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

Attenuation of Syrian hamster 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by in vitro phosphorylation was studied using AMP-activated protein kinase and wild-type and mutant forms of HMG-CoA reductase. The only residue of the wild-type enzyme phosphorylated was Ser871. Substrates protected against kinase-mediated attenuation of activity, consistent with substrate-induced conformational changes at the C-terminal region. Although close to the catalytic histidine His865, Ser871 appears to play no direct role in catalysis or substrate recognition. Mutant enzymes S871A, S871H, S871N, and S871Q exhibited from 62-106% of wild-type activity and had wild-type Km values for HMG-CoA and NADPH. Replacement of Ser871 by aspartate or glutamate, but not by glutamine, asparagine, histidine, or tyrosine, severely attenuated activity. Attenuation of catalytic activity that accompanies phosphorylation thus appears to result primarily from the introduction of negative charge, not merely steric hindrance. Other than the wild-type enzyme, only mutant enzyme S871T was phosphorylated, and phosphorylation was accompanied by attenuation of activity. The AMP-activated kinase thus can also phosphorylate threonyl residues.
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PMID:Modulation of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase activity by phosphorylation. Role of serine 871. 812 43

We have expressed a truncated form of the alpha1 kinase domain of AMP-activated protein kinase (AMPK) in Escherichia coli as a glutathione-S-transferase fusion (GST-KD). A T172D mutant version did not require prior phosphorylation and was utilized for most subsequent studies. We have also created a recombinant substrate (GST-ACC) by expressing 34 residues around the major phosphorylation site (Ser79) on rat acetyl-CoA carboxylase-1/alpha (ACC1) as a GST fusion. This was an excellent substrate that was phosphorylated with similar kinetic parameters to ACC1 by both native AMPK and the bacterially expressed kinase domain. We also constructed a structural model for the binding of the ACC1 sequence to the kinase domain, based on crystal structures for related protein kinases. The model was tested by making a total of 25 mutants of GST-ACC and seven mutants of GST-KD, and measuring kinetic parameters with different combinations. The results reveal that AMPK and ACC1 interact over a much wider region than previously realized (>20 residues). The features of the interaction can be summarised as follows: (i) an amphipathic helix from P-16 to P-5 on the substrate binds in a hydrophobic groove on the large lobe of the kinase; (ii) basic residues at P-6 and P-4 bind to two acidic patches (D215/D216/D217 and E103/D100/E143, respectively), on the large lobe; (iii) a histidine at P+3 interacts with D56 on the small lobe; (iv) the side-chain of P+4 leucine could not be precisely positioned, but a new finding was that asparagine or glutamine could replace a hydrophobic residue at this position. These interactions position the serine residue to be phosphorylated in close proximity to the gamma-phosphate group of ATP. Although based on modelling rather than a determined structure, this represents one of the most detailed studies of the interaction between a kinase and its substrate achieved to date.
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PMID:Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate. 1190 45

Certain amino acids, like glutamine and leucine, induce an anabolic response in liver. They activate p70 ribosomal protein S6 kinase (p70S6K) and acetyl-CoA carboxylase (ACC) involved in protein and fatty acids synthesis, respectively. In contrast, the AMP-activated protein kinase (AMPK), which senses the energy state of the cell and becomes activated under metabolic stress, inactivates by phosphorylation key enzymes in biosynthetic pathways thereby conserving ATP. In this paper, we studied the effect of AMPK activation and of protein phosphatase inhibitors, on the amino-acid-induced activation of p70S6K and ACC in hepatocytes in suspension. AMPK was activated under anoxic conditions or by incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAr) or oligomycin, an inhibitor of mitochondrial oxidative phosphorylation. Incubation of hepatocytes with amino acids activated p70S6K via multiple phosphorylation. It also activated ACC by a phosphatase-dependent mechanism but did not modify AMPK activation. Conversely, the amino-acid-induced activation of both ACC and p70S6K was blocked or reversed when AMPK was activated. This AMPK activation increased Ser79 phosphorylation in ACC but decreased Thr389 phosphorylation in p70S6K. Protein phosphatase inhibitors prevented p70S6K activation when added prior to the incubation with amino acids, whereas they enhanced p70S6K activation when added after the preincubation with amino acids. It is concluded that (a) AMPK blocks amino-acid-induced activation of ACC and p70S6K, directly by phosphorylating Ser79 in ACC, and indirectly by inhibiting p70S6K phosphorylation, and (b) both activation and inhibition of protein phosphatases are involved in the activation of p70S6K by amino acids. p70S6K adds to an increasing list of targets of AMPK in agreement with the inhibition of energy-consuming biosynthetic pathways.
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PMID:Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes. 1215 72

