Báo cáo khoa học: Regulation of the muscle-specific AMP-activated protein kinase a2b2c3 complexes by AMP and implications of the mutations in the c3-subunit for the AMP dependence of the enzyme docx

Regulation of the muscle-specific AMP-activated protein kinase a2b2c3 complexes by AMP and implications of the mutations in the c3-subunit for the AMP dependence of the enzyme Kerstin Lindgren*, Mattias Ormestad*, Ma ˚ rten Persson, Sofia Martinsson, L. Thomas Svensson and Margit Mahlapuu Discovery Research, Biovitrum AB, Go ¨ teborg, Sweden The AMP-activated protein kinase (AMPK) is an evo- lutionarily conserved enzyme that is important for metabolic sensing both within individual cells and at a whole body level [1–3]. AMPK activation has been shown to increase glucose uptake and fatty acid oxida- tion in skeletal muscle, suppress glucose output from the liver, and diminish adiposity [4–7], and as a result AMPK is considered a promising novel drug target for the treatment of diabetes and related metabolic disorders. AMPK is a heterotrimeric complex consisting of a catalytic a-subunit and regulatory b- and c-subunits, Keywords allosteric regulation by AMP; AMP-activated protein kinase; c3 isoform Correspondence M. Mahlapuu, Discovery Research, Biovitrum AB, Biotech Center, Arvid Wallgrens Backe 20, SE-413 46 Go ¨ teborg, Sweden Fax: +46 31 749 1101 Tel: +46 31 749 1126 E-mail: margit.mahlapuu@biovitrum.com *These authors contributed equally to this study (Received 9 February 2007, revised 20 March 2007, accepted 4 April 2007) doi:10.1111/j.1742-4658.2007.05821.x The AMP-activated protein kinase is an evolutionarily conserved hetero- trimer that is important for metabolic sensing in all eukaryotes. The muscle- specific isoform of the regulatory c -subunit of the kinase, AMP-activated protein kinase c3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of c3. We demonstrate that AMP-activated protein kinase a2b2c3 trimers are allosterically activated approximately three-fold by AMP with a half- maximal stimulation (A 0.5 ) at 1.9 ± 0.5 or 2.6 ± 0.3 lm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of c3 (R225Q, H306R and R307G) increased the A 0.5 values for AMP, whereas the fold activation of the enzyme by 200 lm AMP remained unchanged in compari- son to the wild-type complex. The mutations in the C-terminal Bateman domain of c3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 lm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q ⁄ R454G. A V224I mutation in c3, known to result in a reduced gly- cogen content in pigs, did not affect the fold activation or the A 0.5 values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of a2 by the upstream kinases in the presence of increasing con- centrations of AMP. Taken together, the data show that different muta- tions in c3 exert different effects on the allosteric regulation of the a2b2c3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of a2 by upstream kinases. Abbreviations AICAR, 5-aminoimidazole-4-carboxamide-1-b- D-ribonucleoside; AMPK, AMP-activated protein kinase; AMPKK, AMP-activated protein kinase kinase; CAMKKb,Ca 2+ ⁄ calmodulin-dependent protein kinase b; CBS, cystathionine-b-synthase; TAK1, transforming growth factor b-activating kinase 1; ZMP, 5-aminoimidazole-4-carboxamide riboside monophosphate. FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS 2887 all of which are required for its activity [8,9]. In the mammalian genome, two isoforms of the a-subunit (a1 and a2) and the b-subunit (b1 and b2), and three iso- forms of the c-subunit (c1, c2 and c3), are present, with 12 possible heterotrimeric combinations. The cat- alytic a-subunit contains a conventional serine ⁄ threo- nine protein kinase domain [10], and phosphorylation of Thr172 within the activation loop of the a-subunit is essential for the activity of the enzyme. Several upstream kinases have been shown to phosphorylate AMPK at Thr172, including tumor suppressor LKB1 [11–13], Ca 2+ ⁄ calmodulin-dependent protein kinase b (CAMKKb) [14,15] and transforming growth fac- tor b-activating kinase (TAK1) [16]. The three AMPK c-subunit isoforms differ in their N-terminal sequences but share the four tandem repeats of a structural module known as the cystathionine-b-synthase (CBS) domain, located in the C-terminal region [17]. First recognized by Bateman [18], CBS motifs contain about 60 residues and are found in a range of diverse pro- teins. The basic functional unit is believed to contain two CBS motifs, which associate closely together [19], binding ligands containing adenosine [20], and the term ‘Bateman domain’ has been suggested to refer to the structure formed by two tandem repeats [21]. In mammalian AMPK, the CBS domains have been shown to bind the allosteric activator of the kinase, AMP, whereas the two pairs of CBS domains both bind one molecule of AMP [20]. In terms of the struc- ture of AMPK, the b-subunit has been reported to act as a scaffold for binding of the a-subunit and c-sub- unit [22]. Previously, we have shown that AMPK c3 is selec- tively expressed in glycolytic (white, fast-twitch type II) skeletal muscle [23], and is thus the only AMPK sub- unit isoform exhibiting strictly restricted tissue-specific expression. Furthermore, in both human and rodent skeletal muscle, c3 primarily forms complexes with the a2 and b2 isoforms [23,24]. Two naturally occurring missense mutations have been identified in the first CBS domain of the pig c3 gene, resulting in increased (R225Q) or decreased (V224I) skeletal muscle glycogen content [25–27]. R225Q carriers are also characterized by a higher oxidative capacity in white skeletal muscle fibers [28,29]. Transgenic mice with skeletal muscle- specific