Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.31 (AMP-activated protein kinase)
 13,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.
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PMID:Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion. 167 49

Hormone sensitive lipase (HSL) is an enzyme of relatively broad specificity, having the ability to hydrolyze tri-, di- and mono-acylglycerols as well as cholesterol esters and small water-soluble substrates. This broad specificity allows HSL to perform a variety of functions in several tissues. A key feature of HSL is its ability to be activated via phosphorylation by cyclic AMP-dependent protein kinase. In addition it is phosphorylated at a second site by several kinases, notably AMP-activated protein kinase. Phosphorylation of this site apparently plays a role in rendering the enzyme hormone-insensitive, in that prior phosphorylation at site 2 prevents phosphorylation and activation at site 1 by cyclic AMP-dependent protein kinase. Investigation of the protein phosphatases responsible for dephosphorylation of these sites has indicated that phosphatase 2A plays a predominant role but also that protein phosphatase 2C is a significant phosphatase targeted against both phosphorylation sites. Evidence indicates that HSL has at least three functional domains which contain (a) the phosphorylation sites which control activity, (b) the active site responsible for the catalytic activity and (c) a lipid binding site responsible for anchoring the lipase at the water-lipid interface. Using limited proteolytic studies we have found that it is possible to cleave HSL into several fragments including a stable domain of M(r) approximately 17.6 kDa which contains the active site serine residue. Digestion under similar conditions also generates a stable domain of M(r) approximately 11.5 kDa containing both phosphorylation sites. Furthermore, under appropriate conditions it is possible to digest HSL and retain activity against water-soluble substrates but with the concomitant loss of activity against triacylglycerol, implying that a lipid binding domain is lost during this procedure. HSL is responsible for the neutral cholesterol esterase activity in macrophages and it may play a role in the accumulation of cholesterol esters which occur during the development of foam cells. HSL activity is reduced in macrophage foam cells, at least partly due to increased activity of a cytosolic HSL inhibitor protein. A finding unexplained for many years has been that, although lipolysis can be stimulated 50-100-fold in adipocytes by lipolytic hormones, HSL can apparently only be activated 2-3-fold via phosphorylation in vitro by cyclic AMP-dependent protein kinase. One possibility to explain this discrepancy is that an additional anchoring protein is missing from the in vitro system and indirect evidence is now accumulating for such a protein.
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PMID:The multifunctional role of hormone-sensitive lipase in lipid metabolism. 794 81

We recently reported the existence of a protein kinase cascade in higher plants, of which the central component is a 3-hydroxy-3-methylglutaryl(HMG-)-CoA reductase kinase functionally related to mammalian AMP-activated protein kinase [MacKintosh, R. W., Davies, S. P., Clarke P. R., Weekes, J., Gillespie, S. G., Gibb, B. J. & Hardie, D. G. (1992) Eur. J. Biochem. 209, 923-931]. We have now purified this protein kinase 9000-fold from cauliflower inflorescences. During the course of this work we noticed a second minor form (form B) which separated from the major form (A) on ion exchange and gel filtration. Both forms phosphorylate the catalytic fragment of mammalian HMG-CoA reductase. Both forms are markedly inactivated by incubation with the reactive ATP analogue p-fluorosulphonylbenzoyl adenosine (FSO2PhCOAdo), and also by mammalian protein phosphatase 2C, indicating that form B, like form A, is activated by phosphorylation. Form A has an apparent native molecular mass of 200 kDa by gel filtration and, after labelling with [14C]FSO2PhCOAdo, of 150 kDa by electrophoresis in non-denaturing gels. The catalytic subunit was identified as a polypeptide of 58 kDa after labelling with [14C]FSO2PhCOAdo. Form B has an apparent native molecular mass of 45 kDa by gel filtration, and was identified as a polypeptide of 45 kDa after labelling with [14C]FSO2PhCOAdo and [gamma-32P]ATP. Using a series of variants of the synthetic peptide substrate, the substrate specificities of the two forms are similar but not identical. Form B does not appear to be a proteolytic fragment of form A, and we therefore propose that it represents a closely related member of the same protein kinase sub-family.
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PMID:Biochemical characterization of two forms of 3-hydroxy-3-methylglutaryl-CoA reductase kinase from cauliflower (Brassica oleracia). 811 24

