely by trypsin treatment, while the ability to stimulate cGMP-dependent protein kinase activity was resistant to trypsin. Fractionation by a linear potassium phosphate gradient on DEAE-cellulose column did not clearly separate both activities. Phosphorylation of cardiac microsomal component, "phospholamban" (molecular weight = 22,000), was inhibited almost completely by the saturating amounts of protein kinase modulator. This inhibition of phospholamban phosphorylation by protein kinase modulator was accompanied by a decreased Ca uptake rate that had been stimulated by cAMP-dependent protein kinase. These findings indicate that protein kinase modulator is functional in controlling the cAMP-dependent protein kinase-catalyzed phosphorylation of phospholamban and the rate of calcium transport, lending further support for the previously proposed mechanism, in which phospholamban is assumed to serve as a regulator of calcium transport in cardiac sarcoplasmic reticulum.
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PMID:Effect of protein kinase modulator on cAMP-dependent protein kinase-catalyzed phosphorylation of phospholamban and stimulation of calcium transport in cardiac sarcoplasmic reticulum. 20 86

The Ca(2+)-pump ATPases of the plasma membrane and of the endoplasmic reticulum play an important role in controlling the intracellular Ca(2+)-concentration. In this perspective it is not unexpected that these enzymes are modulated by different factors. The activity of the plasmalemmal (Ca2+ +Mg2+)ATPase is modified by the amount of negatively charged phospholipids surrounding the enzyme. Some evidence is presented indicating that in stomach and myometrium smooth muscle agonists inhibit the extrusion of Ca2+ by reducing the negatively charged phospholipids surrounding the plasmalemmal Ca(2+)-pump, while c-GMP dependent protein kinase would activate this Ca(2+)-pump by increasing this amount. The regulation of the Ca(2+)-pump of the endoplasmic reticulum depends on the phosphorylation of phospholamban by cAMP- and cGMP-dependent protein kinase. In the second part of this review, the heterogeneity of the intracellular Ca2+ compartments and a possible connection between the intracellular compartment and the extracellular solution are discussed. In addition, some data on the regulation of Ca2+ inside the nucleus are presented.
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PMID:Ca(2+)-transport ATPases and Ca(2+)-compartments in smooth muscle cells. 166 64

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.
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PMID:Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. 183 34

Cyclic GMP (cGMP) mediates the relaxing action of a variety of vasodilator drugs and endogenous vasodilator substances. Cyclic AMP (cAMP) mediates relaxation by beta-adrenergic agonists as well as other activators of adenylate cyclase. Both second messengers appear to reduce the concentration of intracellular Ca2+ in vascular smooth muscle cells, thus affecting relaxation. The presence of cGMP-dependent protein kinase in vascular smooth muscle cells is required for the reduction of Ca2+ by cAMP and cGMP, suggesting that this enzyme mediates the relaxing effects of both cyclic nucleotides. Although the specific substrate proteins for cGMP-dependent protein kinase are not well characterized in vascular smooth muscle, new evidence indicates that Ca2(+)-ATPase activation by phosphorylation of phospholamban by the kinase may underlie the mechanism of action of cyclic-nucleotide-dependent relaxation.
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PMID:Towards an understanding of the mechanism of action of cyclic AMP and cyclic GMP in smooth muscle relaxation. 184 22

Smooth muscle cells contain two distinct Ca2+-transport ATpases with a different subcellular localization. The plasmalemmal Ca2+ pump has a relative molecular weight (Mr) of 140k and its phospho-intermediate level is increased by La3+. Its resemblance to the erythrocyte Ca2+ pump is further confirmed by its calmodulin-binding capacity and its antigenic properties. A 100k Ca2+-transport ATPase is localized in the endoplasmic reticulum. Its phospho-intermediate level is decreased by La3+, and it is antigenically related to the cardiac sarcoplasmic reticulum Ca2+-transport ATPase. These two different Ca2+-transport ATPases are present in both visceral and vascular smooth muscle, but tissue- and species-dependent differences in their relative amount have been observed. The endoplasmic-reticulum Ca2+-transport ATPase is regulated via phospholamban. Phosphorylation of this regulatory protein by cAMP-dependent as well as by cGMP-dependent protein kinase stimulates the endoplasmic-reticulum Ca2+ pump. The activity of the plasmalemmal Ca2+-transport ATPase can be modulated by calmodulin, negatively charged phospholipids, and by receptor-binding agonists. cGMP-dependent protein kinase also exerts a stimulatory effect on the plasmalemmal Ca2+ pump, but this effect is not mediated via a direct phosphorylation of the Ca2+ pump.
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PMID:The (Ca2+-Mg2+)-ATPases of the plasma membrane and of the endoplasmic reticulum in smooth muscle cells and their regulation. 246 79