expression of the R225Q substituted form of mouse c3 replicate the phenotype observed in pigs, with elevated glycogen levels and increased fat oxida- tion in muscle tissue [30,31]. Recently, R225W and R307C substitutions in the human c3 gene have been reported to result in increased muscle glycogen con- tent, indicating that the function of AMPK c3 is con- served across the mammalian species [32]. The R225Q mutation in c3 is equivalent to the R302Q substitution in c2, the dominant isoform of the c-subunit in the heart. The R302Q mutation, together with several other substitutions identified in CBS domains of the c2 gene, cause Wolff–Parkinson–White syndrome in humans, which is characterized by electrophysiologic abnormalities and hypertrophy of the heart, and also by an increase in cardiac glycogen content [33–36]. Recent studies using transgenic mice overexpressing c2 harboring these mutations showed a disease phenotype that closely mimicked the human condition [37–40]. Direct binding, modeling and mutagenesis studies on AMPK c1 and c2 suggest that the mutations in CBS domains directly interfere with AMP binding, as the mutations are predicted to lie around the mouth of the AMP-binding cleft [20,41]. However, the mechanism by which the mutations lead to the observed pheno- types remains controversial. In this study, we have utilized AMPK a2b2c3 hetero- trimers expressed in Escherichia coli to characterize the enzyme kinetic parameters and regulation mechanisms for these physiologically relevant complexes. We have also evaluated the impact of CBS domain mutations on a-subunit phosphorylation and AMP dependence of a2b2c3, to improve our understanding of the functional consequences of the substitutions. Results In this work, we used AMPK a2b2c3 heterotrimers expressed in E. coli to characterize the regulation of these complexes by AMP as well as to evaluate the effect of substitutions within the AMPK c3 gene on the activity of the enzyme. Neumann et al. [42] des- cribed gaining milligram amounts of the AMPK a1b1c1inE. coli using a tricistronic vector with T7 RNA polymerase controlling transcription of a single a1b1c1 messenger, with each subunit carrying its indi- vidual ribosome-binding site. In our hands, the expres- sion of AMPK a2b2c3 in bacteria by a similar approach gave a lower yield of the trimer, due to solu- bility problems. However, AMPK a2b2c3 complex could be purified from the soluble protein fraction by using a polyhistidine tag fused to a2, and the forma- tion of a stable trimer was demonstrated by SDS ⁄ PAGE and western blot analysis using subunit-specific antibodies (Fig. 1A,B). As E. coli is not capable of phosphorylating Thr172 of the a-subunit, the bacteri- ally expressed trimers were initially inactive, but could be activated through phosphorylation by the upstream kinase LKB1 (Fig. 1B). After incubation with LKB1, the enzymatic activity of the bacterially expressed AMPK a2b2c3 trimers was similar to that of the Regulation of AMPK a2b2c3 complexes by AMP K. Lindgren et al. 2888 FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS a2b2c3 complexes purified from transiently transfected mammalian cells, with respect to both the total activity and the AMP dependence (Fig. 1C). We measured the activity of the recombinant AMPK a2b2c3 complexes over a range of AMP concentrations, and calculated the concentration of AMP giving half-maximal stimu- lation (A 0.5 ). The bacterially expressed phosphorylated AMPK a2b2c3 trimers had an A 0.5 value of 1.9 ± 0.5 lm (Table 1), which is very similar to the A 0.5 for a2b2c3 complexes expressed in COS7 cells (2.6 ± 0.3 lm). In order to study the effect of the mutations within AMPK c3 on the activity of the enzyme, we intro- duced several missense mutations into the c3 gene (Fig. 2), and investigated the effect of these substitu- tions on the allosteric activation of the kinase by AMP. Our main focus was to study the effects of mutations in positions V224, R225 and R307 in the first Bateman domain, given that substitutions in these positions occur in vivo in the pig and ⁄ or human AMPK c3 genes [25,26,32]. Also, the physiologic con- sequence of the R225Q mutation in the AMPK c3 A C D B Fig. 1. Characterization of the AMPK a2b2c3 complexes expressed in E. coli. (A) SDS ⁄ PAGE of bacterially expressed AMPK a2b2c3 complexes after purification by nickel–ion and gel filtration chroma- tography, stained with silver stain. (B) Representative western blot of AMPK a2b2c3 complexes expressed in E. coli and COS7 cells using subunit-specific antibodies raised to either a2, b2, c3or phosphorylated Thr172 in the a-subunit (apThr172). Bacterially expressed complexes were phosphorylated by incubation with an upstream kinase, LKB1. (C) The kinase activity of the AMPK a2b2c3 trimers expressed as pmoles of phosphate transferred to the SAMS peptide in the absence (open bars) or presence (black bars) of AMP (160 l M). Data are presented as mean ± SEM of n ¼ 4. In the activity assay, the equivalent amounts of hetero- trimers expressed in bacteria or mammalian cells were used, as estimated by western blot (B). (D) Western blot analysis of AMPK a2b2c3 complexes expressed in E. coli using antibodies to a2or apThr172. The trimers carry either a wild-type (WT) or R225Q ⁄ R454G version of c3. AMPK complexes were phosphorylated by LKB1 or CAMKKb in the absence or presence of AMP (0, 50, 200 or 500 l M AMP). Table 1. Effect of the mutations in the AMPK c3 gene on the AMP dependence of the enzyme. Fold stimulation reflects the activation of the corresponding AMPK complexes by 200 l M AMP relative to the basal activity in the absence of added AMP (calculated from the data presented in Fig. 3B). Significant