AMP-activated protein kinase, purified from rat liver as far as the diethylaminoethyl-Sepharose step, is inactivated by treatment with protein phosphatase 2C, and reactivated by an endogenous 'kinase kinase'. Further purification of AMP-activated protein kinase on Blue Sepharose removes the kinase kinase, but the system can be reconstituted by adding back the flow-through from the Blue-Sepharose column. The kinase kinase can be further purified by subjecting the flow-through from the Blue-Sepharose column to chromatography on a Mono-Q column. A single peak of kinase kinase activity is obtained. Using this fraction, and the most highly purified preparation of AMP-activated protein kinase, phosphorylation of the 63-kDa polypeptide, previously identified as the catalytic subunit of AMP-activated protein kinase, can be demonstrated. As previously shown in the partially purified system, phosphorylation of the 63-kDa polypeptide is markedly stimulated by AMP. The kinase and kinase kinase reactions exhibit similar dependence on AMP concentration. The structurally related AMP analogue, 8-aza-9-deazaadenosine-5'-monophosphate, mimics the effect of AMP on both allosteric activation and phosphorylation of the kinase, while adenosine (5')tetraphospho(5')adenosine antagonizes both effects. These results suggest that both the allosteric effect of AMP, and the promotion of phosphorylation and activation by the kinase kinase, are due to binding of AMP to a single site on the kinase.
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PMID:Activation of rat liver AMP-activated protein kinase by kinase kinase in a purified, reconstituted system. Effects of AMP and AMP analogues. 811 25

The activity of hepatic carnitine palmitoyltransferase I (CPT-I) may be modulated by interactions with cytoskeletal components [Velasco et al. (1998) J. Biol. Chem. 273, 21497-21504]. We have studied whether the AMP-activated protein kinase (AMPK) is involved in this process. AMPK stimulated CPT-I in permeabilized hepatocytes but not in isolated liver mitochondria. In addition, AMPK abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytoskeletal fraction. These two effects of AMPK were not evident when the kinase was inactivated by pretreatment with protein phosphatase 2C. Cytokeratins 8 and 18 were phosphorylated by AMPK in vitro and by incubation of intact hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside, a cell-permeable activator of AMPK. These results provide the first evidence that AMPK stimulates CPT-I by direct phosphorylation of cytoskeletal components.
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PMID:Evidence that the AMP-activated protein kinase stimulates rat liver carnitine palmitoyltransferase I by phosphorylating cytoskeletal components. 984 45

Rat hearts were perfused for 1 h with 5 mm glucose with or without palmitate or oleate at concentrations characteristic of the fasting state. The inclusion of fatty acids resulted in increased activities of the alpha-1 or the alpha-2 isoforms of AMP-activated protein kinase (AMPK), increased phosphorylation of acetyl-CoA carboxylase and a decrease in the tissue content of malonyl-CoA. Activation of AMPK was not accompanied by any changes in the tissue contents of ATP, ADP, AMP, phosphocreatine or creatine. Palmitate increased phosphorylation of Thr172 within AMPK alpha-subunits and the activation by palmitate of both AMPK isoforms was abolished by protein phosphatase 2C leading to the conclusion that exposure to fatty acid caused activation of an AMPK kinase or inhibition of an AMPK phosphatase. In vivo, 24 h of starvation also increased heart AMPK activity and Thr172 phosphorylation of AMPK alpha-subunits. Perfusion with insulin decreased both alpha-1 and alpha-2 AMPK activities and increased malonyl-CoA content. Palmitate prevented both of these effects. Perfusion with epinephrine decreased malonyl-CoA content without an effect on AMPK activity but prevented the activation of AMPK by palmitate. The concept is discussed that activation of AMPK by an unknown fatty acid-driven signalling process provides a mechanism for a 'feed-forward' activation of fatty acid oxidation.
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PMID:Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids. 1515 11

Elevated levels of tumor necrosis factor (TNFalpha) are implicated in the development of insulin resistance, but the mechanisms mediating these chronic effects are not completely understood. We demonstrate that TNFalpha signaling through TNF receptor (TNFR) 1 suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces ACC phosphorylation, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle, effects observed both in vitro and in vivo. Importantly even at pathologically elevated levels of TNFalpha observed in obesity, the suppressive effects of TNFalpha on AMPK signaling are reversed in mice null for both TNFR1 and 2 or following treatment with a TNFalpha neutralizing antibody. Our data demonstrate that AMPK is an important TNFalpha signaling target and is a contributing factor to the suppression of fatty-acid oxidation and the development of lipid-induced insulin resistance in obesity.
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PMID:Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling. 1714 30

