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1
المؤلفون: Jason R.B. Dyck, D. Grahame Hardie, Amy J. Barr, Naomi Kudo, Gary D. Lopaschuk, Stephen P. Davies
المصدر: European Journal of Biochemistry. 262:184-190
مصطلحات موضوعية: Male, Immunoprecipitation, Protein subunit, Blotting, Western, AMP-Activated Protein Kinases, Protein Serine-Threonine Kinases, Biochemistry, 5'-AMP-Activated Protein Kinase, Rats, Sprague-Dawley, chemistry.chemical_compound, AMP-activated protein kinase, Multienzyme Complexes, Animals, Phosphorylation, Protein kinase A, biology, Myocardium, Acetyl-CoA carboxylase, AMPK, Cyclic AMP-Dependent Protein Kinases, Precipitin Tests, Rats, Isoenzymes, Malonyl-CoA, chemistry, biology.protein, Acetyl-CoA Carboxylase
الوصف: Acetyl-CoA carboxylase (ACC) is regarded in liver and adipose tissue to be the rate-limiting enzyme for fatty acid biosynthesis; however, in heart tissue it functions as a regulator of fatty acid oxidation. Because the control of fatty acid oxidation is important to the functioning myocardium, the regulation of ACC is a key issue. Two cardiac isoforms of ACC exist, with molecular masses of 265 kDa and 280 kDa (ACC265 and ACC280). In this study, these proteins were purified from rat heart and used in subsequent phosphorylation and immunoprecipitation experiments. Our results demonstrate that 5' AMP-activated protein kinase (AMPK) is able to phosphorylate both ACC265 and ACC280, resulting in an almost complete loss of ACC activity. Although cAMP-dependent protein kinase phosphorylated only ACC280, a dramatic loss of ACC activity was still observed, suggesting that ACC280 contributes most, if not all, of the total heart ACC activity. ACC280 and ACC265 copurified under all experimental conditions, and purification of heart ACC also resulted in the specific copurification of the alpha2 isoform of the catalytic subunit of AMPK. Although both catalytic subunits of AMPK were expressed in crude heart homogenates, our results suggest that alpha2, and not alpha1, is the dominant isoform of AMPK catalytic subunit regulating ACC in the heart. Immunoprecipitation studies demonstrated that specific antibodies for both ACC265 and ACC280 were able to coimmunoprecipitate the alternate isoform along with the alpha2 isoform of AMPK. Taken together, the immunoprecipitation and the purification studies suggest that the two isoforms of ACC in the heart exist in a heterodimeric structure, and that this structure is tightly associated with the alpha2 subunit of AMPK.
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2
المؤلفون: Simon A. Hawley, Julia M. Corton, John G. Gillespie, D. Grahame Hardie
المصدر: European Journal of Biochemistry. 229:558-565
مصطلحات موضوعية: Male, Lipolysis, AICA ribonucleotide, AMP-Activated Protein Kinases, Protein Serine-Threonine Kinases, Mitogen-activated protein kinase kinase, Biochemistry, 5'-AMP-Activated Protein Kinase, MAP2K7, chemistry.chemical_compound, AMP-activated protein kinase, Allosteric Regulation, Multienzyme Complexes, Adipocytes, Animals, ASK1, Phosphorylation, Rats, Wistar, Protein kinase A, MAP kinase kinase kinase, biology, Kinase, Chemistry, Fatty Acids, Cyclin-dependent kinase 2, AMPK, Ribonucleotides, Ribonucleoside, Aminoimidazole Carboxamide, Rats, Cell biology, Enzyme Activation, Sterols, Liver, biology.protein, Hydroxymethylglutaryl CoA Reductases, Cyclin-dependent kinase 9, Protein Kinases
الوصف: The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways, the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidaz-ole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade.
