Title: Dissecting the Role of 5′-AMP for Allosteric Stimulation, Activation, and Deactivation of AMP-activated Protein Kinase
Abstract: AMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK α1β1γ1 and α2β2γ1 by their upstream kinases (Ca2+/calmodulin-dependent protein kinase kinase-β and LKB1-MO25α-STRADα), the deactivation by protein phosphatase 2Cα, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 μmol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 μm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK α-subunits at Thr-172 by protein phosphatase 2Cα is attenuated by AMP. Furthermore, it is shown that neither purified NAD+ nor NADH alters the activity of AMPK in a concentration range of 0–300 μm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation. AMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK α1β1γ1 and α2β2γ1 by their upstream kinases (Ca2+/calmodulin-dependent protein kinase kinase-β and LKB1-MO25α-STRADα), the deactivation by protein phosphatase 2Cα, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 μmol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 μm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK α-subunits at Thr-172 by protein phosphatase 2Cα is attenuated by AMP. Furthermore, it is shown that neither purified NAD+ nor NADH alters the activity of AMPK in a concentration range of 0–300 μm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation. AMP-activated protein kinase (AMPK) 2The abbreviations used are: AMPK, 5′-AMP-activated protein kinase; AMPKK, AMPK kinase; AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; Cr, creatine; GST, glutathione S-transferase; LKB1, serine/threonine kinase 11 (STK11); MO25, mouse protein 25; PCr, phospho-creatine; PP2Cα, protein phosphatase-2C α isoform; SAMS, synthetic peptide HMRSAMSGLHLVKRR; STRAD, STE20-related adaptor protein; ZMP, AICAR-monophosphate; HPLC, high pressure liquid chromatography. and its homologues in insects, plants, and yeast are fuel sensors of the eukaryotic cell and master regulators of energy metabolism (1Carling D. Biochimie (Paris). 2005; 87: 87-91Crossref PubMed Scopus (181) Google Scholar, 2Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (655) Google Scholar, 3Kahn B.B. Alquier T. Carling D. Hardie D.G. Cell Metab. 2005; 1: 15-25Abstract Full Text Full Text PDF PubMed Scopus (2347) Google Scholar). AMPK is a heterotrimeric serine/threonine protein kinase consisting of α-, β-, and γ-subunits. In mammals, each subunit exists in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3), which may give rise to 12 different heterotrimeric isoform-subunit combinations. A prerequisite for significant protein kinase activity of AMPK is phosphorylation of the catalytic α-subunit at Thr-172 (4Stein S.C. Woods A. Jones N.A. Davison M.D. Carling D. Biochem. J. 2000; 345: 437-443Crossref PubMed Scopus (499) Google Scholar), but additional phosphorylation sites in α- and β-subunits of AMPK have been reported (5Woods A. Vertommen D. Neumann D. Turk R. Bayliss J. Schlattner U. Wallimann T. Carling D. Rider M.H. J. Biol. Chem. 2003; 278: 28434-28442Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Upstream kinases capable of activating AMPK have been identified recently as LKB1-MO25-STRAD (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, 7Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1453) Google Scholar, 8Woods 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 (1352) Google Scholar) and CaMKKβ (9Hawley 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 (1290) Google Scholar, 10Woods 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 (1085) Google Scholar), collectively called AMPK kinases (AMPKKs). AMP allosterically stimulates AMPK activity by binding to the γ-subunit, which carries four CBS domains organized in two pairs called Bateman domains (11Scott 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 (608) Google Scholar). A mutant truncated form of the catalytic α-subunit, α1-(1–312), still requiring phosphorylation of Thr-172 for enzyme activity, is independent of allosteric activation by AMP (12Crute B.E. Seefeld K. Gamble J. Kemp