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• W2033473463 abstract "AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in the regulation of energy homeostasis. Previously, AMPK was reported to phosphorylate serine 621 of Raf-1 in vitro. In the present study, we investigated a possible role of AMPK in extracellular signal-regulated kinase (Erk) cascades, using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), a cell-permeable activator of AMPK and antisense RNA experiments. Activation of AMPK by AICAR in NIH-3T3 cells resulted in drastic inhibitions of Ras, Raf-1, and Erk activation induced by insulin-like growth factor 1 (IGF-1). Expression of an antisense RNA for the AMPK catalytic subunit decreased the AMPK activity and significantly diminished the AICAR effect on IGF-1-induced Ras activation and the subsequent Erk activation, indicating that its effect is indeed mediated by AMPK. Phosphorylation of Raf-1 serine 621, however, was not involved in AMPK-mediated inhibition of Erk cascades. In contrast to IGF-1, AICAR did not block epidermal growth factor (EGF)-dependent Raf-1 and Erk activation, but our results demonstrated that multiple Raf-1 upstream pathways induced by EGF were differentially affected by AICAR: inhibition of Ras activation and simultaneous induction of Ras-independent Raf activation. The activities of IGF-1 and EGF receptor were not affected by AICAR. Taken together, our results suggest that AMPK differentially regulate Erk cascades by inhibiting Ras activation or stimulating the Ras-independent pathway in response to the varying energy status of the cell. AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in the regulation of energy homeostasis. Previously, AMPK was reported to phosphorylate serine 621 of Raf-1 in vitro. In the present study, we investigated a possible role of AMPK in extracellular signal-regulated kinase (Erk) cascades, using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), a cell-permeable activator of AMPK and antisense RNA experiments. Activation of AMPK by AICAR in NIH-3T3 cells resulted in drastic inhibitions of Ras, Raf-1, and Erk activation induced by insulin-like growth factor 1 (IGF-1). Expression of an antisense RNA for the AMPK catalytic subunit decreased the AMPK activity and significantly diminished the AICAR effect on IGF-1-induced Ras activation and the subsequent Erk activation, indicating that its effect is indeed mediated by AMPK. Phosphorylation of Raf-1 serine 621, however, was not involved in AMPK-mediated inhibition of Erk cascades. In contrast to IGF-1, AICAR did not block epidermal growth factor (EGF)-dependent Raf-1 and Erk activation, but our results demonstrated that multiple Raf-1 upstream pathways induced by EGF were differentially affected by AICAR: inhibition of Ras activation and simultaneous induction of Ras-independent Raf activation. The activities of IGF-1 and EGF receptor were not affected by AICAR. Taken together, our results suggest that AMPK differentially regulate Erk cascades by inhibiting Ras activation or stimulating the Ras-independent pathway in response to the varying energy status of the cell. AMP-activated protein kinase 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside AICA-ribotide insulin-like growth factor 1 epidermal growth factor EGF receptor extracellular signal-regulated kinase mitogen-activated protein kinase MAPK/Erk kinase Ras-binding domain glutathione S-transferase cAMP-dependent protein kinase Dulbecco's modified Eagle's medium IGF-1 receptor. Mammalian AMP-activated protein kinase (AMPK)1 plays a key role in the regulation of energy homeostasis and is highly conserved among animals, plants, and fungi (reviewed in Refs. 1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1275) Google Scholar, 2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.