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:2.7.11.27 (AMPK)
6,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

We have examined the sites phosphorylated on acetyl-CoA carboxylase by three protein kinases which have been shown to inactivate the enzyme, i.e. cyclic-AMP-dependent protein kinase, acetyl-CoA carboxylase kinase-2 (ACK2, purified from rat mammary gland) and the AMP-activated protein kinase (formerly called acetyl-CoA carboxylase kinase-3, purified from rat liver). Each protein kinase phosphorylates two out of three sites (termed 1-3) which have been established by amino acid sequencing. The two sites phosphorylated by each kinase can be recovered on separate peptides, TC1 and TC2, derived by combined digestion of the native enzyme by trypsin and chymotrypsin: TC1 = Ser-2Ser(P)-Met-3Ser(P)-Gly-Leu; TC2 = Arg-Met-1Ser(P)-Phe- Cyclic-AMP-dependent protein kinase phosphorylates sites 1 and 2 exclusively, whereas the AMP-activated protein kinase phosphorylates sites 1 and 3, plus at least one other minor site. ACK2 phosphorylates site 1 and, more slowly, an unidentified site(s) within TC1. We have also established the structures of the single major phosphopeptides (T1 and C1 respectively) which are recovered by HPLC after acetyl-CoA carboxylase phosphorylated by cyclic-AMP-dependent protein kinase is digested with trypsin or chymotrypsin alone. T1 is related to TC1, and has the structure: Ser-Ser(P)-Met-Ser-Gly-Leu-His-Leu-Val-Lys. C1 is identical with TC2. We have carried out studies on the correlation of the activity of acetyl-CoA carboxylase with the occupancy of sites 1, 2 and 3 during phosphorylation by each of the three protein kinases. The results suggest that phosphorylation of site 3 is primarily responsible for the large decrease in Vmax produced by the AMP-activated protein kinase, while phosphorylation of site 1 may be primarily responsible for the increase in A0.5 for citrate and more modest depression of Vmax produced by cyclic-AMP-dependent protein kinase and ACK2. Our results emphasize that amino acid sequence information is essential in the unequivocal interpretation of data from phosphopeptide mapping experiments and allow a more complete interpretation of previous data on phosphorylation of acetyl-CoA carboxylase in intact cells. They also open the way to experiments which could establish the physiological roles of these protein kinases in the control of fatty acid synthesis.
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PMID:Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase. 290 Jan 38

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

Here we report that the beta-adrenergic agonist isoproterenol increases the activity of the stress-activated kinase p38 MAPK over 10-fold in freshly isolated rat epididymal fat cells. Stimulation of the kinase was rapid, sustained for at least 60 min and sensitive to the specific p38 MAPK inhibitor, SB 203580. Half-maximal stimulation of p38 MAPK by isoproterenol occurred at 13 nM isoproterenol. The cell permeable cyclic AMP analogue, chlorophenylthio-cyclic AMP increased p38 MAPK activity to a similar extent to isoproterenol, suggesting that the effect of the beta-adrenergic agonist is mediated via increases in the activity of cyclic-AMP dependent protein kinase. Although it had little or no effect on the activity of c-Jun N-terminal kinase, isoproterenol and a number of other treatments which activated p38 MAPK were found to stimulate AMP-activated protein kinase in fat cells. Activation of AMPK and p38 MAPK were not, however, found to be directly linked.
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PMID:The activation of p38 MAPK by the beta-adrenergic agonist isoproterenol in rat epididymal fat cells. 984 39