B.E. Witters L.A. J. Biol. Chem. 1998; 273: 35347-35354Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The β-subunit carries a glycogen-binding domain (13Hudson E.R. Pan D.A. James J. Lucocq J.M. Hawley S.A. Green K.A. Baba O. Terashima T. Hardie D.G. Curr. Biol. 2003; 13: 861-866Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 14Polekhina G. Gupta A. Michell B.J. van Denderen B. Murthy S. Feil S.C. Jennings I.G. Campbell D.J. Witters L.A. Parker M.W. Kemp B.E. Stapleton D. Curr. Biol. 2003; 13: 867-871Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) and tethers the α- and γ-subunits together (15Iseli T.J. Walter M. van Denderen B.J. Katsis F. Witters L.A. Kemp B.E. Michell B.J. Stapleton D. J. Biol. Chem. 2005; 280: 13395-13400Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Besides directly stimulating AMPK activity, AMP inhibits dephosphorylation of AMPK and in addition was reported to promote phosphorylation of AMPK by upstream kinase(s) (16Davies S.P. Helps N.R. Cohen P.T. Hardie D.G. FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (501) Google Scholar). However, neither CaMKKβ nor LKB1 themselves are directly activated by AMP, and the question of whether binding of AMP to the allosteric site of AMPK renders the enzyme a better substrate for its upstream kinases remains controversial (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, 8Woods 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 (1352) Google Scholar, 9Hawley 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 (1290) Google Scholar, 10Woods 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 (1085) Google Scholar). The degree of stimulation of AMPK by AMP in vitro was of rather moderate extent (1.5–4-fold) (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, 8Woods 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 (1352) Google Scholar, 9Hawley 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 (1290) Google Scholar, 10Woods 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 (1085) Google Scholar), a fact that would seem difficult to reconcile with a system demanding immediate and high responsiveness in vivo to the rapidly changing energy requirement of many cell types. In living cells and organisms, AMPK is activated by metabolic stresses, but many of the downstream effects of AMPK were originally demonstrated using the compound 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) (2Hardie D.G. Hawley S.A. Scott J.W. J. Physiol. (Lond.). 2006; 574: 7-15Crossref Scopus (655) Google Scholar). AICAR is a nucleoside that is taken up by cells and converted to AICAR-monophosphate (ZMP), an AMP analogue that mimics the effects of AMP on the AMPK system (17Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1036) Google Scholar). Despite recent progress in understanding the roles of AMPK in cells, tissues, and in the whole organism, the enzyme characteristics of the AMPK heterotrimer have not been described in much detail. A major reason for this was the lack of sufficient amounts of highly purified AMPK protein available. AMPK has been mainly purified from tissue or cell lysates, but recently we have established an expression system for mammalian AMPK in bacteria, yielding milligram quantities of highly purified AMPK (18Neumann D. Woods A. Carling D. Wallimann T. Schlattner U. Protein Expression Purif. 2003; 30: 230-237Crossref PubMed Scopus (119) Google Scholar), and several groups, including ourselves, have successfully used such recombinant material for a number of different studies (5Woods A. Vertommen D. Neumann D. Turk R. Bayliss J. Schlattner U. Wallimann T. Carling D. Rider M.H. J. Biol. Chem. 2003; 278: 28434-28442Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 8Woods 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 (1352) Google Scholar, 10Woods 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 (1085) Google Scholar, 19Baron S.J. Li J. Russell III, R.R. Neumann D. Miller E.J. Tuerk R. Wallimann T. Hurley R.L. Witters L.A. Young L.H. Circ. Res. 2005; 96: 337-345Crossref PubMed Scopus (88) Google Scholar, 20Carattino M.D. Edinger R.S. Grieser H.J. Wise R. Neumann D. Schlattner U. Johnson J.P. Kleyman T.R. Hallows K.R. J. Biol. Chem. 2005; 280: 17608-17616Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 21Taylor E.B. Ellingson W.J. Lamb J.D. Chesser D.G. Compton C.L. Winder W.W. Am. J. Physiol. 