difference in the AMP-sti- mulated versus basal activity for each complex was determined by two-sided Student’s t-test. A 0.5 (concentration of AMP giving half- maximal stimulation) was calculated from the curves presented in Fig. 3C (curve fitting by KALEIDAGRAPH 4.03 with the Michaelis–Men- ten equation). Values are means ± SEM. NS, not significant; ND, not determined. Mutations in c3 Fold activation by AMP P-value A 0.5 (lM) Wild-type 2.8 ± 0.1 < 0.01 1.9 ± 0.5 V224I 2.6 ± 0.1 < 0.001 2.3 ± 0.7 R225Q 3.5 ± 0.3 < 0.005 131 ± 68 H306R 3.8 ± 0.2 < 0.05 29 ± 6 R307G 3.0 ± 0.2 < 0.001 70 ± 20 H453R 1.6 ± 0.1 < 0.05 ND R454G 1.5 ± 0.1 < 0.05 ND R225Q ⁄ R454G 1.3 ± 0.1 NS ND K. Lindgren et al. Regulation of AMPK a2b2c3 complexes by AMP FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS 2889 gene has been extensively studied using transgenic mouse models [30,31]. Additionally, we generated H306R, H453R, R454G and R225Q ⁄ R454G substitu- tions in c3, which occur at the positions corresponding to R225Q and R307G, in the CBS2 and CBS4 domains (Fig. 2). All complexes were expressed in E. coli and purified from the soluble fraction by nickel–ion chromatography in quantities similar to those of the trimers containing a wild-type c3 (Fig. 3A), suggesting that there was no difference in the assembly of the different mutants into a hetero- trimeric complex. Western blot analysis showed that, in the conditions tested, the phosphorylation level of Thr172 of a2 by the upstream kinase LKB1 did not differ for any of the mutants (Fig. 3A). The V224I, R225Q, H306R and R307G substitutions in AMPK c3 did not affect the fold activation of the enzyme as compared to the wild-type trimers, when measured in the presence of 200 lm AMP (Fig. 3B, Table 1). How- ever, the H453R and R454G substitutions substantially reduced the AMP dependence of the enzyme down to approximately the same level as that of the double mutant R225Q ⁄ R454G (Fig. 3B, Table 1). We calcula- ted the A 0.5 values for the mutant complexes, which showed robust activation by AMP at a concentration of 200 lm (V224I, R225Q, H306R and R307G; Fig. 3C) using the hyperbolic curve model (Hill coeffi- cient ¼ 1). All the substitutions tested, except V224I, increased the A 0.5 value for AMP as compared to the wild-type trimers (Fig. 3C, Table 1; R 2 ‡ 0.97). Pharmacologic activation of AMPK can be achie- ved using 5-aminoimidazole-4-carboxamide-1-b-d-ribo- nucleoside (AICAR). Once taken up by cells, AICAR is phosphorylated to 5-aminoimidazole-4-carboxamide riboside monophosphate (ZMP), which mimics the effects of AMP on AMPK. In our hands, 160 lm ZMP caused a two-fold activation of the bacterially expressed wild-type AMPK a2b2c3 complexes, with half-maximal stimulation (A 0.5 )at61±19lm ZMP. ZMP failed to significantly increase the activity of the AMPK a2b2c3 complexes with an R225Q mutation in c3, when meas- ured at the concentration of 160 lm (Fig. 3C). The presence of AMP (0–500 lm) during the phosphorylation of the bacterially expressed AMPK a2b2c3 trimers with LKB1 or CAMKKb did not increase the phosphorylation level of Thr172 of a2 as shown by western blot analysis (Fig. 1D). Simi- larly, the phosphorylation of AMPK trimers con- taining R225Q ⁄ R454G mutations in c3 by LKB1 or CAMKKb did not differ at varying AMP concentra- tions (Fig. 1D). In line with this, we did not detect any increase in the activity of the AMPK a2b2c3 com- plexes in the presence of AMP in the phosphorylation reaction by LKB1, as measured through their ability to incorporate phosphate into the SAMS substrate, as compared to the complexes phosphorylated in the absence of AMP (data not shown). Discussion We have previously shown that AMPK c3 is the pre- dominant c isoform expressed in glycolytic skeletal muscle, where it primarily forms heterotrimers with the a2 and b2 isoforms [23]. The present study des- cribes bacterial expression of AMPK a2b2c3 trimers, and provides the first characterization of the enzyme kinetic parameters and regulation mechanisms for these physiologically relevant complexes. Fig. 2. An alignment of the amino acids in the four CBS domains in human AMPK c1, c2 and c3. Sequences were aligned using CLUSTALX (1.83). A versatile coloring scheme was incorporated to highlight conserved fea- tures in the alignment, using a default color code of CLUSTALX. Above the alignment is the conservation score generated by the program. The enlargement shows details of the mutations, with red borders indicating the mutations that have been described in vivo in pigs (V224I and R225Q in c3) or humans (mutations in c2, R225W and R307C in c3), and red letters indicating the mutations analyzed in the present study. Regulation of AMPK a2b2c3 complexes by AMP K. Lindgren et al. 2890 FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS The particular subunit isoforms present in the AMPK complex determine the concentration of AMP causing a half-maximal increase in the total activity as well as the fold stimulation by AMP [42–46]. Our experiments show that AMPK a2b2c3 trimers are acti- vated by AMP with a half-maximal stimulation (A 0.5 ) at 1.9 ± 0.5 or 2.6 ± 0.3 lm, as measured for com- plexes expressed in E. coli or mammalian cells, respect- ively (Fig. 3C, Table 1). Typically, 200 lm AMP caused an activation of 2–4-fold of the AMPK a2b2c3 trimers expressed in bacteria or COS7 cells, relative to the activity in the absence of AMP. Previously, allos- teric regulation of c3-containing AMPK complexes by AMP has been questioned, on the basis of activity measurements on rat brain protein immunoprecipitates using antibodies to c3 [17]. One plausible reason for these discrepancies may be a poor specificity of the antibodies