Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.27 (AMPK)
 6,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide ('I'-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.
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PMID:Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action. 197 70

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [gamma 32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3. In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 microM and 1.9 microM respectively. AMP increases the Vmax 3-5-fold without a significant change in the Km for either protein or ATP substrates. 4. The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 microM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.
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PMID:Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities. 259 24

Acetyl-CoA carboxylase has been purified from lactating rat mammary gland using a combination of ammonium sulphate and poly(ethyleneglycol) precipitations. The enzyme was purified from 35--70-fold with a yield of over 50%, the exact figures being difficult to estimate because of activation of the enzyme that occurs during the preparation. The preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis in sodium dodecyl sulphate and had a single subunit of molecular weight 240,000, containing 1.02 +/- 0.04 molecules of biotin and 3.1 +/- 1.7 molecules of alkali-labile phosphate per subunit. The purified enzyme was phosphorylated and inactivated rapidly when incubated in the presence of [gamma 32P]ATP and magnesium ions with the purified catalytic subunit of cyclic-AMP-dependent protein kinase from rabbit skeletal muscle. Both phosphorylation and inactivation are blocked by the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinase, and can be reversed by incubation with purified protein phosphatase-1 from rabbit skeletal muscle. The inactivation by the protein kinase and reactivation by the protein phosphatase correlate with the near-stoichiometric phosphorylation and dephosphorylation of site(s) located in a single tryptic peptide. Phosphorylation does not affect the Km for substrates, but brings about a twofold decrease in V and a twofold increase in the apparent dissociation constant for the allosteric activator, citrate. We also present evidence that the activation of rabbit mammary acetyl-CoA carboxylase by protein phosphatase-1 described previously [Hardie and Cohen (1979) FEBS Lett. 103, 333-338] is due to dephosphorylation at site(s) which are not phosphorylated by either cyclic-AMP-dependent protein kinase or acetyl-CoA carboxylase kinase-2. These results suggest that the rapid inactivation of acetyl-CoA carboxylase, and hence fatty acid synthesis, by adrenaline in adipose tissue, or glucagon in the liver, is due to phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase.
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PMID:Reversible phosphorylation and inactivation of acetyl-CoA carboxylase from lactating rat mammary gland by cyclic AMP-dependent protein kinase. 610 9

The catalytic subunit of cyclic AMP-dependent protein kinase stimulates the inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase. The stimulated inactivation of carboxylase is due to activation of carboxylase kinase by the catalytic subunit. Activation of carboxylase kinase activity is accompanied by the incorporation of 0.6 mol of phosphate per mole of carboxylase kinase. Addition of the regulatory subunit of cyclic AMP-dependent protein kinase prevents the activation of carboxylase kinase. Phosphorylation and activation of carboxylase kinase has no effect on the Km for ATP, but decreases the Km for acetyl-CoA carboxylase from 93 to 45 nM. Inactivation of carboxylase by the carboxylase kinase requires the presence of coenzyme A even when the activated carboxylase kinase is used. Acetyl-CoA carboxylase is not phosphorylated or inactivated by the catalytic subunit of cyclic AMP-dependent protein kinase.
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PMID:Phosphorylation and activation of acetyl-coenzyme A Carboxylase kinase by the catalytic subunit of cyclic AMP-dependent protein kinase. 631 99

Phosphorylation and inactivation of acetyl-coenzyme A (CoA) carboxylase by acetyl-CoA carboxylase kinase in the presence of ATP and Mg2+ requires coenzyme A. Coenzyme A did not enhance the phosphorylation of alternative substrates of the carboxylase kinase such as protamine or histones. Analogs of coenzyme A were also effective in stimulating the inactivation of carboxylase. The KA of CoA for stimulated carboxylase inactivation was 25 microM. The presence of coenzyme A did not alter the Km of the carboxylase kinase for its substrates, ATP and acetyl-CoA carboxylase. Fluorescence binding studies showed that CoA binds to carboxylase but not to the kinase. The KD of CoA binding to carboxylase is 27 microM. These results indicate that coenzyme A, acting on acetyl-CoA carboxylase, may play an important role in the regulation of the covalent modification mechanism for acetyl-CoA carboxylase.
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PMID:Requirement of acetyl-coenzyme A carboxylase kinase for coenzyme A. 662 19