2006; 290: E661-E669Crossref PubMed Scopus (22) Google Scholar, 22Xie Z. Dong Y. Zhang M. Cui M.Z. Cohen R.A. Riek U. Neumann D. Schlattner U. Zou M.H. J. Biol. Chem. 2006; 281: 6366-6375Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 23Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles W.G.T. Schlattner U. Neumann D. Brownlee M. Freeman M.B. Goldman M.H. J. Biol. Chem. 2004; 279: 43940-43951Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Importantly, wild type AMPK produced in bacteria is almost entirely inactive but can be highly activated by upstream kinases that are capable of phosphorylating AMPK α-subunits at Thr-172 (18Neumann D. Woods A. Carling D. Wallimann T. Schlattner U. Protein Expression Purif. 2003; 30: 230-237Crossref PubMed Scopus (119) Google Scholar). AMPK activity is commonly determined by radioactive labeling of artificial peptide substrates, e.g. the so-called SAMS peptide, and spotting of the phosphorylated peptide onto charged membranes, followed by scintillation counting (24Davies S.P. Carling D. Hardie D.G. Eur. J. Biochem. 1989; 186: 123-128Crossref PubMed Scopus (373) Google Scholar). The SAMS peptide is based on the AMPK recognition sequence of acetyl-CoA carboxylase and has been used most frequently for AMPK activity determination, although alternatives like the AMARA peptide (where the AMARA synthetic peptide is AMARAASAAALARRR) have been described (25Dale S. Wilson W.A. Edelman A.M. Hardie D.G. FEBS Lett. 1995; 361: 191-195Crossref PubMed Scopus (269) Google Scholar). GST fusion products of a larger domain of acetyl-CoA carboxylase, comprising the AMPK recognition sequence, have been used to determine the consensus sequence of phosphorylation by AMPK (26Scott J.W. Norman D.G. Hawley S.A. Kontogiannis L. Hardie D.G. J. Mol. Biol. 2002; 317: 309-323Crossref PubMed Scopus (141) Google Scholar), and recently, a pull-down assay using GST-SAMS was developed for AMPK activity determination in crude lysates (27Kishimoto A. Ogura T. Esumi H. Mol. Biotechnol. 2006; 32: 17-21Crossref PubMed Google Scholar). In this study, we developed a dedicated analytical method and scrutinized the roles of AMP in AMPK regulation. Recombinant AMPK—Wild type AMPK α1β1γ1, α2β2γ1, and constitutively active mutant AMPK α1T172Dβ1γ1 were bacterially expressed as published previously (18Neumann D. Woods A. Carling D. Wallimann T. Schlattner U. Protein Expression Purif. 2003; 30: 230-237Crossref PubMed Scopus (119) Google Scholar). All AMPK preparations were pre-purified on nickel-nitrilotriacetic acid superflow columns (Qiagen) and further processed to highest purity. Specific kinase activity and purity, as determined from Coomassie Blue-stained gels after separation by SDS-PAGE, served as criteria for enzyme homogeneity (see “Results”). Stock solutions of the enzyme were kept in 50% glycerol at –20 °C. Activation of AMPK by Upstream Kinases and Activity Assay—AMPK activity was assessed with SAMS-peptide as substrate target and nonradioactive ATP, which was HPLC-purified directly before use or stored frozen until usage (see below). The assay buffer consisted of 40 mm HEPES, pH 7.1, 75 mm NaCl, 2 mm dithiothreitol, 1–10 mm MgCl2. AMP, ATP, SAMS, as well as AMPK (and CaMKKβ or LKB1-MO25α-STRADα for wild type AMPK) were added as indicated in the figure legends. A plasmid encoding the GST fusion construct of CaMKKβ cDNA for bacterial expression was a kind gift from H. Tokumitsu (Kagawa Medical University, Kagawa, Japan), and GST-CaMKKβ was prepared as described previously (28Tokumitsu H. Iwabu M. Ishikawa Y. Kobayashi R. Biochemistry. 2001; 40: 13925-13932Crossref PubMed Scopus (69) Google Scholar). The tricistronic construct, expression, and purification of the heterotrimeric complex LKB1-MO25α-STRADα was prepared in our laboratory. 