Mechanism of Action of A-769662, a Valuable Tool for Activation of AMP-activated Protein Kinase
2007; Elsevier BV; Volume: 282; Issue: 45 Linguagem: Inglês
10.1074/jbc.m706536200
ISSN1083-351X
AutoresOlga Göransson, Andrew McBride, Simon A. Hawley, Fiona A. Ross, Natalia Shpiro, Marc Foretz, Benoı̂t Viollet, D. Grahame Hardie, Kei Sakamoto,
Tópico(s)Glycogen Storage Diseases and Myoclonus
ResumoWe have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators, it directly activates native rat AMPK by mimicking both effects of AMP, i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated α subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the β subunit glycogen-binding domain. Although it mimics actions of AMP, it has no effect on binding of AMP to the isolated Bateman domains of the γ subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC), effects that are completely abolished in AMPK-α1–/–α2–/– cells but not in TAK1–/– mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1, a major upstream kinase for AMPK in this tissue. However, in HeLa cells, which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-β, phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells, the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK. We have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators, it directly activates native rat AMPK by mimicking both effects of AMP, i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated α subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the β subunit glycogen-binding domain. Although it mimics actions of AMP, it has no effect on binding of AMP to the isolated Bateman domains of the γ subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC), effects that are completely abolished in AMPK-α1–/–α2–/– cells but not in TAK1–/– mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1, a major upstream kinase for AMPK in this tissue. However, in HeLa cells, which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-β, phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells, the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK. The AMP-activated protein kinase (AMPK) 3The abbreviations used are: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide riboside; AID, autoinhibitory domain; CaMKK, calmodulin-dependent protein kinase kinase; CBS, cystathionineβ-synthase; GBD, glycogen-binding domain; GST, glutathione S-transferase; MEF, mouse embryo fibroblast; OCT1, organic cation transporter-1; TAK1, TGFβ-activated kinase-1; TGFβ, transforming growth factor-β; TBS, Tris-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; Bis-Tris, 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol. 3The abbreviations used are: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide riboside; AID, autoinhibitory domain; CaMKK, calmodulin-dependent protein kinase kinase; CBS, cystathionineβ-synthase; GBD, glycogen-binding domain; GST, glutathione S-transferase; MEF, mouse embryo fibroblast; OCT1, organic cation transporter-1; TAK1, TGFβ-activated kinase-1; TGFβ, transforming growth factor-β; TBS, Tris-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; Bis-Tris, 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol. is a regulator of energy balance at both the cellular and the whole body levels (1Carling D. Trends Biochem. Sci. 2004; 29: 18-24Abstract Full Text Full Text PDF PubMed Scopus (948) Google Scholar, 2Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2295) Google Scholar, 3Hardie D.G. