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 monoclonal antibody was made using the spleen cells of a mouse immunized with chick synaptic membranes and designated as mAb 1D12. It immunoprecipitated 25% of the omega-conotoxin binding protein but no dihydropyridine binding protein solubilized from chick brain membranes. By immunoblotting, a polypeptide of 58-kDa was identified as the antigen of this antibody in chick, rat, rabbit and guinea pig brain. Immunohistochemical observation indicated the immunoreactivity of mAb 1D12 to be localized in the synaptic regions of central and peripheral neurons. In peripheral organs, there was additional staining in the distal portions of nerve fibers. Immunoelectron microscopy showed immunoreactivity to be located in synaptic vesicle and presynaptic plasma membranes. In the subcellular fractionation of rat brain, 58-kDa protein was recovered in the fractions of synaptic vesicles and plasma membranes but not soluble proteins. This protein could be extracted from membranes by Triton X-100 but treatment with EDTA, acid, base or high salt failed to have such effect. Solubilized 58-kDa protein of rat brain was purified by immunoaffinity chromatography using mAb 1D12. Both protein kinase C and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated purified 58-kDa protein, and maxima of 0.47 and 0.94 mol of phosphates, respectively, were incorporated per mol of 58-kDa protein. 58-kDa protein was not phosphorylated by either cAMP-dependent or cGMP-dependent protein kinase. When present in membranes, it was also phosphorylated by protein kinase C and CaM kinase II. Possible involvement of 58-kDa protein in the protein kinase C and CaM kinase II-mediated regulation of synaptic transmission in central and peripheral neurons is discussed.
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PMID:Protein kinase C and Ca2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles. 165 60

Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles.
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PMID:Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody. 214 59

Cyclic-nucleotide-elevating vasodilators such as prostaglandin E1, prostacyclin, sodium nitroprusside and endothelium-derived relaxing factor inhibit both contraction of vascular smooth muscle cells and the aggregation of platelets at an early step of the activation cascade. Previous studies from this laboratory [Waldmann, R., Nieberding, M. and Walter, U. (1987) Eur. J. Biochem. 167, 441-448) established that in human platelets cyclic-nucleotide-elevating vasodilators stimulated a pattern of protein phosphorylation which was mediated by both cAMP- and cGMP-dependent protein kinases. Of particular interest was a membrane-bound 50-kDa protein whose phosphorylation was increased both by cAMP- and cGMP-elevating vasodilators in intact platelets and by endogenous cAMP- and cGMP-dependent protein kinase in platelet membranes. Since the molecular mechanism of action of cyclic-nucleotide-elevating vasodilators is unknown, this 50-kDa phosphoprotein from human platelets was purified to apparent homogeneity by salt extraction, anion, cation and dye-ligand chromatography. The purified protein migrated as a 46-kDa protein in SDS/PAGE, was an excellent substrate for both cAMP- and cGMP-dependent protein kinases and migrated in SDS/PAGE as a 50-kDa protein after phosphorylation by these protein kinases. Analysis by limited proteolysis, tryptic fingerprinting and of phosphoamino acids established that the purified protein is identical with the 50-kDa protein phosphorylated by both cAMP- and cGMP-dependent protein kinases in platelet membranes and in response to cAMP- and cGMP-elevating vasodilators with intact platelets. Evidence is presented that the purified protein contains at least two phosphorylation sites, each of which is preferentially phosphorylated by either cAMP- or cGMP-dependent protein kinase. The availability of this vasodilator-regulated phosphoprotein as a purified protein should now allow new approaches for investigating the function of this protein and its possible role in the mechanism of action of cyclic-nucleotide-elevating vasodilators.
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PMID:Purification of a vasodilator-regulated phosphoprotein from human platelets. 280 62