applied, in combination with the fact that protein lysate from brain was used, as we and others have not been able to detect any c3 mRNA or protein in this tissue [23,25]. In addition, it is possible that the antibody binding itself interferes with the AMP-bind- ing properties of c3. Additionally, the low A 0.5 value characterizing the activation of a2b2c3 trimer by AMP makes the system highly sensitive to the possible pres- ence of any traces of AMP, either endogenous or as a result of the presence of AMP-generating proteins as contaminants [47]. Several upstream kinases (AMPKKs) have been reported to activate mammalian AMPK through phos- phorylation of Thr172 in the a-subunit, i.e. LKB1 [11–13], CAMKKb [15,48] and TAK1 [16]. LKB1 is believed to be the major upstream kinase for AMPK in skeletal muscle, as knocking out LKB1 almost com- pletely prevents both AICAR- and contraction-induced a2-AMPK signaling [49]. Initially, AMP was thought to increase phosphorylation of AMPK by AMPKK both by direct activation of the upstream kinase and by making the AMPK a better substrate for AMPKK, through binding to the c-subunits [50]. However, sev- eral recent studies have challenged this notion [15,48], and the role of AMP in phosphorylation of AMPK has remained controversial. While this manuscript was A B C Fig. 3. Effect of the substitutions in the AMPK c3 gene on the activity of the enzyme. (A) AMPK a2b2c3 complexes with either wild-type c3 (WT) or mutant c3 harboring the indicated substitu- tions were expressed in E. coli, and equal amounts of the trimers were phosphorylated in vitro by LKB1. Representative western blots probed with antibodies to a2, b2, c3orapThr172 are shown. (B) Activity of the AMPK a2b2c3 complexes was measured by a radioactive filter paper assay using SAMS substrate in the absence (open bars) or presence (black bars) of AMP (200 l M). The data pre- sented are the mean ± SEM from three to four independent phos- phorylation experiments (n ¼ 2 in the activity assay), expressed relative to the activity of the wild-type AMPK complexes measured in the absence of AMP (the activity of the wild-type complex with- out AMP is set to 1). (C) The activity of the wild-type and mutated AMPK a2b2c3 complexes was measured over a range of AMP (from 0 to 160 l M) or ZMP (from 0 to 160 lM) concentrations. The data are expressed relative to the basal activity in the absence of added AMP for each complex (n ¼ 3 in the activity assay; basal activity is set to 0). The graph is plotted in KALEIDAGRAPH 4.03 (Syn- ergy Software), using curve fitting against the one-site Michaelis– Menten equation; V ¼ V max · [S] ⁄ (A 0.5 + [S]), where V max is the maximal activation, A 0.5 ¼ [AMP] at 50% AMPK activation, and V ¼ AMPK activity. Given the very low level or lack of activation by AMP, we were unable to measure A 0.5 values for H453R, R454G and R225Q ⁄ R454G. All the AMPK complexes were expressed in E. coli except for the wild-type trimers denoted by asterisks, which were expressed in COS7 cells. In (B) and (C), equivalent amounts of AMPK were used, as estimated by western blot analysis (A). K. Lindgren et al. Regulation of AMPK a2b2c3 complexes by AMP FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS 2891 in preparation, two studies reported that AMP failed to promote phosphorylation of c1-containing AMPK trimers, when recombinant upstream kinase and AMPK preparations were used [44,47]. In line with these studies, we did not detect any increase in phos- phorylation of a2ina2b2c3 complexes by LKB1 or CAMKKb in the presence of increasing concentrations of AMP (Fig. 1D). Despite the considerable effort invested in this area, the nature of the mutations in the CBS domains of the AMPK c3 gene has remained controversial. A previous report provided evidence that the activity of AMPK was reduced in skeletal muscle of c3 R225Q mutant pigs [25]. In resting muscle from c3 R225Q mutant mice, AMPK activity was reported to be unaltered [30] or reduced [31]. However, in vivo activity measurements are complicated by the potential inhibitory effects of glycogen overload, which characterizes skeletal muscle of mice and pigs carrying the c3 R225Q substitution, on AMPK activation. Therefore, the effect of the mutation has been addressed in mammalian cells. In COS7 cells transfected with plasmids encoding a2b2c3 R225Q or V224I mutants, both substitutions resulted in diminished AMP dependence of AMPK, as com- pared to the wild-type trimers. In addition, AMPK total activity and phosphorylation of a2 were shown to be markedly elevated in cells expressing a2b2c3 R225Q, as compared to the wild-type or V224I-con- taining trimers [30]. However, in our laboratory we have failed to detect any increase in the basal activity for R225Q complexes assayed from material purified from transiently transfected COS7 cells (data not shown). It has to be noted that the activity of the AMPK is highly sensitive to the lysis protocol used (hypoxia, glucose deprivation and mechanical stress during the cell harvest activate AMPK), which poten- tially complicates the comparisons of the results from different research groups and may partly explain the discrepancies observed. In an effort to mimic the R225Q mutation in c3, the equivalent position has been mutated in AMPK c1 (R70Q) as well as in c2 (R302Q) [41,44,45,51]. In the present study, we evalu- ated the impact of CBS domain mutations (locations of the substitutions are shown in Fig. 2) on the AMP dependence of the AMPK a2b2c3 using the enzyme expressed in E. coli, activated in vitro by an LKB1 pre- paration. Importantly, we did not detect any difference in phosphorylation of Thr172 of a2 by LKB1 when we compared the different mutants