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 185-210Crossref PubMed Scopus (352) Google Scholar). Once activated, it effects a metabolic switch from an anabolic to a catabolic state, both by acutely phosphorylating metabolic enzymes and, in the longer term, by regulating gene expression. AMPK is a heterotrimer composed of a catalytic α subunit and regulatory β and γ subunits. Binding of AMP to the two “Bateman domains” formed by four tandem CBS motifs on the γ subunit (4Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar) triggers increased phosphorylation at Thr-172 on the activation loop of the α subunit, causing >100-fold activation (5Suter M. Riek U. Tuerk R. Schlattner U. Wallimann T. Neumann D. J. Biol. Chem. 2006; 281: 32207-32216Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). AMP binding was previously thought both to promote phosphorylation (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar) and to inhibit dephosphorylation (7Davies S.P. Helps N.R. Cohen P.T.W. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (492) Google Scholar), although recent results suggest that the effect is exclusively on dephosphorylation (8Sanders M.J. Grondin P.O. Hegarty B.D. Snowden M.A. Carling D. Biochem. J. 2007; 403: 139-148Crossref PubMed Scopus (517) Google Scholar). AMP binding also causes a further allosteric activation of the phosphorylated kinase by up to 10-fold (5Suter M. Riek U. Tuerk R. Schlattner U. Wallimann T. Neumann D. J. Biol. Chem. 2006; 281: 32207-32216Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Phosphorylation of Thr-172 is in most cells catalyzed by the tumor suppressor kinase LKB1 (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar, 9Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1315) Google Scholar), which appears to be constitutively active (10Lizcano J.M. Göransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Mäkelä T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1042) Google Scholar, 11Sakamoto K. Goransson O. Hardie D.G. Alessi D.R. Am. J. Physiol. 2004; 287: E310-E317Crossref PubMed Scopus (27) Google Scholar). In some cells, Thr-172 can also be phosphorylated in a Ca2+-mediated process catalyzed by calmodulin-dependent protein kinase kinases such as CaMKKβ (12Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar, 13Woods A. Dickerson K. Heath R. Hong S.P. Momcilovic M. Johnstone S.R. Carlson M. Carling D. Cell Metab. 2005; 2: 21-33Abstract Full Text Full Text PDF PubMed Scopus (1053) Google Scholar, 14Hurley R.L. Anderson K.A. Franzone J.M. Kemp B.E. Means A.R. Witters L.A. J. Biol. Chem. 2005; 280: 29060-29066Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). The protein kinase TGFβ-activated kinase-1 (TAK1) can also activate the Saccharomyces cerevisiae homologue of AMPK (the SNF1 complex) when overexpressed in yeast, as well as phosphorylating Thr-172 on mammalian AMPK in cell-free assays (15Momcilovic M. Hong S.P. Carlson M. J. Biol. Chem. 2006; 281: 25336-25343Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). It remains unclear whether this has any physiological relevance in vivo.Most of the metabolic changes induced by AMPK activation, such as increased glucose uptake into muscle (16Merrill G.M. Kurth E. Hardie D.G. Winder W.W. Am. J. Physiol. 1997; 273: E1107-E1112Crossref PubMed Google Scholar), decreased gluconeogenesis in liver (17Lochhead P.A. Salt I.P. Walker K.S. Hardie D.G. Sutherland C. Diabetes. 2000; 49: 896-903Crossref PubMed Scopus (338) Google Scholar, 18Koo S.H. Flechner L. Qi L. Zhang X. Screaton R.A. Jeffries S. Hedrick S. Xu W. Boussouar F. Brindle P. Takemori H. Montminy M. Nature. 2005; 437: 1109-1111Crossref PubMed Scopus (783) Google Scholar), increased fatty acid oxidation in muscle and liver (16Merrill G.M. Kurth E. Hardie D.G. Winder W.W. Am. J. Physiol. 1997; 273: E1107-E1112Crossref PubMed Google Scholar, 19Velasco G. Geelen M.J.H. Guzman M. Arch. Biochem. Biophys. 1997; 337: 169-175Crossref PubMed Scopus (100) Google Scholar), deceased fatty acid synthesis in liver and adipose tissue (20Sullivan J.E. Brocklehurst K.J. Marley A.E. Carey F. Carling D. Beri R.K. FEBS Lett. 1994; 353: 33-36Crossref PubMed Scopus (410) Google Scholar, 21Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1018) Google Scholar), and increased mitochondrial biogenesis (22Zong H. Ren J.M. Young L.H. Pypaert M. Mu J. Birnbaum M.J. Shulman G.