3D. Neumann, M. Suter, R. Tuerk, U. Riek, and T. Wallimann, manuscript in preparation. AMPK assays were performed in Eppendorf tubes on a thermal shaker at 30 °C and 300 rpm. Samples were taken at the indicated time points, and the reaction was stopped by freezing the samples in liquid nitrogen, followed by storage at –20 °C until further analysis. Re-purification of Activated Wild Type AMPK—After activation by either CaMKKβ, the pH of the sample was adjusted to 8.0 by addition of the appropriate amount of phosphate buffer (500 mm sodium phosphate buffer, 250 mm NaCl, pH 9) and loaded onto a 1-ml nickel-Sepharose HiTrap column (GE Healthcare). The column was washed with a minimum of 20 ml of washing buffer (20 mm sodium phosphate buffer, 150 mm NaCl, pH 8.0) containing a low concentration of imidazole (20 mm) and eluted with high imidazole (250 mm imidazole in washing buffer). Samples were analyzed by SDS-PAGE, and selected fractions were stored at –20 °C after addition of glycerol to a final concentration of 50% (w/v). All protein concentrations were determined using protein assay reagent (Bio-Rad) with bovine serum albumin as a standard. HPLC Analysis—AMP, ADP, ATP, NAD+, and NADH were separated and quantified on an anion exchange column (Nucleosil 4000-7 PEI, 50/4 from Macherey-Nagel, Oehnsingen, Switzerland) with a linear gradient (0–1.5 m NaCl in 10 mm Tris-Cl, pH 8.0) using an HPLC system equipped with two independent UV-visible spectrometers (Shimadzu, Reinach, Switzerland). Elution of samples was monitored at 259 and 220 nm. The latter wavelength was used to trace possible contaminants. Commercially available ATP (519979; Roche Diagnostics) always contained traces of AMP (and ADP), even immediately after dissolving from newly opened fresh containers. Therefore, ATP used throughout this study was HPLC-purified by the above method and routinely checked for its AMP content, which was always below 0.1% after HPLC processing. SAMS and SAMS-phosphate were separated and quantified on a cation exchange column (Source 15S, 1 ml, from GE Healthcare) with a linear gradient (0–1.5 m NaCl in 20 mm sodium phosphate, pH 7.5) at 220 nm. SDS-PAGE and Western Blotting—Protein samples were diluted in Laemmli buffer, snap-frozen in liquid nitrogen at indicated time points, and subjected to SDS-PAGE (12% polyacrylamide). After transfer to nitrocellulose membranes (Milian, Meyrin, Switzerland), proteins were immunoblotted using either anti-AMPKα, phospho-specific anti-AMPKα Thr-172 (Cell Signaling Technology, BioConcept, Allschwil, Switzerland, catalogue numbers 2532, 2535, respectively), or anti-AMPKβ primary antibodies (a kind gift of Dr. David Carling, MRC, Hammersmith Hospital, London, UK) and goat-anti rabbit peroxidase conjugated secondary antibodies (catalogue number DC03L; Calbiochem). Signals were detected with enhanced chemiluminescence (Applichem, Axon Lab, Baden-Dättwil, Switzerland) and Kodak x-ray-sensitive films (GE Healthcare). Western blots were quantified by densitometry on a Kodak Imagestation 440CF using Kodak Digital Science Software version 2.0.4 (PerkinElmer Life Sciences). The standard method for AMPK activity determination depends on an artificial peptide substrate, called SAMS that is incubated with AMPK in presence of [γ-32P]ATP (ATP + SAMS → ADP + SAMS-P). The amount of radioactively labeled [32P]SAMS-phosphate is determined by scintillation counting, as the only accessible parameter at the end of the reaction. AMPK, however, interacts with all adenine nucleotides. AMP is an allosteric stimulator; ADP is a product of the kinase reaction, and ATP is a substrate. Additionally, high concentrations of ADP and AMP likely inhibit binding of ATP to the kinase domain by a competitive mechanism. High ATP is also believed to inhibit stimulation of AMPK by AMP by competitive binding to the allosteric site (17Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1036) Google Scholar). We therefore considered accurate determination of the concentrations of all reaction partners, namely adenine nucleotides, SAMS, and SAMS-phosphate. HPLC Detection Enhances Reliability of the AMPK Activity Assay—Separation of adenine nucleotides is commonly performed with anion