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)

PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Nao and 0.7 mM for Ko, is absolutely dependent on Clo and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 approximately 650 nM) or calyculin A (EC50 approximately 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF on cotransport rate is biphasic, with an initial rapid approximately 2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 approximately 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (> 2 days) leads to neurite formation and a maintained approximately 2-fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.
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PMID:Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells. 814 46

Acetylcholine acting via muscarinic cholinoceptors decreased phosphorylation of phospholamban and troponin I without reducing adenosine 3',5'-cyclic monophosphate (cAMP) levels or cAMP-dependent protein kinase activity ratio in the presence of 10-100 nM isoproterenol in guinea pig ventricular myocytes. The effect of acetylcholine was more pronounced when adenosine deaminase (5 U/ml) was present and incubation period was short (10 s). Okadaic acid, an inhibitor of protein phosphatase activity, blocked the acetylcholine-mediated inhibition of isoproterenol-stimulated phosphorylation of phospholamban. It is suggested that acetylcholine reduces protein phosphorylation by a cAMP-independent mechanism in guinea pig ventricular myocytes.
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PMID:M2-specific muscarinic cholinergic receptor-mediated inhibition of cardiac regulatory protein phosphorylation. 816 Aug 16

DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence. The steady-state emission spectra and quenching of the intrinsic (Trp163) and extrinsic fluorescence (acrylodan or lucifer yellow linked to Cys72) were consistent with a complete exposure of these residues to the aqueous environment. The intrinsic fluorescence of DARPP-32 was resolved into three decay components with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime component giving the major contribution. The ratio between the amplitudes associated with the short and long decay constants was decreased upon denaturation. The rotational behavior of DARPP-32 measured by anisotropy decay revealed that Trp163 is located in a highly flexible peptide chain, whereas Cys72 is embedded in a more rigid environment. Phosphorylation by cAMP-dependent protein kinase did not alter any of the fluorescence parameters, whereas only minor effects were associated with casein kinase II phosphorylation. These findings indicate that DARPP-32 contains at least two distinct domains and that phosphorylation has no dramatic effects on its conformation.
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PMID:Study of the conformation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by fluorescence spectroscopy. 822 46

A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the alpha subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric form of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km = 42 microM, Vmax = 10.3 mumol x min-1 x mg-1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km = 2.5 microM, Vmax = 0.008 mumol x min-1 x mg-1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A-like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP-dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification.
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PMID:Dephosphorylation of phosphorylated atrial natriuretic peptide by protein phosphatase 2A. 838 53

We have observed that soluble extracts from the extreme acidothermophilic archaebacterium Sulfolobus solfataricus contained protein phosphatase activity that was greatly stimulated by the divalent metal ions Mn2+, Mg2+, Ni2+, or Co2+. This activity apparently arose from a single enzyme since (a) stimulation by these divalent metal ions was not additive and (b) protein phosphatase activity eluted as a single peak from both a DE52 ion-exchange column and a Sephadex G-100 gel filtration column. Its apparent molecular mass was approximately 28,000 daltons. The enzyme dephosphorylated a variety of phosphoserine-containing substrates including casein, histone H2a, phosphorylase kinase, or glycogen phosphorylase. The enzyme would not dephosphorylate either histone H1 or a number of phosphotyrosine-containing compounds. It removed only half the phosphate bound to histone H2b, which is phosphorylated at two sites by the cAMP-dependent protein kinase. Protein phosphatase activity was inhibited by EDTA, Cu2+, Zn2+, NaF, inorganic phosphate, or pyrophosphate; but was unaffected by other potential activators and inhibitors such as microcystin, okadaic acid, vanadate, polyamines, or sulfhydryl modifying reagents. This enzyme represents the first protein phosphatase to be identified in any member of the third and oldest phylogenetic kingdom in nature, the archaebacteria.
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PMID:Identification of a serine/threonine-specific protein phosphatase from the archaebacterium Sulfolobus solfataricus. 838 14