Membrane proteins of Mr 240,000, 130,000, and 85,000 (GS-proteins) were rapidly and selectively phosphorylated in particulate fractions of rabbit aortic smooth muscle in the presence of [Mg-32P]ATP and low concentrations of cGMP (Ka = 0.01 microM) or cAMP (Ka = 0.2 microM). The effects of both cyclic nucleotides in this preparation were mediated entirely by an endogenous, membrane-bound form of cGMP-dependent protein kinase (G-kinase). The GS-proteins were also phosphorylated by the soluble form of G-kinase purified from bovine lung; this effect was most evident following removal of endogenous G-kinase from the membranes using Na2CO3 and high salt washes. The membrane-bound and cytosolic forms of G-kinase phosphorylated the Mr 130,000 GS-protein with the same specificity as determined by two-dimensional peptide mapping. Despite this functional homology between the two forms of G-kinase, only the particulate enzyme appears to play a role in phosphorylating the GS-proteins. Although little endogenous cAMP-dependent protein kinase (A-kinase) activity was detected in washed aortic smooth muscle membranes, the GS-proteins could be phosphorylated when purified A-kinase catalytic subunit was added to this preparation. Peptide mapping of the Mr 130,000 GS-protein indicated that A-kinase phosphorylated a subset of the same peptides labeled by the two forms of G-kinase. The endogenous A-kinase of rabbit aortic smooth muscle homogenates was also found to phosphorylate the GS-proteins. Since the intracellular concentrations of cGMP or cAMP can be selectively elevated by different stimuli, these results suggest several possible mechanisms by which the phosphorylation state of the GS-proteins may be regulated by cyclic nucleotides: activation of the membrane-bound G-kinase by cGMP or cAMP; and activation of cytosolic A-kinase by cAMP.
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PMID:The cyclic nucleotide-dependent phosphorylation of aortic smooth muscle membrane proteins. 303 5

In a previous study, we demonstrated that a high concentration (> or = 1 microM) of isoproterenol (ISO) produced a dual effect on L-type Ca2+ current (ICa(L)) in vascular smooth muscle (VSM) cells from the portal vein: an initial stimulatory action followed by a sustained inhibition. The first stimulatory phase was fast (presumably more direct) and may reflect G-protein gating of the Ca2+ channels. The second inhibitory phase was slower (presumably more indirect) and may be mediated by the adenylate cyclase/cAMP pathway. In order to define further the mechanism for the ISO inhibition of ICa(L), the effects of cyclic nucleotides and their related protein kinases were examined in freshly isolated single smooth muscle cells from the rabbit portal vein using the whole-cell voltage clamp technique. To isolate ICa(L), the pipette solution contained high Cs+ (to block K+ outward current), and the bath contained physiological salt solution. Upon extracellular application of membrane-permeable cAMP and cGMP analogs (8-Br-cAMP and 8-Br-cGMP, 3 mM), ICa(L) was significantly inhibited by 27.9 +/- 5.0 and 33.5 +/- 4.8%, respectively. Forskolin (100 microM) also depressed ICa(L). The protein kinase inhibitor, H-7, prevented the inhibitory effects of both cyclic nucleotides and forskolin. In addition, intracellular application (via the patch pipettes) of cAMP-dependent protein kinase (PK-A, catalytic subunit; 1.76 microM) and cGMP-dependent protein kinase (PK-G, 50 nM, pre-activated by 10 microM cGMP) significantly inhibited the peak amplitude of ICa(L) by 45.5 +/- 10 and 43.2 +/- 6.2%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of L-type calcium channels by cyclic nucleotides and phosphorylation in smooth muscle cells from rabbit portal vein. 791 17

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

This study was designed to test the hypothesis that 8-Br-cAMP and 8-Br-cGMP dependent relaxation of phorbol dibutyrate stimulated contractions of intact rat aorta are independent of changes in the level of myosin light chain phosphorylation. Phorbol dibutyrate stimulated contraction with a concomitant increase in myosin light chain phosphorylation in normal tissues and without an increase in myosin light chain phosphorylation in calcium-depleted tissues. Phorbol dibutyrate stimulated contractions in normal CaCl2-containing physiological salt solution were relaxed in a concentration-dependent manner by 8-Br-cAMP and 8-Br-cGMP. Phorbol dibutyrate-induced contractions in the absence of Ca2+ were only relaxed by 8-Br-cGMP; 8-Br-cAMP had no effect. The relaxation induced by 8-Br-cGMP was associated with a decrease in myosin light chain phosphorylation suggesting that cGMP-dependent protein kinase may alter the activity of either the myosin light chain kinase or phosphatase. The relaxation induced by 8-Br-cAMP was not associated with a decrease in phosphorylation suggesting that cAMP-dependent protein kinase may uncouple myosin light chain phosphorylation from force.
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PMID:Cyclic AMP and cyclic GMP relax phorbol ester-induced contractions of rat aorta by different mechanisms. 919 89

