Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ability of guanosine-3',5'-cyclic monophosphate (cGMP) to induce increases in the intracellular free calcium ion concentration ([Ca2+]i) was studied at the single cell level in fura-2-loaded rat hepatocytes. Both 8-bromo-cGMP (Br-cGMP) and dibutyryl cGMP (db-cGMP) produced oscillatory [Ca2+]i increases in hepatocytes. In addition, Br-cGMP increased the frequency of agonist-induced spiking or converted [Ca2+]i oscillations into sustained nonoscillatory [Ca2+]i responses. Addition of the nitric oxide donor sodium nitroprusside also produced oscillatory [Ca2+]i increases similar to those generated by cGMP analogues. In the absence of extracellular Ca2+, cGMP-induced [Ca2+]i responses were significantly reduced and mainly appeared as single transient [Ca2+]i increases. The effects of cGMP analogues do not appear to be mediated by a secondary increase in cAMP or activation of cAMP-dependent protein kinase (PKA), since [Ca2+]i responses to cGMP analogues were inhibited by the G-kinase inhibitor 8-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp-Br-cGMP[S]). Both Br-cGMP and db-cGMP also increased [Ca2+]i in the presence of the PKA inhibitor 8-bromoadenosine-3',5'-cyclic monophosphorothioate (Rp-Br-cAMP[S]) and when the cGMP-inhibitable cAMP phosphodiesterase activity was inhibited by pretreatment with siguazodan. Br-cGMP stimulated the Mn2+-induced quench of compartmentalized fura-2 in intact hepatocytes, indicating a site of action at the level of the Ca2+ stores. This locus was further supported by the finding that pretreatment of hepatocytes with Br-cGMP potentiated submaximal inositol 1,4,5-trisphosphate (InsP3)-induced Mn2+ quench in subsequently permeabilized hepatocytes. db-cGMP also decreased PKA-mediated back phosphorylation of the hepatic type-1 InsP3 receptor, indicating that G-kinase phosphorylates the InsP3 receptor at sites targeted by PKA. These data indicate that phosphorylation of the hepatic InsP3 receptor by G-kinase increases the sensitivity to InsP3 for [Ca2+]i release and is associated with the production of [Ca2+]i oscillations in single rat hepatocytes.
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PMID:Cyclic GMP induces oscillatory calcium signals in rat hepatocytes. 870 90

The ability of cAMP to modulate the actions of Ca(2+)-mobilizing agonists was studied in single Fura-2-loaded pig articular chondrocytes in primary culture. Forskolin and 8-Br-cAMP increased both the frequency and amplitude of Ca2+ oscillations induced by ATP, and, in unstimulated cells, induced single Ca2+ transients or even Ca2+ oscillations. The cAMP-dependent protein kinase inhibitor H89 totally prevented the effect of cAMP-elevating agents on Ca2+ signalling. Forskolin and 8-Br-cAMP promptly increased the rate of Mn2+ quenching, when administered in the presence of ATP, suggesting a potentiation of receptor-mediated Ca2+ influx. In Ca(2+)-free medium, ATP-induced Ca2+ oscillations decreased and stopped after a few cycles: subsequent ATP additions temporarily resumed the activity, an effect that could be mimicked by forskolin. The same agent induced single Ca2+ transients in 42% of the cell population maintained in Ca(2+)-free medium. Thapsigargin prevented Ca2+ responses to both ATP and forskolin. The results indicate a dual mechanism for cAMP-induced potentiation of Ca2+ signalling in articular chondrocytes: an increase of receptor-mediated Ca2+ influx and a positive modulation of intracellular Ca2+ release.
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PMID:Dual mechanism for cAMP-dependent modulation of Ca2+ signalling in articular chondrocytes. 880 48

The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.
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PMID:Is there a catalytic base in the active site of cAMP-dependent protein kinase? 906 28

The kinetics of nucleotide binding and phosphoryl group transfer were measured in the catalytic subunit of cAMP-dependent protein kinase using stopped-flow fluorescence spectroscopy and an acrylodan-labeled derivative of this enzyme, which we have previously shown to have kinetic properties similar to those for the wild-type enzyme (Lew et al., 1996). The fluorescence emission spectrum of this enzyme is quenched differentially by ATP and ADP so that both the binding of ligands and phosphoryl group transfer at the active site can be monitored selectively. The association and dissociation rate constants for both nucleotides were measured using two methods: relaxation and competition binding. The ratio of the observed dissociation and association rate constants by both methods are consistent with Kd measurements (25 microM) determined by equilibrium fluorescence quenching. The dissociation rate constant for ADP (100 s(-1)) is approximately 2.5-fold larger than k(cat) (39 s(-1)). A full viscosity effect was measured for k(cat), suggesting that a diffusive step or steps limit maximum turnover. Pre-steady-state kinetic transients are biphasic and were fitted to observed rate constants of 500 s(-1) and 60 s(-1) at 500 microM Kemptide (LRRASLG). Metal substitution studies (Mg2+ vs Mn2+) indicate that this first phase represents the phosphoryl group transfer step. Phosphopeptide release is faster than this second phase since the substrate is in rapid exchange with the enzyme and phosphorylation reduces the affinity of the peptide. The inability to assign this second phase to the chemical event or to product release implies that it reflects a viscosity-sensitive, protein conformational change that occurs after phosphoryl group transfer and prior to product release. Two conformational steps were detected in the binding of both ATP and ADP by relaxation methods that may be related to this second pre-steady-state kinetic phase. We suggest that this additional step in the kinetic mechanism may also occur in the wild-type enzyme and represents a large structural change in the enzyme during normal catalytic cycling.
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PMID:Identification of a partially rate-determining step in the catalytic mechanism of cAMP-dependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy. 918 52