-P. Witters L, A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). AMPK is a heterotrimeric enzyme consisting of a catalytic subunit (α) and two regulatory subunits (β and γ), and it is activated by the cellular stress causing ATP depletion, which in turn leads to elevation of the AMP:ATP ratio (reviewed in Refs. 1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1275) Google Scholar, 2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.-P. Witters L, A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). These stresses include heat shock, ischemia/hypoxia in cardiac muscle, and exercise in skeletal muscle. In addition to allosteric activation by AMP, AMPK is activated by phosphorylation by an upstream kinase termed AMPK kinase (4Hawley 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 (1008) Google Scholar). Once activated, AMPK suppresses the key enzymes involved in ATP-consuming anabolic pathways such as fatty acid and cholesterol synthesis (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1027) Google Scholar, 6Henin N. Vincent M.F. Gruber H.E. Van den Berghe G. FASEB. J. 1995; 9: 541-546Crossref PubMed Scopus (224) Google Scholar). Besides, AMPK initiates a series of compensatory changes that increase cellular ATP supply by activating the rate of fatty acid oxidation (7Velasco G. Geelen M.J.H. Guzman M. Arch. Biochem. Biophys. 1997; 337: 169-175Crossref PubMed Scopus (102) Google Scholar,8Merrill G.F. Kurth E.J. Hardie D.G. Winder W.W. Am. J. Physiol. 1997; 273: E1107-E1112Crossref PubMed Google Scholar) and glucose uptake in cardiac and skeletal muscle (9Bergeron R. Russell III, R.R. Young L.H. Ren J.-M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar, 10Kurth-Kraczek E.J. Hirshman M.F. Goodyear L.J. Winder W.W. Diabetes. 1999; 48: 1667-1671Crossref PubMed Scopus (587) Google Scholar). Thus, AMPK has been speculated to play a role as a “fuel gauge”; that recognizes ATP depletion and maintains ATP level (reviewed in Refs.1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1275) Google Scholar, 2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.-P. Witters L, A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Most of the currently identified substrates of AMPK are metabolic enzymes. However, the recent implications of AMPK in transcriptional control (11Foretz M. Carling D. Guichard C. Ferre P. Foufelle F. J. Biol. Chem. 1998; 273: 14767-14771Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 12Leclerc I. Kahn A. Doiron B. FEBS Lett. 1998; 431: 180-184Crossref PubMed Scopus (129) Google Scholar), insulin secretion in pancreatic β cell (13Salt I.P. Johnson G. Ashcroft S.J.H. Hardie D.G. Biochem. J. 1998; 335: 533-539Crossref PubMed Scopus (339) Google Scholar), and regulation of endothelial NO synthase (14Chen Z.-P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (716) Google Scholar) and Raf-1 kinase (15Sprenkle A.B. Davies S.P. Carling D. Hardie D.G. Sturgill T.W. FEBS Lett. 1997; 403: 254-258Crossref PubMed Scopus (52) Google Scholar) suggest that it might be involved in the regulation of many cellular processes other than those that have been identified. Thus, it appears that numerous novel targets remain to be discovered. Recently, Sprenkle et al. (15Sprenkle A.B. Davies S.P. Carling D. Hardie D.G. Sturgill T.W. FEBS Lett. 1997; 403: 254-258Crossref PubMed Scopus (52) Google Scholar) identified the principal Raf-1 Ser621 kinase activity present in cytosolic extracts of NIH-3T3 cells as AMPK by analyzing cytosolic fractions for Ser621 peptide kinase activity. They also demonstrated that AMPK phosphorylated Ser621 of Raf-1, which was expressed inEscherichia coli or Sf9 insect cell (15Sprenkle A.B. Davies S.P. Carling D. Hardie D.G. Sturgill T.W. FEBS Lett. 1997; 403: 254-258Crossref PubMed Scopus (52) Google Scholar). The Ser/Thr Raf-1 kinase is a key intermediate in the transduction of growth factor signals from the cell membrane to the nucleus (16Moelling K. Heimann B. Beimling P. Rapp U.R. Sander T. Nature. 