To examine the role of AMP-activated protein kinase (AMPK; EC 2.7.1. 109) in the regulation of autophagy, rat hepatocytes were incubated with the AMPK proactivators, adenosine, 5-amino-4-imidazole carboxamide riboside (AICAR), or N6-mercaptopurine riboside. Autophagic activity was inhibited by all three nucleosides, AICAR and N6-mercaptopurine riboside being more potent (IC50 = 0.3 mM) than adenosine (IC50 = 1 mM). 2'-Deoxycoformycin, an adenosine deaminase (EC 3.5.4.4) inhibitor, increased the potency of adenosine 5-fold, suggesting that the effectiveness of adenosine as an autophagy inhibitor was curtailed by its intracellular deamination. 5-Iodotubercidin, an adenosine kinase (EC 2.7.1.20) inhibitor, abolished the effects of all three nucleosides, indicating that they needed to be phosphorylated to inhibit autophagy. A 5-iodotubercidin-suppressible phosphorylation of AICAR to 5-aminoimidazole-4-carboxamide riboside monophosphate was confirmed by chromatographic analysis. AICAR, up to 0.4 mM, had no significant effect on intracellular ATP concentrations. Because activated AMPK phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.88), the rate-limiting enzyme in cholesterol synthesis, the strong inhibition of hepatocytic cholesterol synthesis by all three nucleosides confirmed their ability to activate AMPK under the conditions used. Lovastatin and simvastatin, inhibitors of HMG-CoA reductase, strongly suppressed cholesterol synthesis while having no effect on autophagic activity, suggesting that AMPK inhibits autophagy independently of its effects on HMG-CoA reductase and cholesterol metabolism.
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PMID:Inhibition of hepatocytic autophagy by adenosine, aminoimidazole-4-carboxamide riboside, and N6-mercaptopurine riboside. Evidence for involvement of amp-activated protein kinase. 972 84

The AMP-activated protein kinase cascade is a sensor of cellular energy charge, and its existence provides strong support for the energy charge hypothesis first proposed by Daniel Atkinson in the 1960s. The system is activated in an ultrasensitive manner by cellular stresses that deplete ATP (and consequently elevate AMP), either by inhibiting ATP production (e.g., hypoxia), or by accelerating ATP consumption (e.g., exercise in muscle). Once activated, it switches on catabolic pathways, both acutely by phosphorylation of metabolic enzymes and chronically by effects on gene expression, and switches off many ATP-consuming processes. Recent work suggests that activation of AMPK is responsible for many of the effects of physical exercise, both the rapid metabolic effects and the adaptations that occur during training. Dominant mutations in regulatory subunit isoforms (gamma2 and gamma3) of AMPK, which appear to increase the basal activity in the absence of AMP, lead to hypertrophy of cardiac and skeletal muscle respectively.
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PMID:AMP-activated protein kinase: the energy charge hypothesis revisited. 1174 30

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.
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PMID:Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. 1219 24

StubGAL83 is a potato gene that encodes the beta-subunit of a protein kinase complex similar to the yeast SNF1, and the mammalian AMPK complexes that are modulated by changes in the cellular AMP/ATP ratio and are important regulators of metabolic and stress responses. Here we show that the expression of StubGAL83 in potato foliage is much higher in the dark than in the light and can be repressed by metabolisable sugars in the dark. The amounts of StubGAL83 mRNA are higher in sink than in source leaves. To unravel the role of StubGAL83, transgenic potato plants expressing a part of the StubGAL83 cDNA in antisense orientation under the control of the constitutive CaMV35S promoter were generated. Northern analysis revealed a reduction up to 90-95% in StubGAL83 mRNA accumulation in leaves of seven lines. Five out of these seven lines exhibited a reduction of StubGAL83 mRNA levels also in root and tuber tissues. Independent on the type of repression, the transgenic lines showed a delay in rooting and an increased sensitivity to salt stress. The roots were stunted and possessed less pronounced tap roots than the controls albeit with different severity in the different transgenic lines. The root cells were smaller and some of them had irregular shape. Tuberisation of the antisense-StubGAL83 lines was delayed, the size of the tubers was reduced while the number of tubers per plant was increased. These results together suggest that StubGAL83 affects root and tuber development probably by altering the metabolic status of the leaves.
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PMID:Antisense repression of StubGAL83 affects root and tuber development in potato. 1294 48

Several links relate mitochondrial metabolism and type 2 diabetes or chronic hyperglycaemia. Among them, ATP synthesis by oxidative phosphorylation and cellular energy metabolism (ATP/ADP ratio), redox status and reactive oxygen species (ROS) production, membrane potential and substrate transport across the mitochondrial membrane are involved at various steps of the very complex network of glucose metabolism. Recently, the following findings (1) mitochondrial ROS production is central in the signalling pathway of harmful effects of hyperglycaemia, (2) AMPK activation is a major regulator of both glucose and lipid metabolism connected with cellular energy status, (3) hyperglycaemia by inhibiting glucose-6-phosphate dehydrogenase (G6PDH) by a cAMP mechanism plays a crucial role in NADPH/NADP ratio and thus in the pro-oxidant/anti-oxidant cellular status, have deeply changed our view of diabetes and related complications. It has been reported that metformin has many different cellular effects according to the experimental models and/or conditions. However, recent important findings may explain its unique efficacy in the treatment of hyperglycaemia- or insulin-resistance related complications. Metformin is a mild inhibitor of respiratory chain complex 1; it activates AMPK in several models, apparently independently of changes in the AMP-to-ATP ratio; it activates G6PDH in a model of high-fat related insulin resistance; and it has antioxidant properties by a mechanism (s), which is (are) not completely elucidated as yet. Although it is clear that metformin has non-mitochondrial effects, since it affects erythrocyte metabolism, the mitochondrial effects of metformin are probably crucial in explaining the various properties of this drug.
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PMID:Mitochondrial metabolism and type-2 diabetes: a specific target of metformin. 1450 5


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