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15983-15987Crossref PubMed Scopus (816) Google Scholar), would be desirable outcomes for the treatment of type 2 diabetes and the metabolic syndrome. AMPK was therefore proposed (23Winder W.W. Hardie D.G. Am. J. Physiol. 1999; 277: E1-E10PubMed Google Scholar) to be a promising target for drugs aimed at treatment of these common conditions, which are essentially disorders of energy balance. The first drug shown to activate AMPK in intact cells was 5-aminoimidazole-4-carboxamide riboside (AICAR) (20Sullivan J.E. Brocklehurst K.J. Marley A.E. Carey F. Carling D. Beri R.K. FEBS Lett. 1994; 353: 33-36Crossref PubMed Scopus (410) Google Scholar, 21Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1018) Google Scholar, 24Henin N. Vincent M.F. Gruber H.E. Van den Berghe G. FASEB J. 1995; 9: 541-546Crossref PubMed Scopus (220) Google Scholar). When administered to rodent models of obesity and insulin resistance such as the ob/ob mouse, the fa/fa rat, and the high fat-fed rat, AICAR was shown to reverse many of their metabolic abnormalities (25Song X.M. Fiedler M. Galuska D. Ryder J.W. Fernström M. Chibalin A.V. Wallberg-Henriksson H. Zierath J.R. Diabetologia. 2002; 45: 56-65Crossref PubMed Scopus (171) Google Scholar, 26Bergeron R. Previs S.F. Cline G.W. Perret P. Russell R.R. II I Young L.H. Shulman G.I. Diabetes. 2001; 50: 1076-1082Crossref PubMed Scopus (252) Google Scholar, 27Iglesias M.A. Ye J.M. Frangioudakis G. Saha A.K. Tomas E. Ruderman N.B. Cooney G.J. Kraegen E.W. Diabetes. 2002; 51: 2886-2894Crossref PubMed Scopus (251) Google Scholar, 28Buhl E.S. Jessen N. Pold R. Ledet T. Flyvbjerg A. Pedersen S.B. Pedersen O. Schmitz O. Lund S. Diabetes. 2002; 51: 2199-2206Crossref PubMed Scopus (206) Google Scholar). At around the same time, the biguanide metformin was shown to activate AMPK in intact cells and in vivo (29Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Investig. 2001; 108: 1167-1174Crossref PubMed Scopus (4335) Google Scholar). Metformin is currently the drug of first choice for the treatment of type 2 diabetes, being prescribed to at least 120 million people worldwide. The therapeutic effects of the drug are primarily on the liver, probably because hepatocytes express the organic cation transporter OCT1, resulting in more rapid uptake of the drug into hepatocytes than other cells (30Wang D.S. Jonker J.W. Kato Y. Kusuhara H. Schinkel A.H. Sugiyama Y. J. Pharmacol. Exp. Ther. 2002; 302: 510-515Crossref PubMed Scopus (374) Google Scholar, 31Shu Y. Sheardown S.A. Brown C. Owen R.P. Zhang S. Castro R.A. Ianculescu A.G. Yue L. Lo J.C. Burchard E.G. Brett C.M. Giacomini K.M. J. Clin. Investig. 2007; 117: 1422-1431Crossref PubMed Scopus (738) Google Scholar). Recent studies involving mice in which AMPK could not be activated in liver due to a tissue-specific knock-out of the upstream kinase, LKB1, revealed that the anti-hyperglycemic effects of metformin were abolished, suggesting that the major effect of the drug is to repress gluconeogenesis via activation of liver AMPK (32Shaw R.J. Lamia K.A. Vasquez D. Koo S.H. Bardeesy N. Depinho R.A. Montminy M. Cantley L.C. Science. 2005; 310: 1642-1646Crossref PubMed Scopus (1538) Google Scholar). It is possible that an AMPK activator that was also effective in organs other than the liver would have additional efficacy.Although metformin is relatively safe, it is not effective in all patients, perhaps due to variability in the efficiency of hepatic uptake by the OCT1 transporter (31Shu Y. Sheardown S.A. Brown C. Owen R.P. Zhang S. Castro R.A. Ianculescu A.G. Yue L. Lo J.C. Burchard E.G. Brett C.M. Giacomini K.M. J. Clin. Investig. 2007; 117: 1422-1431Crossref PubMed Scopus (738) Google Scholar). It does not activate AMPK or affect the phosphorylation or dephosphorylation of the kinase by upstream kinases and phosphatases in cell-free assays (33Hawley S.A. Gadalla A.E. Olsen G.S. Hardie D.G. Diabetes. 