to the wild-type trimers (Fig. 3A), although we have to acknowledge that the semiquantitative nature of the western blot technique makes the exact measurement difficult. It is noteworthy that for the mutations in the N-terminal Bateman domain formed by CBS1–2 (V224I, R225Q, H306R and R307G), the fold activation of the complex by 200 lm AMP was similar to that of wild-type com- plexes (Fig. 3B, Table 1). Mutations in the C-terminal Bateman domain formed by CBS3–4 (H453R and R454G), on the other hand, significantly reduced AMP stimulation and resulted in AMP dependence curves highly similar to that of the double mutant R225Q ⁄ R454G (Fig. 3B, Table 1). The R225Q, H306R and R307G mutations substantially increased the A 0.5 value for AMP as compared to the wild-type trimers (Fig. 3C, Table 1). The V224I mutation, on the other hand, resulted in AMP dependence curves that were very similar to those of wild-type complexes (Fig. 3C, Table 1). According to the current model of the allos- teric control of mammalian AMPK, the N-terminal and C-terminal pairs of CBS domains both bind one molecule of AMP [20,41]. However, the different effects on AMP activation observed in this study of substitu- tions in corresponding positions of the two Bateman domains (H306R and R307G in CBS2 are equivalent to H453R and R454G in CBS4, respectively, Fig. 2) indicate that the two suggested AMP-binding sites in c3 are not functionally equivalent. Notably, the AMP dependence curves of the AMPK a2b2c3 complexes appear hyperbolic rather than sigmoid (Fig. 3C). While this manuscript was in preparation, a crystal struc- ture of the Schizosaccharomyces pombe AMPK was reported, demonstrating that one AMP molecule bound to a single site formed by the CBS domains of the c-subunit [52]. Nevertheless, the absence of regula- tion of Saccharomyces cerevisiae AMPK activity by AMP [53,54] raises the possibility that nucleotide bind- ing in Sc. pombe AMPK may differ from that in the human enzyme. The present study does not explain the mechanisms by which the mutations in CBS domains of AMPK c3 interfere with activation by AMP. However, our data would be consistent with a single AMP-binding site being present in c3. The A 0.5 value for ZMP was increased for the R225Q AMPK complexes as compared to the wild- type heterotrimers, which was expected, as ZMP is thought to bind to the same site as AMP and activate AMPK in a similar manner. This result may explain the impairment of AICAR-stimulated muscle glucose uptake described in R225Q transgenic mice, as com- pared to the wild-type littermates [30]. The present study does not answer the question of how the reduced AMP dependence of the R225Q vari- ants of c3 would lead to the increased glycogen content described in the skeletal muscle of the carriers of this mutation [25,30]. Also, we did not detect any difference in the regulation of AMPK complexes containing a Regulation of AMPK a2b2c3 complexes by AMP K. Lindgren et al. 2892 FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS V224I form of c3 by AMP, whereas this mutation is known to lead to reduced skeletal muscle glycogen stores [26]. Clearly, an important challenge is to deter- mine the effect of the mutations in the CBS domains on AMPK activity in the presence of physiologic AMP and ATP concentrations, which is technically difficult. The in vitro AMPK activity assay is commonly per- formed at 200 lm ATP, utilizing radiolabeled ATP, which causes technical problems when the concentra- tion is increased to close to physiologic levels [55,56]. The possibility of altered functionality of the AMPK a2b2c3 trimers expressed in E. coli, as compared to the in vivo complexes, also has to be acknowledged. Native AMPK is known to be post-translationally modified at multiple sites, other than Thr172, in the a-subunit [13,46,57], and the possibility that the absence of these modifications has an impact on the activity of the mutant complexes cannot be excluded. Another possi- bility would be that interactions other than with AMP ⁄ ATP, occurring in eukaryotic cells only, have an impact on the activity of the enzyme. Further studies in relevant cell systems as well as in animal models are required to investigate these issues. Experimental procedures Expression and purification of recombinant AMPK a2b2c3inE. coli The tricistronic plasmids containing the three AMPK sub- units a2 (mouse, accession number NP_835279) with N-ter- minal polyhistidine tag, b2 (human, accession number O43741) and c3 (human, accession number Q9UGI9) in pET vector (Novagen, Madison, WI), were transformed into competent host cells (E. coli BL21 DE3 pLysS; Nov- agen). Single colonies were used to inoculate 5 mL of LB medium containing 100 lgÆ mL )1 carbenicillin, 50 lgÆmL )1 chloramphenicol and 0.5% glucose. Following incubation in a shaker incubator (250 r.p.m.) overnight at 37 °C, the starter cultures were used to inoculate 0.5 L of the above medium. Protein expression was induced with 1 mm isopro- pyl thio-b-d-galactoside (final concentration) at an A 600 of 0.5–0.7, and cultures were grown for an additional 3 h at 37 °C. Cells were harvested (4000 r.p.m. for 15 min at 4 °C; Allegra 6R), and the cell pellet was resuspended in the lysis buffer containing 50 mm Hepes (pH 7.5), 50 mm NaF, 5 mm sodium pyrophosphate, 1 mm EDTA, 1 mm di- thiothreitol, 10% glycerol, 1% Triton X-100, lysozyme (48 lgÆmL )1 ), and complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), and placed on ice for 1 h before being sonicated (Dr Hielscher GmbH, Teltow, Germany) for 3 min. Insoluble material was removed by centrifugation [20 400 g for 20 min at 4 °C using an Avanti J-20 XP centrifuge (Beckman Coulter), rotor type JA25.50]. The