In smooth muscle cells two distinct Ca2+-pumps with a different subcellular localization can be demonstrated. A plasma-membrane localized Ca2+-pump with a relative molecular weight (Mr) of 140 kDa resembles the Ca2+-pump of the erythrocyte plasma membrane in the sensitivity of its phospho-intermediate towards La3+, in its calmodulin-binding capacity and in its antigenic properties. A second Ca2+-pump with a Mr of 100 kDa is situated in the endoplasmic reticulum. On the basis of its antigenicity and the degradation pattern of its phospho-intermediate the endoplasmic-reticulum Ca2+-pump is found to be homologous to the sarcoplasmic-reticulum Ca2+-pump of cardiac muscle and slow twitch skeletal muscle, but it clearly differs from the Ca2+-pump present in the sarcoplasmic reticulum of fast skeletal muscle. The endoplasmic-reticulum and the plasma-membrane Ca2+-pumps are present in both visceral and vascular smooth muscle, but tissue-and species-dependent differences in their relative amount have been observed. The endoplasmic-reticulum Ca2+-pump is regulated via phospholamban. Phosphorylation of this regulatory protein by cAMP-dependent as well as by cGMP-dependent protein kinase stimulates the endoplasmic-reticulum Ca2+-pump. On the other hand, the activity of the plasmalemmal Ca2+-pump is modulated by calmodulin, negatively charged phospholipids and membrane-receptor-binding agonists. cGMP-dependent protein kinase also exerts a stimulatory effect on the plasmalemmal Ca2+-pump. However, cGMP-dependent protein kinase does not directly phosphorylate the plasmalemmal Ca2+-pump, but by activating a phosphatidyl-inositol kinase it promotes the formation of phosphatidyl-inositol monophosphate which then acts as the final stimulator of the Ca2+-pump.
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PMID:Ca2+-transport by smooth muscle membranes and its regulation. 254 62

The atrial natriuretic peptide (ANP) stimulates cGMP production and protein phosphorylation in a particulate fraction of cultured rat aortic smooth muscle cells. Three proteins of 225, 132, and 11 kDa were specifically phosphorylated in response to ANP treatment, addition of cGMP (5 nM), or addition of purified cGMP-dependent protein kinase. The cAMP-dependent protein kinase inhibitor had no effect on the cGMP-stimulated phosphorylation of the three proteins but inhibited cAMP-dependent phosphorylation of a 17-kDa protein. These results demonstrate that the particulate cGMP-dependent protein kinase mediates the phosphorylation of the 225-, 132-, and 11-kDa proteins. The 11-kDa protein is phospholamban based on the characteristic shift in apparent Mr from 11,000 to 27,000 on heating at 37 degrees C rather than boiling prior to electrophoresis. ANP (1 microM) increased the cGMP concentration approximately 4-fold in the particulate fractions, from 4.3 to 17.7 nM, as well as the phosphorylation of the 225-, 132-, and 11-kDa proteins. In contrast, the biologically inactive form of ANP, carboxymethylated ANP (1 microM), did not stimulate phosphorylation of any proteins nor did the unrelated peptide hormone, angiotensin II (1 microM). These results demonstrate the presence of the cGMP-mediated ANP signal transduction pathway in a particulate fraction of smooth muscle cells and the specific phosphorylation of three proteins including phospholamban, which may be involved in ANP-dependent relaxation of smooth muscle.
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PMID:Atrial natriuretic peptide-dependent phosphorylation of smooth muscle cell particulate fraction proteins is mediated by cGMP-dependent protein kinase. 257 2