Prolonged culture in low-glucose concentrations (</=5mM) induces apoptosis in pancreatic beta-cells by a poorly defined mechanism. We now show that, in both purified rat beta-cells and isolated rat islets, culture in the presence of 3 or 5mM (G3-G5) instead of 10mM glucose (G10) induces a large increase in c-myc expression before onset of a caspase-dependent apoptosis. These effects were prevented by addition of leucine and glutamine to G3 and G5, and were mimicked by addition of the mitochondrial poison azide to G10. In contrast, inhibition of Ca(2+) influx and insulin secretion with diazoxide under control conditions did not stimulate islet c-myc expression nor beta-cell apoptosis. In rat beta-cells, adenovirus-mediated c-myc overexpression increased their rate of apoptosis, whereas antisense-c-myc expression reduced low-glucose-induced apoptosis by approximately 50%. In the insulin producing MIN6 cell line, apoptosis induction by either low glucose or an activator of AMP-activated protein kinase (AMPK) was associated with c-myc mRNA and protein upregulation. In conclusion, stimulation of beta-cell apoptosis by prolonged culture at low glucose partly results from early and sustained induction of beta-cell c-myc expression. These effects may be due to sustained restriction in nutrient-derived metabolic signals.
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PMID:Prolonged culture in low glucose induces apoptosis of rat pancreatic beta-cells through induction of c-myc. 1465 61

SNARK is a member of the AMPK subfamily of serine/threonine protein kinases. In this study, we examined the regulation of SNARK activity in kidney (BHK, HEK293), pancreatic beta-cell insulinoma (INS-1), hepatocarcinoma (H4IIE) and keratinocyte (NRKC)-derived cell lines in response to diverse cellular stresses. We show that SNARK activity is regulated by glucose- or glutamine-deprivation, induction of endoplasmic reticulum stress by homocysteine or DTT, elevation of cellular AMP and/or depletion of ATP, hyperosmotic stress, salt stress, ultraviolet B radiation and oxidative stress caused by hydrogen peroxide. Moreover, the regulation of SNARK activity in response to cellular stresses depends greatly upon cell type. Furthermore, SNARK activity is downregulated by metformin in a dose- and time-dependent manner in H4IIE cells. These observations support a role for SNARK as a molecular component of the cellular stress response.
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PMID:Regulation of SNARK activity in response to cellular stresses. 1589 79

Troglitazone (Tro) and pioglitazone (Pio) activation of peroxisome proliferator-activated receptor (PPAR)-gamma and PPAR-gamma-independent pathways was studied in cell lines derived from porcine renal tubules. PPAR-gamma-dependent activation of PPAR response element-driven luciferase gene expression was observed with Pio at 1 microM but not Tro at 1 microM. On the other hand, PPAR-gamma-independent P-ERK activation was observed with 5 microM Tro but not with Pio (5-20 microM). In addition, Pio (1-10 microM) increased metabolic acid production and activated AMP-activated protein kinase (AMPK) associated with decreased mitochondrial membrane potential, whereas Tro (1-20 microM) did not. These results are consistent with three pathways through which glitazones may act in effecting metabolic processes (ammoniagenesis and gluconeogenesis) as well as cellular growth: 1) PPAR-gamma-dependent and PPAR-gamma-independent pathways, 2) P-ERK activation, and 3) mitochondrial AMPK activation. The pathways influence cellular acidosis and glucose and glutamine metabolism in a manner favoring reduced plasma glucose in vivo. In addition, significant interactions can be demonstrated that enhance some physiological processes (ammoniagenesis) and suppress others (ligand-mediated PPAR-gamma gene expression). Our findings provide a model both for understanding seemingly opposite biological effects and for enhancing therapeutic potency of these agents.
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PMID:Troglitazone and pioglitazone interactions via PPAR-gamma-independent and -dependent pathways in regulating physiological responses in renal tubule-derived cell lines. 1706 4

The AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic beta-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of beta-cell function. Here, we show that inhibition of AMPK in beta-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the beta-cell.
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PMID:The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells. 1728 12