1984; 312: 558-561Crossref PubMed Scopus (149) Google Scholar, 17Morrison D.K. Cutler R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (537) Google Scholar). Activation of receptor tyrosine kinases stimulates the small GTP-binding protein Ras, which interacts with cytoplasmic inactive Raf and recruits it to the plasma membrane where further activation steps occur (18Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (885) Google Scholar, 19Stokoe D. MacDonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (845) Google Scholar). Activated Raf in turn phosphorylates and activates MAPK/Erk kinase 1 (MEK1), which phosphorylates and stimulates mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/Erks). This pathway (Ras → Raf → MEK → Erk) has been known to play a significant role in the transmission of cellular proliferation and developmental signals (20Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar). Although the exact regulatory mechanisms for Raf-1 activity are not fully understood despite extensive investigations, they include phosphorylation of the enzyme (17Morrison D.K. Cutler R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (537) Google Scholar). The role of Raf-1 Ser621 phosphorylation has been controversial. Ser621 was reported to be a constitutive phosphorylation site, and the mutation of this site to alanine resulted in a total loss of the kinase activity (21Morrison D.K. Heidecker G. Rapp U.R. Copeland T.D. J. Biol. Chem. 1993; 268: 17309-17316Abstract Full Text PDF PubMed Google Scholar). On the other hand, it was also claimed that phosphorylation of Ser621 by cAMP-dependent protein kinase (PKA) confers negative regulation (22Mischak H. Seitz T. Janosch P. Eulitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar). In the present study, we have taken a pharmacological and molecular approach to determine whether AMPK plays a role in Raf-1-involved signaling pathways. Our results suggest that AMPK can attenuate Ras activation induced by IGF-1 or EGF and that AMPK can also further stimulate Ras-independent Raf activation induced by EGF. Phosphorylation of Raf-1 Ser621, however, was not involved in the AMPK-mediated regulation of Erk cascades. To our knowledge, this is the first report demonstrating that AMPK is involved in the regulation of growth factor-induced multiple signaling pathways. Dulbecco's modified Eagle's medium (DMEM), DMEM/Ham's F-12 medium, and the other cell culture products were purchased from Life Technologies, Inc. Forskolin and PD098059 were obtained from Calbiochem. EGF and IGF-1 were from Calbiochem and Sigma, respectively. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) and other chemicals were from Sigma. [γ-32P]ATP (6000 Ci/mmol) and [methyl-3H]thymidine (6.7 Ci/mmol) were purchased from PerkinElmer Life Sciences. NIH-3T3, 3T3-L1 preadipocytes, and COS-7 cells were maintained in DMEM containing 10% calf serum. H9c2 cardiomyoblasts were proliferated in DMEM/Ham's F-12 medium supplemented with 10% calf serum. When confluent, cells were induced to differentiate for 6 days with medium containing 1% horse serum. About 80–90% confluent cells or H9c2 cardiomyotube were serum-starved for 16 h, and then the indicated pretreatment (1 mm AICAR, 50 μmPD098059, or 50 μm forskolin) was performed for 1 h in Krebs-Ringer buffer (25 mm HEPES, pH 7.4, 118 mm NaCl, 4.8 mm KCl, 1.3 mmCaCl2, 1.2 mm KH2PO4, 1.3 mm MgSO4, 5 mmNaHCO3, 0.07% bovine serum albumin, and 5.5 mmglucose). Then cells were stimulated with 50 nm IGF-1 or 100 ng/ml EGF for the indicated time period. The anti-dual phospho-specific antibody that recognizes the active Erk1/2 was from New England Biolabs, Inc. Erk1/2 antibody that recognizes the total Erk1/2 regardless of their phosphorylation was from Transduction Laboratories. Antibodies for Raf-1 (C-12), EGF receptor (EGFR, 1005), IGF-1 receptor β subunit (IGF-1R, C-20), Grb2 (C-23), phosphotyrosine (PY20), and c-Myc (9E10) were obtained from Santa Cruz Biotechnology Inc. Phosphospecific Raf Ser621 antibody was kindly provided by Dr. Andrey S. Shaw (Center for Immunology and Department of Pathology, Washington