2002; 51: 2420-2425Crossref PubMed Scopus (570) Google Scholar). However, metformin and the more potent biguanide, phenformin, have both been reported to be inhibitors of complex I of the respiratory chain (34Owen M.R. Doran E. Halestrap A.P. Biochem. J. 2000; 348: 607-614Crossref PubMed Scopus (1590) Google Scholar, 35El-Mir M.Y. Nogueira V. Fontaine E. Averet N. Rigoulet M. Leverve X. J. Biol. Chem. 2000; 275: 223-228Abstract Full Text Full Text PDF PubMed Scopus (1041) Google Scholar), which suggests that they may activate AMPK indirectly by decreasing cellular ATP and increasing AMP. Significant changes in the cellular AMP: ATP ratio are indeed readily detected after treatment of cultured cells with phenformin (12Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar), and decreases in ATP have recently been reported in primary rodent hepatocytes treated with metformin (36Guigas B. Bertrand L. Taleux N. Foretz M. Wiernsperger N. Vertommen D. Andreelli F. Viollet B. Hue L. Diabetes. 2006; 55: 865-874Crossref PubMed Scopus (158) Google Scholar). Intestinal epithelial cells also express transporters of the OCT1 family (Oct1–3 (37Koepsell H. Lips K. Volk C. Pharm. Res. 2007; PubMed Google Scholar)), and inhibition of the respiratory chain in the gut may be responsible for the unpleasant gastrointestinal side effects of biguanides, as well as the more dangerous side effect of lactic acidosis that led to the withdrawal of phenformin. There is evidence that much of the lactic acid produced in animals treated with biguanides is derived from the gut (38Bailey C.J. Wilcock C. Day C. Br. J. Pharmacol. 1992; 105: 1009-1013Crossref PubMed Scopus (103) Google Scholar).It therefore seems quite likely that a drug that activated the AMPK system more directly would be more efficacious in the treatment of type 2 diabetes and the metabolic syndrome than the biguanides, while avoiding some of their side effects, which may be related to inhibition of the respiratory chain rather than to activation of AMPK per se. The results of a screen of >700,000 compounds designed to detect AMPK activators were reported recently (39Cool B. Zinker B. Chiou W. Kifle L. Cao N. Perham M. Dickinson R. Adler A. Gagne G. Iyengar R. Zhao G. Marsh K. Kym P. Jung P. Camp H.S. Frevert E. Cell Metab. 2006; 3: 403-416Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). The thienopyridone A-592017 emerged from the initial screen, and after optimization, the more potent activator, A-769662, was developed. When administered to ob/ob mice, A-769662 had many of the effects expected for an AMPK activator, including decreases in plasma glucose and triglyceride, decreases in hepatic triglyceride, decreases in expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase and the lipogenic enzyme fatty acid synthase, and even decreases in weight gain (39Cool B. Zinker B. Chiou W. Kifle L. Cao N. Perham M. Dickinson R. Adler A. Gagne G. Iyengar R. Zhao G. Marsh K. Kym P. Jung P. Camp H.S. Frevert E. Cell Metab. 2006; 3: 403-416Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). Although the utility of the compound as a drug may be limited by its poor oral availability, it does hold considerable promise as an experimental tool for the study of the downstream consequences of AMPK activation. However, the original study provided little information as to the exact mechanism of activation of AMPK by A-769662. We have therefore synthesized A-769662 and have now addressed its mechanism of action in cell-free assays and in intact cells.EXPERIMENTAL PROCEDURESMaterials and Antibodies—A-769662 was synthesized as described previously (40Iyengar, R. R., Judd, A. S., Zhao, G., Kym, P. R., Sham, H. L., Gu, Y., Liu, G., Zhao, H., Clark, R. E., Frevert, E. U., Cool, B. L., Zhang, T., Keyes, R. F., Hansen, T. M., and