Considerable controversy exists in the literature with regard to the nature of the agent mediating the biological effects of nitroxyl (NO-) donors. Here it is demonstrated that Angeli's salt (AS), a generator of NO-, enhanced human neutrophil migration. Under aerobic conditions, AS was converted to peroxynitrite to a small extent. However, using methionine, a scavenger of peroxynitrite, it was shown that peroxynitrite was not involved in AS-induced migration. AS equally enhanced human neutrophil migration under aerobic and anaerobic conditions, which strongly suggests that extracellular conversion of NO- to .NO by oxygen was not required. Furthermore, metHb and L-cysteine, which react more readily with NO- than with .NO, inhibited AS-induced migration, whereas the response towards gaseous .NO remained unaffected. AS induced an increase in the intracellular level of cGMP, although the curves for migration and cGMP level appeared to be slightly different in their concentration dependence. An inhibitor of soluble guanylate cyclase and antagonists of cGMP-dependent protein kinase had a more pronounced inhibitory effect on .NO-induced migration than on AS-induced migration. This suggests that the cGMP signalling cascade is partially, but not solely, responsible for AS-induced migration. As it has been demonstrated that soluble guanylate cyclase can only be activated by .NO, and not by NO-, these data indicate that NO- is at least partly converted intracellularly to .NO.
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PMID:Intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration. 948 Aug 81

1. We used patch clamp to study whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells freshly dissociated from pig coronary arteries. 2. CGRP (50 nM) activated an inward current at -60 mV in symmetrical 140 mM K+ that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive potassium (KATP) channels. CGRP-induced currents were larger in cells dialysed with 0.1 mM ATP than with 3.0 mM ATP. 3. Forskolin (10 microM) activated a glibenclamide-sensitive current, as did intracellular dialysis with cAMP (100 microM). The catalytic subunit of cAMP-dependent protein kinase (protein kinase A, PKA), added to the pipette solution, activated equivalent currents in five out of twelve cells. 4. CGRP-induced currents were reduced by the PKA inhibitors adenosine 3',5'-cyclic monophosphorothioate, RP-isomer, triethylammonium salt (Rp-cAMPS; 100 microM) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide+ ++ dihydrochloride (H-89; 1 microM), and abolished by inclusion of a PKA inhibitor peptide in the pipette solution. 5. The beta-adrenergic agonist isoprenaline (10 microM) also activated a glibenclamide-sensitive K+ current. 6. CGRP-induced currents were unaffected by the inhibitor of cGMP-dependent protein kinase (PKG) KT5823 (1 microM). Sodium nitroprusside (10 microM) did not activate a glibenclamide-sensitive current in cells held at -60 mV, but did activate an outward current at +60 mV that was abolished by KT5823, or by 100 nM iberiotoxin (an inhibitor of BKCa channels). 7. Our findings suggest that CGRP activates coronary KATP channels through a pathway that involves adenylyl cyclase and PKA, but not PKG.
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PMID:ATP-sensitive K+ channel activation by calcitonin gene-related peptide and protein kinase A in pig coronary arterial smooth muscle. 949 Aug 26

Hepatotoxicity of allyl alcohol involves its bioactivation to acrolein and subsequent protein sulfhydryl loss and lipid peroxidation. However, the links between these events and hepatocellular death are not known. The purpose of these studies was to examine whether specific signal transduction pathways are associated with allyl alcohol toxicity in hepatocytes. Inhibition or augmentation of cyclic AMP and/or protein kinase A (PKA) by Rp-Ado-3N,5N-cyclic monophosphorothioate triethylamine salt or 3-isobutyl-1-methylxanthine had no effect on allyl alcohol-induced cell death. H-7, an inhibitor of PKA, PKC, and PKG, partially inhibited cell killing by allyl alcohol, whereas chelerythrine chloride, a nonselective PKC inhibitor, almost completely abolished allyl alcohol cytotoxicity. Neither 2,2N,3,3N,4,4N-hexahydroxy-1,1N,-biphenyl-6,6N-dimethanol-dimethyl ether, a selective PKC alpha and beta inhibitor, nor bisindolylmaleimide I, an inhibitor of PKC alpha, beta, and epsilon, had any effect on allyl alcohol cytotoxicity. In contrast, rottlerin, a selective PKCdelta inhibitor, blocked hepatocellular killing by allyl alcohol. Cytoprotection by chelerythrine chloride and rottlerin was not the result of inhibition of bioactivation of allyl alcohol because each inhibitor also prevented cell death from acrolein. Western blotting and immunohistochemical techniques revealed that allyl alcohol stimulated phosphorylation and translocation of PKCdelta to hepatocyte membranes (i.e., activation), and this activity was inhibited by rottlerin. Cell death appeared to occur via oncotic necrosis rather than apoptosis based on single-stranded DNA ELISA and propidium iodide staining. Together, these results indicate that activation of PKCdelta is a critical, early event in initiating hepatocyte injury and death from allyl alcohol.
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PMID:Allyl alcohol activation of protein kinase C delta leads to cytotoxicity of rat hepatocytes. 1275 90


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