exchange chromatography. The SAMS-peptide with a theoretical pI of 12.3 was found to interact strongly with cation exchange material at pH 7.5, and the introduction of negative charges through phosphorylation reduces its binding to the negatively charged column matrix. Thus, a salt gradient allowed for separation of SAMS from SAMS-phosphate. Samples were taken from an AMPK activity assay, and two sequential runs on the different HPLC columns were analyzed. Fig. 1A shows a representative overlay of two HPLC runs showing separation of adenine nucleotides, as well as SAMS and SAMS-phosphate. The applicability of the analytical procedure for activity determination of AMPK is demonstrated in Fig. 1B. Samples of an activity assay were taken at different time points, divided, snap-frozen, and stored for later injection. The data show the reciprocal decrease or increase of the ATP/ADP and SAMS-phosphate/SAMS ratios, respectively. AMP was added at constant concentrations to stimulate AMPK activity. A comparatively greater decrease in [ATP] compared with [SAMS] was observed consistently with different samples of AMPK after a longer incubation of samples. This might be due to “energy waste” by an inherent ATPase activity of AMPK rather than to unspecific decay of ATP or ADP over time, as it appears to be associated with substrate turnover. AMPK in the absence of SAMS did not degrade ATP (data not shown). As shown in Fig. 1B, the formation of ADP and SAMS-phosphate perfectly correlates within the first 10 min of the assay. As AMPK assays were generally stopped and analyzed after an incubation time of 5–10 min, [SAMS-phosphate] and [ADP] equally reflect AMPK activity. Furthermore, the concentrations of all educts and products in a single sample are interrelated and can be determined individually, thereby increasing the accuracy of the method. We generally used SAMS ([SAMS] + [SAMS-phosphate] = constant) and adenine nucleotides ([ADP] + [ATP] = constant) to evaluate the data, but in most cases determination of [ADP] or [SAMS] was found satisfactory. Thus, a single data point is sufficiently exact and can be used for calculation of specific activity. Specific Activity of Fully Activated Recombinant AMPK—Highly purified heterotrimeric wild type AMPK α1β1γ1 and α2β2γ1 was activated with recombinant preparations of either of the two known upstream kinases of AMPK. CaMKKβ (GST-tagged) or the LKB1-MO25α-STRADα heterotrimer was used at saturating concentrations. To reach full activation with CaMKKβ, a 1:1 ratio (w/w) of upstream kinase to AMPK had to be used, whereas with the heterotrimeric LKB1-MO25α-STRADα complex a ratio of 1:50 was sufficient to reach full activity. The specific activity of AMPK after activation and full stimulation by AMP (Fig. 2A) was in the range of 6 μmol of SAMS-phosphate/mg of wild type AMPK/min at 30 °C. This value was similar for both isoforms of AMPK and much higher than reported previously with recombinant AMPK, e.g. 0.3 μmol/mg/min, using recombinant AMPK activated by partially purified upstream kinase preparation from liver containing LKB1 (5Woods A. Vertommen D. Neumann D. Turk R. Bayliss J. Schlattner U. Wallimann T. Carling D. Rider M.H. J. Biol. Chem. 2003; 278: 28434-28442Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) or 0.2 μmol/mg/min activated by recombinant CaMKKβ (10Woods 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 (1085) Google Scholar). Native AMPK α1 complexes have been purified from tissue to near homogeneity and exhibited similar high specific activities of 8.2 μmol/mg/min using the radioactive detection method and SAMS as a substrate (29Michell B.J. Stapleton D. Mitchelhill K.I. House C.M. Katsis F. Witters L.A. Kemp B.E. J. Biol. Chem. 1996; 271: 28445-28450Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To compare the HPLC-based method with the most widely used radioactive detection method of AMPK activity determination, we purchased a partially purified preparation of rat liver AMPK (Upstate, Lucerna-Chem, Lucerne, Switzerland) with a declared specific activity (0.8 μmol/mg/min at 30 °C, as determined by SAMS assay, using the