Xin, Z. (February 17, 2005) U. S. Patent 0038068 A1Google Scholar). STO-609 was from Tocris (Ellisville, Missouri). Protein G-Sepharose and [γ-32P]ATP were from Amersham Biosciences (Little Chalfont, UK), Protease inhibitor mixture tablets from Roche Applied Science (Lewes, UK), precast SDS-polyacrylamide gels from Invitrogen (Paisley, UK), phosphocellulose P81 paper from Whatman, and ionomycin and phenformin were from Sigma (Poole, Dorset, UK). Anti pan-AMPKα (anti-AMPK, catalog number 2532), phospho-AMPK (anti-pT172, catalog number 2535), pan-ACC (anti-ACC, catalog number 3661), and phospho-ACC (anti-pACC, catalog number 3662) antibodies were from Cell Signaling Technology (New England Biolabs, UK). Anti-TAK1 was from Santa Cruz Biotechnology (catalog number sc-7162). Anti-GST antibodies were purified as a by-product of production of antibodies against GST·LKB1 (41Sapkota G.P. Boudeau J. Deak M. Kieloch A. Morrice N. Alessi D.R. Biochem. J. 2002; 362: 481-490Crossref PubMed Scopus (79) Google Scholar). Antibodies recognizing glutathione S-transferase (GST) were removed from the anti-GST·LKB1 antiserum using an immobilized GST column. GST fusions of the rat α1 kinase domain (1–312, wild type, and T172D mutant) (42Scott J.W. Norman D.G. Hawley S.A. Kontogiannis L. Hardie D.G. J. Mol. Biol. 2002; 317: 309-323Crossref PubMed Scopus (138) Google Scholar) and human CaMKKβ in bacteria (12Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1259) Google Scholar) were expressed and purified as described. Rat α1 kinase domain (residues 1–310) and rat α1 kinase domain plus the autoinhibitory domain (residues 1–333) were amplified by PCR from a plasmid expressing the full-length α1 subunit (sense oligonucleotide, 5′-acctcggaattcgcgagaagcagaagc-3′; 312 antisense oligonucleotide, 5′-tggtttctgctcgagaggcagctgagg-3′; 332 antisense oligonucleotide, 5′-ggctctcgagattattctcctgttgtc-3′) and inserted into the EcoRI/XhoI sites of the pGEX6P2 vector (GE Healthcare). Positive clones were confirmed by DNA sequencing. Both GST recombinant proteins were expressed and purified as described (4Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar). Recombinant protein phosphatase-2Cα was obtained as described (7Davies S.P. Helps N.R. Cohen P.T.W. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (492) Google Scholar).Animals—All animal studies were approved by the University of Dundee Ethics Committee and performed according to the UK Animals (Scientific Procedures) Act 1986. Muscle-specific LKB1-deficient mice were generated, bred, and genotyped as described previously (43Sakamoto K. McCarthy A. Smith D. Green K.A. Hardie D.G. Ashworth A. Alessi D.R. EMBO J. 2005; 24: 1810-1820Crossref PubMed Scopus (438) Google Scholar).Preparations of AMPK and Upstream Kinases and AMPK Assays—Rat liver AMPK (a mixture of the α1 and α2 isoforms with β1 and γ1 (44Woods A. Salt I. Scott J. Hardie D.G. Carling D. FEBS Lett. 1996; 397: 347-351Crossref PubMed Scopus (230) Google Scholar)) was purified as far as the gel filtration step (45Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. J. Biol. Chem. 1996; 271: 27879-27887Abstract Full Text Full Text PDF PubMed Scopus (995) Google Scholar), and rat testis LKB1 complex was purified as described for the rat liver complex (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. 2003; 2: 28Crossref PubMed Google Scholar). AMPK activity was assayed as described previously (46Hardie D.G. Salt I.P. Davies S.P. Methods Mol. Biol. 2000; 99: 63-75PubMed Google Scholar). Immunoprecipitate assays of AMPK in cell lysates were performed as described previously (11Sakamoto K. Goransson O. Hardie D.G. Alessi D.R. Am. J. Physiol. 