supernatant was collected and diluted in buffer A containing 20 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl, 15 mm imidazole, and 10% glycerol, and loaded onto a 5 mL His-Trap column (GE Healthcare, Uppsala, Sweden). Bound protein was eluted using a gradi- ent with buffer B (identical to buffer A except for contain- ing 500 mm imidazole), and stored at ) 20 °C until use. In some cases, the AMPK complex was further purified by gel filtration chromatography with a Superdex 200 pg HiLoad 16 ⁄ 60 column (GE Healthcare) connected to A ¨ kta FPLC (GE Healthcare). Expression and purification of recombinant AMPK a2b2c3 in COS7 cells COS7 cells were cotransfected with cDNAs encoding mouse AMPK a2, b2 (human) and c3 [human, all cloned into pcDNA3.1(+); Invitrogen, Paisley, UK] using Lipofectin reagent, according to the manufacturer’s instructions (Invi- trogen). Immediately prior to the lysis, the cells were sub- mitted to hyperosmotic stress by incubating them for 30 min with sorbitol (final concentration 0.6 m) in the culture med- ium. Cells were harvested 48 h post-transfection by rapid lysis: the medium was removed, and ice-cold lysis buffer (see above) was added. Insoluble material was removed by centrifugation [15 700 g for 20 min at 4 °C using a 5415R centrifuge (Eppendorf), rotor type, 24 places fixed angle], and AMPK was partially purified from the soluble fraction using a DEAE–sepharose ion-exchange step (GE Health- care). The major part of the endogenous AMPK was deple- ted by immunoprecipitation of the relevant fractions with antibody to a1 prebound to protein A–sepharose as described previously [23]. Site-directed mutagenesis The mutations V224I, R225Q, H306R, R307G, H453R, R454G and R225Q ⁄ R454G were introduced into AMPK c3 using the Quick Change II site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutagenesis primers were designed using Stratagene’s Tm calculator (primer seq- uences are available on request). Point mutations generated in vitro were confirmed by DNA sequencing. Western blotting Quantitative analysis of the expression of different AMPK subunits was performed as described previously [23]. Phosphorylation of bacterial trimer and AMPK activity assay To activate the bacterial AMPK, recombinant trimers were incubated with LKB1 or CAMKKb in the presence of K. Lindgren et al. Regulation of AMPK a2b2c3 complexes by AMP FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS 2893 0.2 mm ATP, 5 mm MgCl 2 and 1 mm dithiothreitol for 1 h at 32 °C in a thermostated shaker. Rat liver LKB1 was purified as described previously [12] up to the Q-sepharose step, except that the poly(ethylene glycol) precipitation of the liver lysate was omitted. Bacterially expressed CAMKKb was a gift from D. Carling [14,15]. To determine the relevant amount of upstream kinase versus AMPK to be used in the phosphorylation reaction, the AMPK trimers were incubated with increasing concentrations of AMPKK, and the amount of upstream kinase corresponding to the highest possible level of Thr172 phosphorylation was used in the reaction. AMPK activity was determined by in vitro phosphorylation of the SAMS (HMRSAMSGLHLVKRR) synthetic peptide substrate as previously described [58]. Briefly, kinase reactions were initiated by adding 4 lLof the phosphorylated AMPK to 21 lL of assay buffer con- taining 50 mm Hepes (pH 7.5), 80 mm NaCl, 8% glycerol, 5mm MgCl 2 , 0.8 mm EDTA, 0.8 mm dithiothreitol, 0.2 mm ATP, 0.2 mm SAMS peptide, and [ 32 P]ATP (final concentration 0.016 lCiÆl L )1 ) in the presence or absence of varying concentrations of AMP, as described in the figure legends. Reactions were incubated on a vibrating platform for 3 h at 37 °C. The reactions were terminated by adding trichloroacetic acid to a final concentration of 13%, fol- lowed by centrifugation. Immediately thereafter, aliquots were spotted onto Whatman P81 paper, and washed with 1% phosphoric acid, and the incorporation of 32 P into peptide substrate was measured in a liquid scintillation counter. Acknowledgements We gratefully acknowledge Professor David Carling (Medical Research Council Clinical Sciences Center, Imperial College, Hammersmith Hospital, London, UK) for the recombinant CAMKKb preparation used in this study and for discussions concerning the work presented. This work was supported by an Integrated Project from the European Commission (LSHG-CT- 2005-518181). References 1 Carling D (2005) AMP-activated protein kinase: balan- cing the scales. Biochimie 87, 87–91. 2 Hardie DG, Hawley SA & Scott JW (2006) AMP-acti- vated protein kinase-development of the energy sensor concept. J Physiol 574, 7–15. 3 Kahn BB, Alquier T, Carling D & Hardie DG (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1, 15–25. 4 Hayashi T, Hirshman MF, Kurth EJ, Winder WW & Goodyear LJ (1998) Evidence for 5¢-AMP-activated protein kinase mediation of the effect of muscle contrac- tion on glucose transport. Diabetes 47, 1369–1373. 5 Merrill GF, Kurth EJ, Hardie DG & Winder WW (1997) AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol 273, E1107–E1112. 6 Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D & Kahn BB (2002) Leptin stimulates fatty-acid oxidation by activating AMP-activated pro- tein kinase. Nature 415, 339–343. 7 Ruderman NB, Saha AK & Kraegen EW (2003) Mini- review: malonyl CoA, AMP-activated protein kinase, and adiposity. Endocrinology 144, 5166–5171. 8 Kemp BE, Stapleton D, Campbell DJ, Chen ZP, Murthy S, Walter M, Gupta A, Adams JJ, Katsis F, van Denderen B et al. (2003) AMP-activated protein kinase, super metabolic regulator. Biochem Soc Trans 31, 162–168. 