Over the past two decades increases in obesity, due to high caloric intakes and immobilizing technologies, has led to a surge in type 2 diabetes. In obesity elevated circulating fatty acids set-off a pro-inflammatory cascade that increases the production of tumour necrosis factor-alpha(TNFalpha) from macrophages. Obesity is associated with blunted skeletal muscle fatty acid oxidation, accumulation of bioactive lipids and insulin resistance. The factors contributing to defects in fatty acid metabolism are not understood but new data demonstrates that increased TNFalpha in obesity increases protein phosphatase 2C (PP2C), which in turn suppresses the activity of AMP-activated protein kinase (AMPK), a critical regulator of energy metabolism.(1) These data identify a novel mechanism by which inflammation in obesity is a precursor to defects in skeletal muscle fatty acid oxidation that generates a vicious cycle exacerbating the development of insulin resistance.
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PMID:Inflammation in obesity is the common link between defects in fatty acid metabolism and insulin resistance. 1743 70

Brain-specific kinases 1 and 2 (BRSK1/2) are AMP-activated protein kinase (AMPK)-related kinases that are highly expressed in mammalian forebrain. Studies using transgenic animal models have implicated a role for these kinases in the establishment of neuronal polarity. BRSK1 and BRSK2 are activated by phosphorylation of a threonine residue in the T-loop activation segment of the kinase domain. In vitro studies have demonstrated that LKB1, an upstream kinase in the AMPK cascade, can catalyze this phosphorylation. However, to date, a detailed comparative analysis of the molecular regulation of BRSK1/2 has not been undertaken. Here we present evidence that excludes another upstream kinase in the AMPK cascade, Ca(2+)/calmodulin-dependent protein kinase kinase beta, from a role in activating BRSK1/2. We show that equivalent mutations in the ubiquitin-associated domains of the BRSK isoforms produce differential effects on the activation of BRSK1 and BRSK2. Contrary to previous reports, activation of cAMP-dependent protein kinase does not affect BRSK1 or BRSK2 activity in mammalian cells. Furthermore, stimuli that activate AMPK had no effect on BRSK1/2. Finally, we provide evidence suggesting that protein phosphatase 2C is a likely candidate for catalyzing the dephosphorylation and inactivation of BRSK1/2.
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PMID:Investigating the regulation of brain-specific kinases 1 and 2 by phosphorylation. 1833 22

Maternal obesity and over-nutrition give rise to both obstetric problems and neonatal morbidity. The objective of this study was to evaluate effects of maternal obesity and over-nutrition on signalling of the AMP-activated protein kinase (AMPK) pathway in fetal skeletal muscle in an obese pregnant sheep model. Non-pregnant ewes were assigned to a control group (Con, fed 100% of NRC nutrient recommendations, n = 7) or obesogenic group (OB, fed 150% of National Research Council (NRC) recommendations, n = 7) diet from 60 days before to 75 days after conception (term 150 days) when fetal semitendinosus skeletal muscle (St) was sampled. OB mothers developed severe obesity accompanied by higher maternal and fetal plasma glucose and insulin levels. In fetal St, activity of phosphoinositide-3 kinase (PI3K) associated with insulin receptor substrate-1 (IRS-1) was attenuated (P < 0.05), in agreement with the increased phophorylation of IRS-1 at serine 1011. Phosphorylation of AMP-activated protein kinase (AMPK) at Thr 172, acetyl-CoA carboxylase at Ser 79, tuberous sclerosis 2 at Thr 1462 and eukaryotic translation initiation factor 4E-binding protein 1 at Thr 37/46 were reduced in OB compared to Con fetal St. No difference in energy status (AMP/ATP ratio) was observed. The expression of protein phosphatase 2C was increased in OB compared to Con fetal St. Plasma tumour necrosis factor alpha (TNFalpha) was increased in OB fetuses indicating an increased inflammatory state. Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) was higher in OB St, indicating enhanced adipogenesis. The glutathione: glutathione disulphide ratio was also lower, showing increased oxidative stress in OB fetal St. In summary, we have demonstrated decreased signalling of the AMPK system in skeletal muscle of fetuses of OB mothers, which may play a role in altered muscle development and development of insulin resistance in the offspring.
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PMID:AMP-activated protein kinase signalling pathways are down regulated and skeletal muscle development impaired in fetuses of obese, over-nourished sheep. 1848 Mar 84


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