University, St. Louis, MO). The AMPK α1-specific antibody and the AMPK pan-α antibody were kindly provided by Dr. Ian Salt and Dr. D. Grahame Hardie (Biochemistry Department, The University, Dundee, UK). Raf-1 mutant constructs (Raf-wt, RafS621A, and RafR89L in pEFm vector fused with Myc epitope at its N terminus) were generous gifts from Dr. Richard Marais and Christopher J. Marshall (Cancer Research Campaign Center for Cell and Molecular Biology, Institute of Cancer Research, UK). GST-MEK(−) plasmid was provided by Dr. Kun-Liang Guan (Department of Biochemistry, University of Michigan, town, MI). A dominant negative Ras (RasS17N) was subcloned into pCMV-tag2B vector. cDNA corresponding to a portion of the coding region of AMPK α1 (amino acids 1–392) was amplified by polymerase chain reaction. It was subcloned into pcDNA 3 in such an orientation as to express an antisense RNA. The plasmid was transfected into NIH-3T3 cells using LipofectAMINE (Life Technologies, Inc.), and stable transfectants were obtained in the presence of G418. Individual clones were further isolated and examined for the effect of an antisense RNA expression. Cells were lysed with digitonin buffer (50 mm Tris-HCl, pH 7.3, 50 mm NaF, 30 mm glycerol phosphate, 250 mm sucrose, 1 mm sodium metavanadate, and 0.4 mg/ml digitonin) on ice for 2 min. AMPK was partially purified from cell lysates by adding saturated ammonium sulfate to final 35% (v/v) concentration on ice for 15 min. AMPK activity was determined as previously described (23Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (373) Google Scholar) with these fractionated proteins in kinase assay buffer (62.5 mmHEPES, pH 7.0, 62.5 mm NaCl, 62.5 mm NaF, 6.25 mm sodium pyrophosphate, 1.25 mm EDTA, 1.25 mm EGTA, and 1 mm dithiothreitol) containing 200 μm AMP, ATP mixture (200 μm ATP and 1.5 μCi of [γ-32P]ATP), with or without 250 μm SAMS peptide (HMRSAMSGLHLVKRR) at 30 °C for 10 min. The reaction was terminated by spotting the reaction mixture on phosphocellulose paper (P81), and the paper was extensively washed with 150 mm phosphoric acid. The radioactivity was measured with a scintillation counter. Raf-1 kinase assay was performed essentially as described by Ziogas et al. (24Ziogas A. Lorenz I.C. Moelling K. Radziwill G. J. Biol. Chem. 1998; 273: 24108-24114Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) with the following modifications. After cell lysis, 500 μg of protein extracts were subjected to immunoprecipitation with a Raf-1 antibody (C-12) or c-Myc antibody (9E10) coupled to protein G-agarose. After extensive washing of the immunoprecipitates, the kinase assay was performed at 30 °C for 30 min in a kinase assay buffer containing the Raf-1 immune complex, 10 μm ATP, 10 μCi of [γ-32P]ATP, and 1 μg of kinase defective GST-MEK(−) as a specific substrate of Raf-1. After reaction, samples were centrifuged, and the supernatants were separated on 10% SDS/polyacrylamide gel electrophoresis. Proteins were blotted on nitrocellulose membranes, and the radioactivity incorporated into the substrates was measured by PhosphorImager. The resultant pellets were also resolved on the 10% SDS/polyacrylamide gel electrophoresis for immunoblotting with a Raf-1 antibody to compare the amount of Raf-1 present in the immune complex. Raf Ras-binding domain (RBD) fragment (Raf-1 amino acid residues 1–149) fused to GST was purchased from Upstate Biotechnology. The fusion protein was immobilized on glutathione-agarose. The activated Ras affinity precipitation assay was performed as described according to the manufacturer's protocol. Briefly, 500 μg of cell extracts were incubated with 5 μg of GST-RBD complexes for 30 min at 4 °C. After extensive washing of the agarose beads five times with immunoprecipitation washing buffer (25 mm HEPES, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 10 mmMgCl2, 1 mm EDTA, and 2% glycerol), the active Ras (Ras-GTP) bound to GST-RBD complexes was released by addition of 