9 Stapleton D, Woollatt E, Mitchelhill KI, Nicholl JK, Fernandez CS, Michell BJ, Witters LA, Power DA, Sutherland GR & Kemp BE (1997) AMP-activated pro- tein kinase isoenzyme family: subunit structure and chromosomal location. FEBS Lett 409, 452–456. 10 Kemp BE, Mitchelhill KI, Stapleton D, Michell BJ, Chen ZP & Witters LA (1999) Dealing with energy demand: the AMP-activated protein kinase. Trends Bio- chem Sci 24, 22–25. 11 Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, Depinho RA & Cantley LC (2004) The tumor suppressor LKB1 kinase directly activates AMP- activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci USA 101, 3329–3335. 12 Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR & Hardie DG (2003) Complexes between the LKB1 tumor suppressor, STRAD alpha ⁄ beta and MO25 alpha ⁄ beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2, doi:10.1186/1475-4924-2-28. 13 Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U, Wallimann T, Carlson M & Carling D (2003) LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13, 2004–2008. 14 Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR & Witters LA (2005) The Ca2+ ⁄ calmodu- lin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280, 29060–29066. 15 Woods A, Dickerson K, Heath R, Hong SP, Momcilo- vic M, Johnstone SR, Carlson M & Carling D (2005) Ca2+ ⁄ calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mam- malian cells. Cell Metab 2, 21–33. 16 Momcilovic M, Hong SP & Carlson M (2006) Mamma- lian TAK1 activates Snf1 protein kinase in yeast and Regulation of AMPK a2b2c3 complexes by AMP K. Lindgren et al. 2894 FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS phosphorylates AMP-activated protein kinase in vitro. J Biol Chem 281, 25336–25343. 17 Cheung PC, Salt IP, Davies SP, Hardie DG & Carling D (2000) Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem J 346 (Part 3), 659–669. 18 Bateman A (1997) The structure of a domain common to archaebacteria and the homocystinuria disease pro- tein. Trends Biochem Sci 22, 12–13. 19 Zhang R, Evans G, Rotella FJ, Westbrook EM, Beno D, Huberman E, Joachimiak A & Collart FR (1999) Characteristics and crystal structure of bacterial inosine- 5¢-monophosphate dehydrogenase. Biochemistry 38, 4691–4700. 20 Scott JW, Hawley SA, Green KA, Anis M, Stewart G, Scullion GA, Norman DG & Hardie DG (2004) CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations. J Clin Invest 113, 274–284. 21 Kemp BE (2004) Bateman domains and adenosine derivatives form a binding contract. J Clin Invest 113, 182–184. 22 Woods A, Cheung PC, Smith FC, Davison MD, Scott J, Beri RK & Carling D (1996) Characterization of AMP-activated protein kinase beta and gamma subu- nits. Assembly of the heterotrimeric complex in vitro. J Biol Chem 271, 10282–10290. 23 Mahlapuu M, Johansson C, Lindgren K, Hjalm G, Barnes BR, Krook A, Zierath JR, Andersson L & Marklund S (2004) Expression profiling of the gamma- subunit isoforms of AMP-activated protein kinase suggests a major role for gamma3 in white skeletal muscle. Am J Physiol Endocrinol Metab 286, E194–E200. 24 Birk JB & Wojtaszewski JF (2006) Predominant {alpha}2 ⁄ {beta}2 ⁄ {gamma}3 AMPK activation during exercise in human skeletal muscle. J Physiol 577, 1021–1032. 25 Milan D, Jeon JT, Looft C, Amarger V, Robic A, Thelander M, Rogel-Gaillard C, Paul S, Iannuccelli N, Rask L et al. (2000) A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Sci- ence 288, 1248–1251. 26 Ciobanu D, Bastiaansen J, Malek M, Helm J, Woollard J, Plastow G & Rothschild M (2001) Evidence for new alleles in the protein kinase adenosine monophosphate- activated gamma(3)-subunit gene associated with low glycogen content in pig skeletal muscle and improved meat quality. Genetics 159, 1151–1162. 27 Estrade M, Vignon X, Rock E & Monin G (1993) Glycogen hyperaccumulation in white muscle fibres of RN- carrier pigs. A biochemical and ultrastructural study. Comp Biochem Physiol B 104, 321–326. 28 Lebret B, Le Roy P, Monin G, Lefaucheur L, Caritez JC, Talmant A, Elsen JM & Sellier P (1999) Influence of the three RN genotypes on chemical composition, enzyme activities, and myofiber characteristics of por- cine skeletal muscle. J Anim Sci 77, 1482–1489. 29 Estrade M, Ayoub S, Talmant A & Monin G (1994) Enzyme activities of glycogen metabolism and mito- chondrial characteristics in muscles of RN- carrier pigs (Sus scrofa domesticus). Comp Biochem Physiol Biochem Mol Biol 108, 295–301. 30 Barnes BR, Marklund S, Steiler TL, Walter M, Hjalm G, Amarger V, Mahlapuu M, Leng Y, Johansson C, Galuska D et al. (2004) The 5¢-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J Biol Chem 279, 38441–38447. 31 Yu H, Hirshman MF, Fujii N, Pomerleau JM, Peter LE & Goodyear LJ (2006) Muscle-specific overexpression of wild-type and R225Q mutant AMP-activated protein kinase gamma3-subunit differentially regulates glycogen accumulation. Am J Physiol Endocrinol Metab 291, E557–E565. 32 Costford SR, Chaudhry SN, Kavaslar N, Ahitus N, Pennacchio LA, Dent R, Mcpherson R & Harper M-E (2006) Novel mutations in the gamma-3 subunit of AMP-activated protein kinase affect protein function in humans. In 13th Biochemistry of Exercise International Conference, Seoul, Korea, Abstract P010. 33 Gollob MH, Seger JJ, Gollob TN, Tapscott T, Gonzales O, Bachinski L & Roberts R (2001) Novel PRKAG2 mutation responsible for the genetic syndrome of ventri- cular preexcitation and conduction system disease with childhood onset and absence of cardiac hypertrophy. Circulation 104, 3030–3033. 34 Gollob MH, Green MS, Tang AS, Gollob T, Karibe A, Ali Hassan AS, Ahmad F, Lozado R, Shah G, Fananapazir L et al. (2001) Identification of a gene responsible for familial Wolff–Parkinson–White syn- drome. N Engl J Med 344, 1823–1831. 