radioactive method), and we determined its activity by our HPLC method. Under conditions identical to the company's kinase assay protocol (100 μm ATP, 100 μm AMP, and 100 μm SAMS), we measured 0.2 μmol/mg/min or using optimized assay conditions (200 μm ATP, 50 μm AMP, and 500 μm SAMS) reached a value of 0.4 μmol/mg/min, suggesting that the HPLC-based analytical method is, if anything, underestimating the specific activities by a factor of 2–4 in comparison to the commonly used radioactive assay procedure. This would indicate that the specific activity of highly purified recombinant AMPK is even higher, although no attempts were undertaken to determine activity using traditional approaches. Furthermore, the re-purification step of fully activated AMPK resulted in no significant loss of specific activity (Fig. 2A), suggesting that once AMPK has been activated by upstream kinases these high specific activities can be preserved in vitro in the absence of AMPKKs. Autophosphorylation May Reduce AMP Sensitivity of AMPK—The same AMPK samples as shown in Fig. 2A were also analyzed by SDS-PAGE (Fig. 2B). Upon activation by AMPKK, shifts of electrophoretic mobility were observed in α-subunits as a consequence of phosphorylation of AMPK at Thr-172 by AMPKKs, as has been observed previously (10Woods 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 (1085) Google Scholar). The immunostaining of the β-subunit in Western blots also revealed changes after activation by AMPKK (Fig. 2C), suggesting that β-subunits partly shift to higher apparent molecular weight in SDS-PAGE. In contrast, the β-subunits of the enzymatically inactive α1D157Aβ1γ1 and α2D157Aβ2γ1 mutants did not shift toward higher molecular weight in SDS-PAGE and are not incorporating radioactivity from [γ-32P]ATP upon activation by CaMKKβ or LKB1-MO25α-STRADα (data not shown), strongly suggesting that the altered electrophoretic mobility of the β-subunit is caused by autophosphorylation. However, a certain proportion of the β-subunit-specific signal did not shift toward higher molecular weight (Fig. 2C). At present, we do not have an explicatory interpretation for this phenomenon. The appearance of multiple AMPK β-subunit immunoreactive signals, which may represent different phospho-species of the β-subunits, prompted us to investigate a time course of AMPK activation. The kinetics of the activation process with AMPK α2β2γ1 is shown in Fig. 3A, reaching saturation after 30 min. Western blotting of samples taken at different time points of incubation with upstream kinases (up to 4 h) demonstrates that the β2-subunit phosphorylation still increased after full activity was reached (Fig. 3B). Interestingly, the time course of increasing autophosphorylation, as reflected by the lowered electrophoretic mobility of the β2-subunit in SDS-PAGE, is concomitant with a decrease of the stimulatory effect by AMP (Fig. 3C). Activation of AMPK with Upstream Kinases Is Not Stimulated by AMP—AMP does not directly regulate the activities of the upstream kinases LKB1-MO25α-STRADα or CaMKKβ (6Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Makela T.P. Alessi D.R. Hardie D.G. J. Biol. (Bronx N. Y.). 2003; 2: 28Google Scholar, 8Woods 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 (1352) Google Scholar, 9Hawley 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 (1290) Google Scholar, 10Woods 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 (1085) Google Scholar). However, the question of whether binding of AMP to AMPK renders the enzyme a better substrate for upstream kinases is still a matter of discussion. We therefore used bacterially expressed truncated α1-(1–312) comprising only the kinase domain of AMPK and thus lacking allosteric effects by AMP, in comparison with the two heterotrimeric isoforms of AMPK carrying their functional γ-subunits and allowing for allosteric regulation. AMPK was incubated in the presence and absence of AMP with the two upstream kinases (LKB1-MO25α-STRADα or CaMKKβ) and analyzed by Western blotting. Identical signal intensities derived from the phospho-specific anti-AMPKα Thr-172 antibody were obtained