2× SDS/polyacrylamide gel electrophoresis loading buffer. The amount of active Ras was determined by immunoblotting with an anti-pan Ras monoclonal antibody. To investigate a possible involvement of AMPK in the Ras/Raf/MEK/Erk signaling pathway, dose- and time-dependent effects of AICAR on IGF-1-induced Erk1 and Erk2 activation were first examined (Fig. 1, A andB). AICAR becomes a potent activator of AMPK after its intracellular phosphorylation to AMP-mimetic AICA-ribotide (ZMP) (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1027) Google Scholar). NIH-3T3 cells were serum-starved for 16 h, pretreated with AICAR, and then challenged with 50 nm IGF-1 for 10 min. In the absence of AICAR, IGF-1 rapidly stimulated phosphorylation of Erk1 (p44 MAPK) and Erk2 (p42 MAPK) when examined by immunoblotting with an antibody specific for the active biphosphorylated form of Erk1 and Erk2. The pretreatment with AICAR inhibited IGF-1-dependent Erk activation in a dose- and time-dependent manner, and a maximum effect was observed at 1 mm of AICAR for 1 h of preincubation (Fig. 1, A and B). The total amount of Erk proteins was essentially the same at each condition. The kinetics of Erk phosphorylation correlated well with the enzyme activity, which was directly measured by an immune complex using mylein basic protein as a substrate (data not shown). As expected, AMPK was activated by AICAR in a dose- and time-dependent manner, and the maximum 3-fold activation was observed in 1 h at 1 mm AICAR (Fig. 1, C and D). These results suggest that AMPK is probably involved in the negative regulation of Erk cascades. The AMPK activation conditions found in this study are consistent with other reports showing that the maximum effect of AICAR on AMPK activity or cellular processes regulated by AMPK was observed in the range of 0.5–1 mm (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1027) Google Scholar, 6Henin N. Vincent M.F. Gruber H.E. Van den Berghe G. FASEB. J. 1995; 9: 541-546Crossref PubMed Scopus (224) Google Scholar, 7Velasco G. Geelen M.J.H. Guzman M. Arch. Biochem. Biophys. 1997; 337: 169-175Crossref PubMed Scopus (102) Google Scholar, 8Merrill G.F. Kurth E.J. Hardie D.G. Winder W.W. Am. J. Physiol. 1997; 273: E1107-E1112Crossref PubMed Google Scholar, 9Bergeron R. Russell III, R.R. Young L.H. Ren J.-M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar, 10Kurth-Kraczek E.J. Hirshman M.F. Goodyear L.J. Winder W.W. Diabetes. 1999; 48: 1667-1671Crossref PubMed Scopus (587) Google Scholar, 11Foretz M. Carling D. Guichard C. Ferre P. Foufelle F. J. Biol. Chem. 1998; 273: 14767-14771Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 12Leclerc I. Kahn A. Doiron B. FEBS Lett. 1998; 431: 180-184Crossref PubMed Scopus (129) Google Scholar). Therefore, this pretreatment condition (1 mm AICAR/1 h of incubation) was used throughout the present study. Because of the lack of molecular approaches for manipulating AMPK activity or a specific inhibitor, AICAR has been widely used to demonstrate a role of AMPK in various cell lines and tissues (5Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1027) Google Scholar, 6Henin N. Vincent M.F. Gruber H.E. Van den Berghe G. FASEB. J. 1995; 9: 541-546Crossref PubMed Scopus (224) Google Scholar, 7Velasco G. Geelen M.J.H. Guzman M. Arch. Biochem. Biophys. 1997; 337: 169-175Crossref PubMed Scopus (102) Google Scholar, 8Merrill G.F. Kurth E.J. Hardie D.G. Winder W.W. Am. J. Physiol. 1997; 273: E1107-E1112Crossref PubMed Google Scholar, 9Bergeron R. Russell III, R.R. Young L.H. Ren J.-M. Marcucci M. Lee A. Shulman G.I. Am. J. Physiol. 1999; 276: E938-E944Crossref PubMed Google Scholar, 10Kurth-Kraczek E.J. Hirshman M.F. Goodyear L.J. Winder W.W. Diabetes. 1999; 48: 1667-1671Crossref PubMed Scopus (587) Google Scholar, 11Foretz M. Carling D. Guichard C. Ferre P. Foufelle F. J. Biol. Chem. 1998; 273: 14767-14771Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 12Leclerc I. Kahn A. Doiron B. FEBS Lett. 1998; 431: 180-184Crossref PubMed Scopus (129) Google Scholar). However, AMPK-independent effects of AICAR were also reported in some cases (25Vincent M.F. Marangos P.J. Gruber H.E. Van den Berghe G. Diabetes. 