2004; 287: E310-E317Crossref PubMed Scopus (27) Google Scholar).Preparations and Assays of Other Kinases—The panel of 76 protein kinases was prepared and assayed in the presence and absence of A-769662 as described previously (47Bain J. Cummings L. Elliot M. Shpiro N. Hastie J. McLaughlan H. Klevernic I. Arthur S. Alessi D. Cohen P. Biochem. J. 2007; (in press)Google Scholar).Creation of a GST·GBD Fusion and Assays of Glycogen Binding—The glycogen-binding domain (GBD) of the rat β1 subunit (residues 65–182) was amplified from a pcDNA3-rat β1 plasmid using primers incorporating SalI and EcoRI restriction sites (sense 5′-ctagaattcacgacctcgaggtgaatgag-3′, antisense 5′-ccaagactggacagctcagatacatcgg-3′). The SalI/EcoRI digested product was cloned into the pGEX6P2 vector (GE Healthcare), and positive clones were confirmed by DNA sequencing. The GST·GBD fusion was expressed in Escherichia coli (BL21, Invitrogen). Cells were cultured in LB ampicillin at 37 °C prior to induction of protein expression, at an A600 of 0.6, using 1 mm isopropyl-1-thio-β-d-galactopyranoside (Melford Laboratories). Cells were cultured for a further 4 h at 37°C prior to harvesting by centrifugation. The cell pellet was lysed by rapid freezing and subsequent grinding to a fine powder in liquid N2. The cell lysate was then resuspended in a minimal volume of sucrose buffer (0.27 mm sucrose, 50 mm Tris, pH 7.5, 1 mm sodium vanadate, 1 mm EDTA, 1 mm EGTA, 10 mm sodium-β-glycerophosphate, 50 mm NaF, 1 mm dithiothreitol, with one EDTA-free protease inhibitor mixture tablet (Roche Applied Science) per 50 ml). The lysate was clarified by centrifugation and applied to a 5-ml GSTrap FF column (GE Healthcare) pre-equilibrated with sucrose buffer. Nonspecifically bound proteins were removed by extensive washing in wash buffer (50 mm Tris, pH 7.5, 200 mm NaCl, 1 mm dithiothreitol) and protein eluted in 20 mm reduced glutathione. Fractions containing GST·GBD were identified by protein assay and SDS-PAGE analysis. GST·GBD was dialyzed overnight into wash buffer with two buffer changes. To make the glycogen-Sepharose column, CNBr-activated Sepharose 4 Fast Flow (GE Healthcare) was washed with 10 volumes of cold 1 mm HCl. Glycogen was then directly coupled by incubation with 1 volume of 50 mg/ml bovine liver (Type IX) glycogen (Sigma-Aldrich) in 10 mm KH2PO4, pH 8.0, overnight at 4 °C. Excess glycogen was removed by washing the beads with 5 volumes of 10 mm KH2PO4, pH 8.0. Unreacted sites on the Sepharose were blocked by incubation with 1 volume of 0.1 m Tris-HCl, pH 8.0, at room temperature for 2 h. The beads were then washed with 6 × 3 volumes of 0.1 m Tris-HCl, pH 9.0, 0.5 m NaCl, and 6 × 3 volumes of 0.1 m sodium acetate, pH 4.0, 0.5 m NaCl. The beads were finally resuspended and stored in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl. For the binding assay, 50 μl of beads were incubated with 2 μg of protein in a final volume of 150 μl of 50 mm Tris-HCl, pH 7.5, 150 mm NaCl at 4 °C for 1 h. The glycogen-Sepharose beads were then pelleted at 13,000 rpm for 30 s, and 10 μl of supernatant were retained for analysis. The glycogen-Sepharose beads were then washed with 500 μl of 50 mm Tris-HCl, pH 7.5, 150 mm NaCl prior to resuspension in the original volume of the same buffer. The bead suspension prior to incubation (10 μl), the supernatant after incubation, and the resuspended pellet were analyzed on 4–12% Bis-Tris gels in a MOPS buffer system (Invitrogen). Proteins were transferred to a nitrocellulose membrane, which was blocked for 1 h at room temperature in TBS (50 mm Tris-HCl, pH 7.5, 150 mm NaCl) + 5% nonfat milk powder. The membrane was washed in 4 × 10 ml of TBS. Anti-GST antibody (in 10 ml of TBS + 1% milk powder and 0.2% (v/v) Tween 20) was added and incubated for a further 1 h at room temperature. The membrane was washed 3 × 5 min with TBS + 0.2% v/v Tween 20. The membrane was then incubated for a further 1 h with sheep IgG conjugated to IR dye 680 (Molecular Probes). The membrane was finally washed 3 × 5 min with TBS + 0.2% v/v Tween 20 and 1 × 5 min in TBS. The membrane was scanned in the 680 channel of the Odyssey IR imager.Scintillation Proximity Assay for AMP Binding—Human γ2 (CBS motifs 1–4) was expressed as a GST fusion as described previously (4Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. J. Clin. Investig. 