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3
المؤلفون: David Carling, Angela Woods, Simon A. Hawley, Stephen P. Davies, Timothy A.J. Haystead, D. Grahame Hardie
المصدر: European Journal of Biochemistry. 223:351-357
مصطلحات موضوعية: Glycerol, Protein subunit, Blotting, Western, Allosteric regulation, Centrifugation, AMP-Activated Protein Kinases, Protein Serine-Threonine Kinases, Biology, Biochemistry, Chromatography, Affinity, Sepharose, AMP-Activated Protein Kinase Kinases, Affinity chromatography, Multienzyme Complexes, Phosphoprotein Phosphatases, Amino Acid Sequence, Protein Phosphatase 2, Binding site, Protein kinase A, Polyacrylamide gel electrophoresis, Binding Sites, Kinase, Molecular biology, Adenosine Monophosphate, Molecular Weight, Electrophoresis, Polyacrylamide Gel, Protein Kinases
الوصف: The AMP-activated protein kinase has been purified by affinity chromatography on ATP-gamma-Sepharose. A proportion of the activity can be eluted using AMP, while the remainder is eluted using ATP. The AMP eluate contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was previously identified as the AMP-binding catalytic subunit [Carling, D., Clarke, P. R., Zammit, V. A. & Hardie, D. G. (1989) Eur. J. Biochem. 186, 129-136]. All three polypeptides exactly comigrate both on native gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4-5.8 nm) by gel filtration, and sedimentation coefficient (7.9-8.4 S) by glycerol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 +/- 10 kDa. Thus the native enzyme appears to be a heterotrimer with a p63/p38/p35 (1:1:1) structure. Despite the fact that the ATP eluate has a higher specific activity than the AMP eluate (3.5 +/- 0.2 vs 2.3 +/- 0.2 mumol.min-1.mg-1), it appears to be less pure, containing p63, p38 and p35 plus other polypeptides. Experiments examining the effects of protein phosphatase-2A and kinase kinase, and analysis by Western blotting with anti-p63 antibody, suggests that the AMP eluate is entirely in the low-activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high-activity phosphorylated form. As well as establishing the subunit structure of the AMP-activated protein kinase, these results suggest that the kinase can bind to ATP-gamma-Sepharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases the affinity for the latter site.
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4
المؤلفون: John G. Gillespie, Paul Clarke, B J Gibb, John Weekes, D G Hardie, Stephen P. Davies, R W Mackintosh
المصدر: European Journal of Biochemistry. 209:923-931
مصطلحات موضوعية: Molecular Sequence Data, AMP-Activated Protein Kinases, Protein Serine-Threonine Kinases, Biology, Mitogen-activated protein kinase kinase, Peptide Mapping, Biochemistry, Substrate Specificity, MAP2K7, Calmodulin, Multienzyme Complexes, Phosphoprotein Phosphatases, ASK1, Amino Acid Sequence, c-Raf, Phosphorylation, Protein kinase A, MAP kinase kinase kinase, Cyclin-dependent kinase 2, food and beverages, Plants, Adenosine Monophosphate, Enzyme Activation, biology.protein, Calcium, Cyclin-dependent kinase 9, Hydroxymethylglutaryl-CoA Reductase Inhibitors, Protein Kinases, Acetyl-CoA Carboxylase
الوصف: Protein phosphorylation is well established as a regulatory mechanism in higher plants, but only a handful of plant enzymes are known to be regulated in this manner, and relatively few plant protein kinases have been characterized. AMP-activated protein kinase regulates key enzymes of mammalian fatty acid, sterol and isoprenoid metabolism, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. We now show that there is an activity in higher plants which, by functional criteria, is a homologue of the AMP-activated protein kinase, although it is not regulated by AMP. The plant kinase inactivates mammalian HMG-CoA reductase and acetyl-CoA carboxylase, and peptide mapping suggests that it phosphorylates the same sites on these proteins as the mammalian kinase. However, with the target enzymes purified from plant sources, it inactivates HMG-CoA reductase but not acetyl-CoA carboxylase. The kinase is located in the soluble, and not the chloroplast, fraction of leaf cells, consistent with the idea that it regulates HMG-CoA reductase, and hence isoprenoid biosynthesis, in vivo. The plant kinase also appears to be part of a protein kinase cascade which has been highly conserved during evolution, since the kinase is inactivated and reactivated by mammalian protein phosphatases (2A or 2C) and mammalian kinase kinase, respectively. This contrasts with the situation for many other mammalian protein kinases involved in signal transduction, which appear to have no close homologue in higher plants. To our knowledge, this represents the first direct evidence for a protein kinase cascade in higher plants.
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