1991; 40: 1259-1266Crossref PubMed Google Scholar, 26Sabina R.L. Patterson D. Holmes E.W. J. Biol. Chem. 1985; 260: 6107-6114Abstract Full Text PDF PubMed Google Scholar). Thus, to substantially demonstrate the role of AMPK in Erk cascades, we established NIH-3T3 cells stably expressing an antisense RNA for the catalytic core region of AMPK catalytic α1 subunit, a predominantly expressed isoform in these cells, as described under “Experimental Procedures.”; Two isoforms of the AMPK catalytic subunit (α1 and α2) were identified, and α1 is ubiquitously expressed, whereas α2 is highly expressed in skeletal muscle, heart, and liver (27Carling D. Aguan K. Woods A. Verhoeven A.J.M. Beri R.K. Brennan C.H. Sidebottom C. Davison M.D. Scott J. J. Biol. Chem. 1994; 269: 11442-11448Abstract Full Text PDF PubMed Google Scholar, 28Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Both isoforms show an identical molecular mass (63 kDa) and have 90% amino acid sequence identity within the catalytic core (28Stapleton D. Mitchelhill K.I. Gao G. Widmer J. Michell B.J. Teh T. House C.M. Fernandez C.S. Cox T. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 611-614Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). For simplicity, we use anti-α1 to refer to NIH-3T3 cells expressing an antisense RNA. The immunoblot analysis using either an AMPK α1-specific antibody or an AMPK pan-α antibody that recognizes both α1 and α2 revealed that the expression level of AMPK α was substantially reduced in anti-α1 cells compared with the vector-transfected control cells (Fig.2 A). The reduced expression level of the α subunit resulted in a ∼30–40% decrease in the basal and the AICAR-stimulated AMPK activity (Fig. 2 B). When anti-α1 cells were pretreated with AICAR and then challenged with IGF-1, the inhibitory effect of AICAR on Erk activation was significantly diminished (Fig. 2 C). These results indicate that the effect of AICAR is indeed mediated by AMPK. In contrast to IGF-1, AICAR did not block EGF (100 ng/ml)-induced Erk activation, whereas PD098059, a specific inhibitor of MEK, almost completely inhibited Erk activation by two growth factors (Fig.3 A). One explanation for the selective effects of AICAR is that EGF could suppress the AICAR-stimulated AMPK activity. To this end, the effects of IGF-1 and EGF on AMPK activity were examined (Fig. 3 B). However, the results revealed that these two growth factors practically had no effect on the basal and the AICAR-stimulated AMPK activities (Fig.3 B), supporting the general perspective that AMPK system is stress-sensitive but not hormone-sensitive (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1275) Google Scholar, 2Hardie D.G. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1141) Google Scholar, 3Kemp B.E. Mitchelhill K.I. Stapleton D. Michell B.J. Chen Z.-P. Witters L, A. Trends Biochem. Sci. 1999; 24: 22-25Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Therefore, these results suggest that the differential effects of AICAR are due to the intrinsic differences between signal pathways leading to Erk activation by IGF-1 and EGF. Next, to determine whether AMPK is involved in the regulation of Erk pathways in other cell types, we examined the AICAR effects in the COS-7, H9c2 cardiomyotube, and 3T3-L1 preadipocyte cell lines (Fig.4). Although these cells showed slight differences, IGF-1-induced Erk activation was inhibited, whereas EGF-induced Erk activation was not affected by AICAR treatment in all cell lines tested (Fig. 4). Therefore, the effect of AMPK on Erk cascades is likely to be quite a widespread phenomena rather than certain cell type-specific. Erk cascades are among the most intensively studied signal transduction systems, and they have shown to participate in a diverse array of cellular