Phospholamban is a negative regulator of the sarcoplasmic reticulum Ca(2+)-pumping ATPase. Phosphorylation of phospholamban activates the ATPase and decreases the level of cytosolic calcium. Phospholamban is phosphorylated in heart by cAMP-dependent protein kinase, cGMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II (CM-kinase-II) and in smooth muscle cells by cGMP-dependent protein kinase. In contrast to heart muscle, phospholamban is poorly phosphorylated by CM-kinase-II in extracts of rat aortic smooth muscle cells. Rat aorta phospholamban amino acid sequence was identical to dog heart. The peptide substrate specificity of CM-kinase-II from rat aorta was the same as that from rat heart. The lack of phosphorylation of rat aorta phospholamban by the CM-kinase-II appears to result from the relatively low abundance of phospholamban in smooth muscle.
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PMID:Phosphorylation of phospholamban in aortic smooth muscle cells and heart by calcium/calmodulin-dependent protein kinase II. 785 66

Sarcoplasmic reticulum (SR) vesicles were prepared from either canine or sheep heart and fused into lipid bilayers to study their ionic channels. A 92 +/- 5 pS anion-selective channel was recorded in asymmetric 50 mM trans/250 mM cis CsCl buffer system. Reversal potentials and theoretical equilibrium potentials for Cl-ions obtained under various experimental conditions allowed us to confirm the Cl- selectivity of this SR channel. The majority (69%) of channel recordings (n = 45) displayed steady-state kinetics and a slight voltage dependency of the open probability. However, 31% of the channels inactivated after their incorporation. We now report that the channel might be reactivated by depolarizing voltage steps. Furthermore, the use of either PKA or PKG in association with adequate phosphorylating buffers lengthens the deactivation process at the end of the voltage pulses, but does not prevent the inactivation. It was assumed that the change in gating mode was due to a voltage-sensitive association/dissociation mechanism with a phosphorylated protein of the SR membrane such as phospholamban (PL). We demonstrated that a specific monoclonal antibody raised against canine PL inhibited the activity of the channel and prevented its reactivation by depolarizing steps. 400 to 800 ng/ml of Anti-PL Ab consistently and sequentially turned off the channel activities. In contrast, heat inactivated Anti-PL Ab had no effect. We propose that phospholamban may be a primer of the SR Cl- channel whereby Cl- anions would play the role of counter-charge carrier during rapid Ca2+ release and Ca2+ uptake by the SR.
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PMID:Examination of the role of phosphorylation and phospholamban in the regulation of the cardiac sarcoplasmic reticulum Cl- channel. 856 46

Elevation of intracellular cGMP and activation of cGMP-dependent protein kinase (PKG) in vascular smooth-muscle cells produces relaxation, but mechanisms distal to PKG activation are not well understood. Few PKG substrates have been described in smooth muscle that may mediate the action of PKG, including P240, P132 and phospholamban. None of them is a specific PKG substrate, raising the question of whether any specific PKG substrates possibly exist in vascular smooth muscle that may play roles in relaxation. In this study PKG substrates were detected in aortic smooth muscle by adding purified exogenous PKG and [gamma-32P]-ATP. Very few PKG substrates were detectable in whole-tissue homogenates or detergent-solubilized fractions, due to the high basal activity of other protein kinases and the large numbers of other phosphoproteins. Heat or acid treatment of such fractions, to remove any endogenous protein kinase activity and achieve partial protein purification, revealed many potential PKG substrates. Of the 3 substrates identified previously, P240 and P132 were partly heat-stable. Thirty-one new PKG substrates were found: 14 in the initial heat-stable extract and 9 in the heat- and acid-soluble extract, whereas the others were revealed only after chromatography. All of the heat-stable PKG substrates were bound and salt-eluted from a DEAE-cellulose column in 2 major peaks called pool I and II. After sequential application to Q-Sepharose and S-Sepharose columns, 7 PKG substrates were found in pool I, in particular a group of 4 substrates of 40, 33, 28 and 22 kD virtually coeluted through all 3 columns. The former 3 produced similar phosphopeptide maps, suggesting a relationship. All the new substrates from pool I were relatively specific for PKG because they were poorly phosphorylated with exogenous cAMP-dependent protein kinase and not with Ca2+/phospholipid-dependent protein kinase. Further chromatography of the proteins in pool II resulted in an extensive purification of P132 as well as a group of 4 PKG substrates of 33-30 kD. Phosphopeptide mapping of the 132-kD protein revealed a close homology to the 132-kD PKG substrate previously described in rat aortic smooth muscle. These data demonstrate the presence of multiple substrates for PKG in aortic smooth-muscle tissue.
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PMID:Multiple substrates for cGMP-dependent protein kinase from bovine aortic smooth muscle: purification of P132. 863 Mar 52


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