2004; 113: 274-284Crossref PubMed Scopus (599) Google Scholar). GST·γ2 was purified using a 5-ml GST FF column (GE Healthcare) followed by size exclusion chromatography as described previously (48Scott J.W. Ross F.A. Liu J.K. Hardie D.G. EMBO J. 2007; 26: 806-815Crossref PubMed Scopus (42) Google Scholar). The protein was incubated with glutathione-coupled scintillation proximity assay yttrium silicate beads (GE Healthcare), preblocked with 5% gelatin from cold water fish skin (Sigma). The beads were washed with 50 mm sodium Hepes, pH 7.4, 200 mm NaCl and resuspended to 10 mg/ml. A 96-well plate was set up with 0.1 mg of scintillation proximity assay beads and 120 μm [3H]AMP (GE Healthcare) and made up to 90 μl with buffer. The plate was shaken for 15 min at room temperature, and varying concentrations of A-7969662 or AMP were added, to a final volume of 100 μl. The plate was shaken for 15 min, beads were allowed to settle, and the plate was read using a 1450 MicroBeta counter (PerkinElmer Life Sciences).Cell Culture—Mouse embryonic fibroblasts (MEFs) from AMPK-α1+/+α2+/+ and AMPK-α1–/–α2–/– mice were generated as described previously (49Laderoute K.R. Amin K. Calaoagan J.M. Knapp M. Le T. Orduna J. Foretz M. Viollet B. Mol. Cell Biol. 2006; 26: 5336-5347Crossref PubMed Scopus (362) Google Scholar). MEFs were cultured in standard Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin, non-essential amino acids, and 1 mm sodium pyruvate. HeLa cells were cultured in minimum essential Eagle's medium supplemented with 10% fetal bovine serum, non-essential amino acids, and 100 units/ml penicillin and 100 mg/ml streptomycin. MEFs from TAK1+/+ and TAK1–/– mice were generated (50Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (740) Google Scholar) and cultured as for AMPK-α1–/–α2–/– MEFs. Cells cultured in 10-cm dishes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum were treated as described in the figure legends, rinsed with phosphate-buffered saline, and lysed in 500 μl of ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm EDTA, 1% (w/v) Nonidet P-40, 1 mm sodium orthovanadate, 10 mm sodium β-glycerophosphate, 50 mm NaF, 5 mm sodium pyrophosphate, 0.27 m sucrose, 1 mm dithiothreitol, and complete proteinase inhibitor mixture (Roche Applied Science, one tablet per 50 ml)). Lysates were centrifuged at 4 °C for 15 min at 13,000 rpm, and the supernatant was collected. Total protein concentration was determined by the Bradford method using bovine serum albumin as standard.Primary Mouse Hepatocytes—Primary hepatocytes were prepared from wild-type (AMPK-α1+/+α2+/+) or conditional double knock-out (AMPK-α1–/–α2–/–) mice and incubated as described previously (36Guigas B. Bertrand L. Taleux N. Foretz M. Wiernsperger N. Vertommen D. Andreelli F. Viollet B. Hue L. Diabetes. 2006; 55: 865-874Crossref PubMed Scopus (158) Google Scholar).Incubation of Isolated Mouse Skeletal Muscle—Mice were fasted overnight and sacrificed by cervical dislocation. Muscles (extensor digitorum longus) were rapidly removed, and tendons from both ends were tied with suture and mounted on an incubation apparatus. The muscles were incubated as described previously (11Sakamoto K. Goransson O. Hardie D.G. Alessi D.R. Am. J. Physiol. 2004; 287: E310-E317Crossref PubMed Scopus (27) Google Scholar) in Krebs-Ringer bicarbonate buffer (117 mm NaCl, 2.5 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 24.6 mm NaHCO3, pH 7.4) containing 2 mm sodium pyruvate for 1 h at 37°C in the presence or absence of 100 μm A-769662 (100 mm stock prepared in Me2SO) or 2 mm AICAR. Me2SO (0.1% final concen
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