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

The insulin resistance syndrome is characterized by several risk factors for cardiovascular disease. Chronic chemical activation of AMP-activated protein kinase by the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside (AICAR) has been shown to augment insulin action, upregulate mitochondrial enzymes in skeletal muscles, and decrease the content of intra-abdominal fat. Furthermore, acute AICAR exposure has been found to reduce sterol and fatty acid synthesis in rat hepatocytes incubated in vitro as well as suppress endogenous glucose production in rats under euglycemic clamp conditions. To investigate whether chronic AICAR administration, in addition to the beneficial effects on insulin sensitivity, is capable of improving other phenotypes associated with the insulin resistance syndrome, obese Zucker (fa/fa) rats (n = 6) exhibiting insulin resistance, hyperlipidemia, and hypertension were subcutaneously injected with AICAR (0.5 mg/g body wt) daily for 7 weeks. Obese control rats were either pair-fed (PF) (n = 6) or ad libitum-fed (AL) (n = 6). Lean Zucker rats (fa/-) (n = 8) served as a reference group. AICAR administration significantly reduced plasma triglyceride levels (P < 0.01 for AICAR vs. AL, and P = 0.05 for AICAR vs. PF) and free fatty acids (P < 0.01 for AICAR vs. AL, and P < 0.05 for AICAR vs. PF) and increased HDL cholesterol levels (P < 0.01 for AICAR vs. AL and PF). AICAR treatment also lowered systolic blood pressure by 14.6 +/- 4.3 mmHg (P < 0.05), and AICAR-treated animals exhibited a tendency toward decreased intra-abdominal fat content. Furthermore, AICAR administration normalized the oral glucose tolerance test and decreased fasting concentrations of glucose and insulin close to the level of the lean animals. Finally, in line with previous findings, AICAR treatment was also found to enhance GLUT4 protein expression and to increase maximally insulin-stimulated glucose transport in primarily white fast-twitch muscles. Our data provide strong evidence that long-term administration of AICAR improves glucose tolerance, improves the lipid profile, and reduces systolic blood pressure in an insulin-resistant animal model. The present study gives additional support to the hypothesis that AMPK activation might be a potential future pharmacological strategy for treating the insulin resistance syndrome.
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PMID:Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome. 1208 50

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

AMPK (5'-AMP-activated protein kinase) is emerging as a metabolic master switch, by which cells in both mammals and lower organisms sense and decode changes in energy status. Changes in AMPK activity have been shown to regulate glucose transport in muscle and glucose production by the liver. Moreover, AMPK appears to be a key regulator of at least one transcription factor linked to a monogenic form of diabetes mellitus. As a result, considerable efforts are now under way to explore the usefulness of AMPK as a therapeutic target for other forms of this disease. Here we review this topic, and discuss new findings which suggest that AMPK may play roles in regulating insulin release and the survival of pancreatic islet beta-cells, and nutrient sensing by the brain.
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PMID:Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. 1283 90

The AMPK (AMP-activated protein kinase) cascade plays a key role in regulating energy metabolism. Conditions which cause a decrease in the ATP/AMP ratio lead to activation of AMPK. Once activated, AMPK initiates a series of responses that act to restore the energy balance of the cell. In skeletal muscle, activation of AMPK increases both glucose uptake and fatty acid oxidation, raising the possibility that AMPK can bypass the glucose/fatty acid cycle. This review focuses on the role of AMPK in the regulation of glucose and fatty acid metabolism in muscle. Recently, naturally occurring mutations within the gamma isoforms have been identified which lead to altered metabolic regulation in cardiac and skeletal muscle and suggest an important role for the kinase in regulating glycogen metabolism.
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PMID:Bypassing the glucose/fatty acid cycle: AMP-activated protein kinase. 1464 Oct 16

The AMPK (5'AMP-activated protein kinase) is becoming recognized as a critical regulator of energy metabolism. However, many of these effects in muscle metabolism have been ascribed to AMPK based on the use of the unspecific activator AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside). Using mouse models in which AMPK activity has been specifically blocked (kinase dead) or knocked out we and others have been able to conduct studies gaining more conclusive data on the role of AMPK in muscle metabolism. In this mini-review focus is on AMPK and its regulatory role for glucose transport and GS (glycogen synthase) activity in skeletal muscle, indicating that AMPK is a GS kinase in vivo which might influence GS activity during exercise and that AMPK is involved in AICAR/hypoxia-induced glucose transport but not or only partially in contraction-stimulated glucose transport.
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PMID:Transgenic models--a scientific tool to understand exercise-induced metabolism: the regulatory role of AMPK (5'-AMP-activated protein kinase) in glucose transport and glycogen synthase activity in skeletal muscle. 1464 Oct 45


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