Antiobesity drugs that target peripheral metabolism may avoid some of the problems that have been encountered with centrally acting anorectic drugs. Moreover, if they cause weight loss by increasing fat oxidation, they not only address a cause of obesity but also should promote loss of fat rather than lean tissue and improve insulin sensitivity. Weight loss may be slow but more sustained than with anorectic drugs, and thermogenesis may be insufficient to cause any discomfort. Some thermogenic approaches are the activation of adrenergic, thyroid hormone or growth hormone receptors and the inhibition of glucocorticoid receptors; the modulation of transcription factors [e.g. peroxisome proliferator-activated receptor delta (PPARdelta) activators] or enzymes [e.g. glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors] that promote mitochondrial biogenesis, and the modulation of transcription factors (PPAR alpha activators) or enzymes (AMP-activated protein kinase) that promote fatty acid oxidation. More surprisingly, studies on genetically modified animals and with enzyme inhibitors suggest that inhibitors of fatty acid synthesis [e.g. ATP citrate lyase, fatty acid synthase, acetyl-CoA carboxylase (ACC)], fatty acid interconversion [stearoyl-CoA desaturase (SCD)] and triglyceride synthesis (e.g. acyl-CoA : diacylglycerol acyltransferase) may all be thermogenic. Some targets have been validated only by deleting genes in the whole animal. In these cases, it is possible that deletion of the protein in the brain is responsible for the effect on adiposity, and therefore a centrally penetrant drug would be required. Moreover, whilst a genetically modified mouse may display resistance to obesity in response to a high fat diet, it requires a tool compound to demonstrate that a drug might actually cause weight loss. Even then, it is possible that differences between rodents and humans, such as the greater thermogenic capacity of rodents, may give a misleading impression of the potential of a drug.
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PMID:Thermogenic and metabolic antiobesity drugs: rationale and opportunities. 1739 Nov 51

Sir David Cuthbertson was the first to define metabolic alterations in post-aggression syndrome (PAS). From basic measurements of nitrogen loss and total protein synthesis/degradation, the current research has moved to genomics, proteomics and metabolomics. In this respect, first evidence was accumulated for the influence of acute catabolism, immobilisation by bed rest and sarcopenia of old age on the muscle-cell genome and proteome. Moreover, in post-aggression syndrome specific amino acids such as glutamine, arginine, glycine, taurine, tryptophan and cysteine are used for cell and immune modulation. Our laboratory has focused on the regulative capacity of glutamine. Glutamine deficiency as found in post-aggression syndrome reduces lymphocyte proliferation, alters monocyte/macrophage activity, decreases the formation of heat-shock proteins, stimulates cell apoptosis, shifts the cellular redox potential by altering the glutathione synthesis and increases the activity of the AMPK system. Investigating the molecular effect of glutamine on Hsp 70 induction, we tested the glutamine dependence on the formation of transfer-RNA and of heat-shock factor 1 (HSF 1), and on transcription and translation of Hsp 70. We could demonstrate that glutamine stabilises the mRNA of Hsp 70 thereby prolonging its half-life. The lecture also discusses the principal molecular targets of administered arginine, glycine, cysteine, taurine and tryptophan.
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PMID:Immune and cell modulation by amino acids. 1759 24

Glutamine : fructose-6-phosphate amidotransferase 1 (GFAT1) was identified as a protein phosphorylated in glucose-deprived cells by immunoprecipitation using the anti-phospho Akt substrates (PAS) antibody, which recognizes the phosphorylation motif site by AMP-activated protein kinase (AMPK), followed by mass fingerprinting analysis. Glucose depletion-induced phosphorylation of endogenous GFAT was potentiated by 2-deoxyglucose (2-DG), an AMPK activator, and the 2-DG-stimulated phosphorylation of FLAG-tagged GFAT1 in transfected cells was suppressed by Compound C, an AMPK inhibitor. The 2-DG induced phosphorylation of GFAT1 was attenuated by the introduction of the kinase-negative mutant of AMPK, and the phosphorylation was observed in the cells expressing the constitutively active mutant of AMPK even in the absence of 2-DG. Subsequent analysis revealed that the PAS antibody recognized GFAT1 phosphorylated at Ser243, which is conserved among different species. The assay of the GFAT enzymatic activity in the cell lysates indicated that the 2-DG-treatment inhibited the enzymatic activity, and Compound C-preincubation partially prevented the 2-DG-induced decrease of the activity. Furthermore, the mutant replacing Ser243 by alanine partially prevented the decrease of GFAT activity by 2-DG treatment. These results indicate that the phosphorylation of GFAT1 at Ser243 by AMPK has an important role in the regulation of the GFAT1 enzymatic activity.
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PMID:AMP-activated protein kinase phosphorylates glutamine : fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity. 1917 Jul 65


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