CDC2 kinase activity was decreased by up to 75% when mitotic cell free extracts from mouse fibroblasts were incubated with cAMP and ATP. This effect was blocked by PKI, the heat stable inhibitor of cAMP-dependent protein kinase (PKA). An acidic, heat stable protein from G1 cells, consistent with inhibitor-1 of protein phosphatase 1, mimicked the effect of cAMP, but was not antagonized by PKI. Okadaic acid, another inhibitor of protein phosphatase 1, also downregulated CD2 activity, and the effect was independent of both cAMP and PKI. The evidence suggests that PKA exerts its effect by activating inhibitor-1 by phosphorylation, and that the next step in the regulatory pathway requires the inactivation of one or more protein phosphatase 1 isoenzymes. Non-denaturing gel electrophoresis suggested that the size and/or charge density of the CDC2 kinase complex was changed when the activity was downregulated by cAMP or G1 extracts.
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PMID:Mitotic CDC2 kinase is negatively regulated by cAMP-dependent protein kinase in mouse fibroblast cell free extracts. 838 4

Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin-LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine-sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non-lysosomal (propylamine-resistant) degradation was unaffected. The autophagy-inhibitory effect of okadaic acid was not affected by inhibitors of cAMP-dependent protein kinase or protein kinase C (H-7, H-89, calphostin C) but eliminated by the non-specific inhibitor K-252a and its analogues (KT-5720, KT-5823, KT-5926) and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic-lysosomal degradation pathway.
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PMID:Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors. 839 87

Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
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PMID:Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A. 855 May 70

Glycogen synthase, the regulatory enzyme of glycogen synthesis undergoes multisite phosphorylation leading to its inactivation. The kinases responsible for this covalent modification (ex. cAMP-dependent protein kinase, protein kinase C and glycogen synthase kinase-3) are controlled by the second messengers generated by different hormones. The isolated hepatocytes has been used as one of the experimental models for studying this complex regulatory process. Inactivation of glycogen synthase by glucagon and vasopressin has been shown to be accompanied with incorporation of phosphate into the enzyme protein. Insulin has been shown to activate glycogen synthase by inhibition of kinases and activation of synthase phosphatase. Glycogen synthase is activated by several gluconeogenic substrates, in addition to glucose. Studies in hepatocytes with activators and inhibitors of protein kinase C show that this enzyme negatively controls glycogen synthase. The differential effects of the phosphatase inhibitors, calyculin A and okadaic acid in liver cells provide supporting evidence that protein phosphatase type-1 plays a major role in the regulation of glycogen synthase. Hepatocytes isolated from diabetic rats of both types (insulin-dependent and non-insulin-dependent) mimic the defective glycogen synthase activation seen in vivo.
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PMID:Regulation of glycogen synthase activation in isolated hepatocytes. 856 54

The mechanism of dephosphorylation of multiphosphorylated proteins in the brain is not well understood. We have used the multiphosphorylated protein, phosvitin as a model substrate and undertaken the purification and characterization of brain phosphatases that preferentially dephosphorylate multiphosphorylated proteins. Two phosvitin phosphatase activities, termed Phosvitin Phosphatase 1 and 2 (PvP1, PvP2), which show acidic pH optima were resolved from the 33,000g supernatant fraction from rat brain by a procedure employing successive DEAE-cellulose, Sepharose 6B, second DEAE-cellulose and FPLC/Superose 6 chromatography steps. Following FPLC/Superose 6 size exclusion chromatography of PvP1 and PvP2, single peaks of phosvitin phosphatase activities were eluted in the range of 160-220 kDa with acidic pH optima. When FPLC/Sepharose 6 chromatography was performed in the presence of 0.5 M NaCl and 0.1% Triton X-100, low molecular mass protein phosphatase forms were produced in addition to the high-M, activity peak, ranging from 25 to 35 kDa (PvP1) and from 15 to 25 kDa (PvP2). Under these conditions, both high- and low-M, forms of PvP1 and PvP2 exhibited neutral pH optima. Both phosphatases dephosphorylate also (i) phosphorylase a, (ii) the alpha and beta subunits of phosphorylase kinase, and (iii) the microtubule-associated protein tau, phosphorylated by cAMP-dependent protein kinase. The present results suggest that two forms of protein phosphatases, displayed molecular and biochemical characteristics both similar and distinct from type 1 and type 2A protein phosphatases, are present in rat brain.
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PMID:Partial purification and characterization of two phosvitin phosphatases from rat brain. 862 49


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