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
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PMID:A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II. 944 23

The catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is more stable by several criteria when it is part of a holoenzyme complex. By measuring the thermal stability of the free C subunit in the presence and absence of nucleotides and/or divalent metal ions, it was found that most of the stabilizing effects associated with the type I holoenzyme could be attributed to the nucleotide. The specific requirements for this enhanced stability were further dissected: Adenosine stabilized the C subunit up to 5 degrees C; however, divalent cations (i.e., Mg2+, Ca2+, and Mn2+) do not increase heat stability in combination with adenosine and adenine (1). Divalent cations as well as ATP and ADP have no effect by themselves (2). The enhanced stability derived from both ATP and ADP requires divalent cations. MnATP (12 degrees C) shows a much stronger effect than CaATP (7 degrees C) and MgATP (5 degrees C) (3). In the holoenzyme complex or the protein kinase inhibitor/C subunit complex, metal/ATP is also required for enhanced stability; neither the RI or RII subunits nor PKI alone stabilize the C subunit significantly (4). For high thermal stability, the occupation of the second, low-affinity metal-binding site is necessary (5). From these results, we concluded that the adenine moiety works independently from the metal-binding sites, stabilizing the free C subunit by itself. When the beta- and gamma-phosphates are present, divalent metals are required for positioning these phosphates, and two metals are required to achieve thermostability comparable to adenosine alone. The complex containing two metals is the most stable. A comparison of several conformations of the C subunit derived from different crystal structures is given attributing open and closed forms of the C subunit to less and more thermostable enzymes, respectively.
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PMID:Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase. 1032 Mar 66

Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S. cerevisiae was almost equally activated by cAMP and cGMP in 3 x 10(-6) M concentrations of either nucleotide in the presence of Mg2+ ions. Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.
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PMID:PKA from Saccharomyces cerevisiae can be activated by cyclic AMP and cyclic GMP. 1034 18

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated glucose transport. Repeated contractions increased 2-deoxyglucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by approximately 50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a approximately 25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation approximately 10-fold, and NAC blocked approximately 50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of glucose transport in fast-twitch muscle.
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PMID:Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle. 1680 55

The immune system is necessary for protecting against various pathogens. However, under certain circumstances, self-reactive immune cells can drive autoimmunity, like that exhibited in type 1 diabetes (T1D). CD4+ T cells are major contributors to the immunopathology in T1D, and in order to drive optimal T cell activation, third signal reactive oxygen species (ROS) must be present. However, the role ROS play in mediating this process remains to be further understood. Recently, cellular metabolic programs have been shown to dictate the function and fate of immune cells, including CD4+ T cells. During activation, CD4+ T cells must transition metabolically from oxidative phosphorylation to aerobic glycolysis to support proliferation and effector function. As ROS are capable of modulating cellular metabolism in other models, we sought to understand if blocking ROS also regulates CD4+ T cell activation and effector function by modulating T cell metabolism. To do so, we utilized an ROS scavenging and potent antioxidant manganese metalloporphyrin (MnP). Our results demonstrate that redox modulation during activation regulates the mTOR/AMPK axis by maintaining AMPK activation, resulting in diminished mTOR activation and reduced transition to aerobic glycolysis in diabetogenic splenocytes. These results correlated with decreased Myc and Glut1 upregulation, reduced glucose uptake, and diminished lactate production. In an adoptive transfer model of T1D, animals treated with MnP demonstrated delayed diabetes progression, concurrent with reduced CD4+ T cell activation. Our results demonstrate that ROS are required for driving and sustaining T cell activation-induced metabolic reprogramming, and further support ROS as a target to minimize aberrant immune responses in autoimmunity.
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PMID:Reactive oxygen species are required for driving efficient and sustained aerobic glycolysis during CD4+ T cell activation. 2842 86

Protein kinases are key enzymes in the regulation of eukaryotic signal transduction. As metalloenzymes they employ divalent cations for catalysis and regulation. We used the catalytic (C) subunit of cAMP-dependent protein kinase (PKA) as a model protein to investigate the role of a variety of physiologically or pathophysiologically relevant divalent metal ions in distinct steps within the catalytic cycle. It is established that divalent metal ions play a crucial role in co-binding of nucleotides and also assist in catalysis. Our studies reveal that besides the physiologically highly relevant magnesium, metals like zinc and manganese can assist in phosphoryl transfer, however, only a few support efficient substrate turnover (turnover catalysis). Those trace metals allow for substrate binding and phosphotransfer but hamper product release. We further established the unique role of magnesium as the common biologically relevant divalent metal ideally enabling (co-) substrate binding and orientation. Magnesium allows stable substrate binding and, on the other hand accelerates product release, thus being extremely efficient in turnover catalysis. We extended our studies to non-catalytic functions of protein kinases looking at pseudokinases, a subfamily of protein kinases inherently lacking critical residues for catalysis. Recently, pseudokinases have been linked to human diseases. Some pseudokinases are still capable of binding metal ions, yet have lost the ability to transfer the phosphoryl group from ATP to a given substrate. Here metal ions stabilize an active like, though catalytically unproductive conformation and are therefore crucial to maintain non-catalytic function. Finally, we demonstrate for the canonical kinase PKA that the trace metal manganese alone can stabilize protein kinases in an active like conformation allowing them to bind substrates even in the absence of nucleotides.
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PMID:Divalent metal ions control activity and inhibition of protein kinases. 2904 44


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