activities such as cell proliferation, development, cell survival, and death (20Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar). To investigate a physiological significance of the AMPK-mediated Erk regulation, the effect of AICAR on the growth factor-induced DNA synthesis was examined. IGF-1 and EGF induced DNA synthesis ∼2-fold after 9 h of incubation and 4-fold after 19 h of incubation (Fig.5 A). In close correlation with the Erk phosphorylation level, AICAR treatment led to ∼70% inhibition of IGF-induced DNA synthesis after 9 h and 50% inhibition after 19 h, whereas EGF-induced DNA synthesis was relatively less affected, resulting in about 25% reduction (Fig.5 A). Furthermore, the inhibitory effect of AICAR on IGF-1-dependent DNA synthesis was distinctively diminished in the anti-α1 cell (Fig. 5 B), in which IGF-1-induced Erk activation is quite resistant to the AICAR-dependent down-regulation (Fig. 2 C). Under these conditions, no cytotoxic effects were observed (data not shown). A MEK inhibitor, 50 μm PD098059, almost completely blocked DNA synthesis induced by both growth factors, indicating that Erk activation is critical for cell proliferation. As shown in Fig. 3 A, AICAR exerted practically no effect on the basal Erk activity (first and fourth lanes) or EGF-induced Erk activation (third and sixth lanes). Nevertheless, this treatment resulted in statistically significant inhibition of the basal as well as EGF-induced DNA synthesis (Fig. 5). Thus, besides Erk cascades, some other mechanism(s) required for DNA synthesis seems to be affected by AICAR. This possibility is considered later under “Discussion.”; The previous report demonstrated that AMPK can phosphorylate Ser621 of Raf-1 in vitro(15Sprenkle A.B. Davies S.P. Carling D. Hardie D.G. Sturgill T.W. FEBS Lett. 1997; 403: 254-258Crossref PubMed Scopus (52) Google Scholar). The sequence around Raf-1 Ser621 exactly matches the recognition motif of AMPK (29Dale S. Wilson W.A. Edelman A.M. Hardie D.G. FEBS Lett. 1995; 361: 191-195Crossref PubMed Scopus (268) Google Scholar). Because it was also demonstrated that phosphorylation of this residue by PKA leads to inhibition of Raf-1 kinase (22Mischak H. Seitz T. Janosch P. Eulitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar), we next examined the possibility that phosphorylation of Raf-1 Ser621 is a mechanism responsible for AMPK-mediated inhibition of Erk activation induced by IGF-1. To this end, we first analyzed the effect of AICAR on the phosphorylation level of Ser621 of Raf-1 (Fig.6). The Myc-tagged Raf-wild type (mycRaf-wt) plasmid was transiently transfected in NIH-3T3 cells, and in 48 h post-transfection, cells were treated for 1 h with 1 mm AICAR or 50 μm forskolin, which enhances cAMP accumulation and in turn activates PKA. The expressed mycRaf-wt protein was immunoprecipitated with a c-Myc epitope-specific monoclonal antibody 9E10 and then immunoblotted with a phosphospecific Raf Ser621 antibody. The basal phosphorylation level of Ser621 of mycRaf-wt was hardly detectable under our experimental conditions, and AICAR did not exert any effect on this residue, whereas forskolin markedly increased the phosphorylation level of Ser621 (Fig. 6, upper panel). Under the identical condition, forskolin did not exert any effect on mycRafS621A, in which Ser621 is replaced with alanine. There was essentially no difference in the amount of the immunoprecipitated mycRaf-wt and mycRafS621A (Fig. 6, lower panel). This result supports the previous finding that Ser621 of Raf-1 can be phosphorylated by PKA (22Mischak H. Seitz T. Janosch P. Eulitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar). However, in contrast to the report describing that AMPK in vitro phosphorylated Ser621 of the overexpressed Raf-1 from bacteria and insect cell (15Sprenkle A.B. Davies S.P. Carling D. Hardie D.G. Sturgill T.W. FEBS Lett. 1997; 403: 254-258Crossref PubMed Scopus (" @default.
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