35 Arad M, Benson DW, Perez-Atayde AR, Mckenna WJ, Sparks EA, Kanter RJ, Mcgarry K, Seidman JG & Seidman CE (2002) Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy. J Clin Invest 109, 357–362. 36 Blair E, Redwood C, Ashrafian H, Oliveira M, Brox- holme J, Kerr B, Salmon A, Ostman-Smith I & Watkins H (2001) Mutations in the gamma(2) subunit of AMP- activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet 10, 1215–1220. 37 Arad M, Moskowitz IP, Patel VV, Ahmad F, Perez- Atayde AR, Sawyer DB, Walter M, Li GH, Burgon PG, Maguire CT et al. (2003) Transgenic mice over- expressing mutant PRKAG2 define the cause of Wolff–Parkinson–White syndrome in glycogen storage cardiomyopathy. Circulation 107, 2850–2856. K. Lindgren et al. Regulation of AMPK a2b2c3 complexes by AMP FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS 2895 38 Sidhu JS, Rajawat YS, Rami TG, Gollob MH, Wang Z, Yuan R, Marian AJ, Demayo FJ, Weilbacher D, Taffet GE et al. (2005) Transgenic mouse model of ventricular preexcitation and atrioventricular reentrant tachycardia induced by an AMP-activated protein kinase loss-of-function mutation responsible for Wolff– Parkinson–White syndrome. Circulation 111, 21–29. 39 Davies JK, Wells DJ, Liu K, Whitrow HR, Daniel TD, Grignani R, Lygate CA, Schneider JE, Noel G, Watkins H et al. (2006) Characterization of the role of gamma2 R531G mutation in AMP-activated protein kinase in cardiac hypertrophy and Wolff–Parkinson–White syn- drome. Am J Physiol Heart Circ Physiol 290, H1942– H1951. 40 Zou L, Shen M, Arad M, He H, Lofgren B, Ingwall JS, Seidman CE, Seidman JG & Tian R (2005) N488I mutation of the gamma2-subunit results in bidirectional changes in AMP-activated protein kinase activity. Circ Res 97, 323–328. 41 Adams J, Chen ZP, Van Denderen BJ, Morton CJ, Parker MW, Witters LA, Stapleton D & Kemp BE (2004) Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site. Protein Sci 13, 155–165. 42 Neumann D, Woods A, Carling D, Wallimann T & Schlattner U (2003) Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr Purif 30, 230–237. 43 Scott JW, Norman DG, Hawley SA, Kontogiannis L & Hardie DG (2002) Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate. J Mol Biol 317, 309–323. 44 Sanders MJ, Grondin PO, Hegarty BD, Snowden MA & Carling D (2006) Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem J 403, 139–148. 45 Daniel T & Carling D (2002) Functional analysis of mutations in the gamma 2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff–Parkinson–White syndrome. J Biol Chem 277, 51017–51024. 46 Stein SC, Woods A, Jones NA, Davison MD & Carling D (2000) The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345 (Part 3), 437–443. 47 Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T & Neumann D (2006) Dissecting the role of 5¢-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase. J Biol Chem 281, 32207–32216. 48 Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG & Hardie DG (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2, 9–19. 49 Sakamoto K, Mccarthy A, Smith D, Green KA, Grahame Hardie D, Ashworth A & Alessi DR (2005) Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction. EMBO J 24, 1810–1820. 50 Hawley SA, Selbert MA, Goldstein EG, Edelman AM, Carling D & Hardie DG (1995) 5¢-AMP activates the AMP-activated protein kinase cascade, and Ca2+ ⁄ calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J Biol Chem 270, 27186–27191. 51 Hamilton SR, Stapleton D, O’Donnell JB Jr, Kung JT, Dalal SR, Kemp BE & Witters LA (2001) An activating mutation in the gamma1 subunit of the AMP-activated protein kinase. FEBS Lett 500, 163–168. 52 Townley R & Shapiro L (2007) Crystal structures of the adenylate sensor from fission yeast AMP-activated pro- tein kinase. Science 315, 1726–1729. 53 Wilson WA, Hawley SA & Hardie DG (1996) Glucose repression ⁄ derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepress- ing conditions, and this correlates with a high AMP:ATP ratio. Curr Biol 6, 1426–1434. 54 Woods A, Munday MR, Scott J, Yang X, Carlson M & Carling D (1994) Yeast SNF1 is functionally related to mammalian AMP-activated protein kinase and regulates acetyl-CoA carboxylase in vivo. J Biol Chem 269, 19509–19515. 55 Bergeron R, Russell RR 3rd, Young LH, Ren JM, Marcucci M, Lee A & Shulman GI (1999) Effect of AMPK activation on muscle glucose metabolism in conscious rats. Am J Physiol 276, E938–E944. 56 Sabina RL, Kernstine KH, Boyd RL, Holmes EW & Swain JL (1982) Metabolism of 5-amino-4-imidazole- carboxamide riboside in cardiac and skeletal muscle. Effects on purine nucleotide synthesis. J Biol Chem 257, 10178–10183. 57 Mitchelhill KI, Michell BJ, House CM, Stapleton D, Dyck J, Gamble J, Ullrich C, Witters LA & Kemp BE (1997) Posttranslational modifications of the 5¢-AMP- activated protein kinase beta1 subunit. J Biol Chem 272, 24475–24479. 58 Derave W, Ai H, Ihlemann J, Witters LA, Kristiansen S, Richter EA & Ploug T (2000) Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slow-twitch muscle. Diabetes 49, 1281–1287. Regulation of AMPK a2b2c3 complexes by AMP K. Lindgren et al. 2896 FEBS Journal 274 (2007) 2887–2896 ª 2007 Biovitrum AB. Journal compilation ª 2007 FEBS . Regulation of the muscle-specific AMP- activated protein kinase a2b2c3 complexes by AMP and implications of the mutations in the c3-subunit for the AMP dependence of the enzyme Kerstin Lindgren*,. important for metabolic sensing in all eukaryotes. The muscle- specific isoform of the regulatory c -subunit of the kinase, AMP- activated protein kinase c3, has a key role in glucose and fat metabolism. kinase; AMPKK, AMP- activated protein kinase kinase; CAMKKb,Ca 2+ ⁄ calmodulin-dependent protein kinase b; CBS, cystathionine-b-synthase; TAK1, transforming growth factor b-activating kinase 1;