AMPK and TOR: The Yin and Yang of Cellular Nutrient Sensing and Growth Control

The AMPK (AMP-activated protein kinase) and TOR (target-of-rapamycin) pathways are interlinked, opposing signaling pathways involved in sensing availability of nutrients and energy and regulation of cell growth. AMPK (Yin, or the “dark side”) is switched on by lack of energy or nutrients and inhibits cell growth, while TOR (Yang, or the “bright side”) is switched on by nutrient availability and promotes cell growth. Genes encoding the AMPK and TOR complexes are found in almost all eukaryotes, suggesting that these pathways arose very early during eukaryotic evolution. During the development of multicellularity, an additional tier of cell-extrinsic growth control arose that is mediated by growth factors, but these often act by modulating nutrient uptake so that AMPK and TOR remain the underlying regulators of cellular growth control. In this review, we discuss the evolution, structure, and regulation of the AMPK and TOR pathways and the complex mechanisms by which they interact. The AMPK (AMP-activated protein kinase) and TOR (target-of-rapamycin) pathways are interlinked, opposing signaling pathways involved in sensing availability of nutrients and energy and regulation of cell growth. AMPK (Yin, or the “dark side”) is switched on by lack of energy or nutrients and inhibits cell growth, while TOR (Yang, or the “bright side”) is switched on by nutrient availability and promotes cell growth. Genes encoding the AMPK and TOR complexes are found in almost all eukaryotes, suggesting that these pathways arose very early during eukaryotic evolution. During the development of multicellularity, an additional tier of cell-extrinsic growth control arose that is mediated by growth factors, but these often act by modulating nutrient uptake so that AMPK and TOR remain the underlying regulators of cellular growth control. In this review, we discuss the evolution, structure, and regulation of the AMPK and TOR pathways and the complex mechanisms by which they interact. All eukaryotic cells are now thought to have arisen via a single endosymbiotic event when an archaeal host cell engulfed bacteria that were capable of oxidative metabolism, the latter eventually becoming mitochondria (Lane, 2006Lane N. Power, sex and suicide: mitochondria and the meaning of life. Oxford University Press, 2006Google Scholar, Sagan, 1967Sagan L. On the origin of mitosing cells.J. Theor. Biol. 1967; 14: 255-274Crossref PubMed Google Scholar). This event was followed by the transfer of most of the genes from the genome of the endosymbiont to that of the host—it has been argued that this separation of energy-generating capacity from gene expression allowed a large increase in the energy available per gene, thus permitting a major expansion in gene number in the host (Lane and Martin, 2010Lane N. Martin W. The energetics of genome complexity.Nature. 2010; 467: 929-934Crossref PubMed Scopus (695) Google Scholar). This may in turn have enabled major enhancements in the complexity of eukaryotic cells compared with their prokaryotic counterparts, including the development of endomembrane systems such as lysosomes or vacuoles (de Duve, 2005de Duve C. The lysosome turns fifty.Nat. Cell Biol. 2005; 7: 847-849Crossref PubMed Scopus (173) Google Scholar), and the associated trafficking of materials between these internal compartments and the plasma membrane via membrane-bound vesicles. New cellular functions this led to were phagocytosis and pinocytosis, used by many protists today as mechanisms of feeding, and autophagy, used by all eukaryotic cells for recycling of cellular components that are damaged or surplus to requirements, or as an emergency measure during nutrient starvation. Phagocytosis, pinocytosis, and autophagy deliver proteins, lipids, and carbohydrates, or even whole organelles, such as mitochondria, to lysosomes or vacuoles; the latter are acidic compartments, where the engulfed materials are broken down to recycle their components either for catabolism or re-use. Lysosomes or vacuoles can therefore be considered to be the “gut” or digestive systems of unicellular eukaryotes, particularly in amoeboid protists that feed by phagocytosis or pinocytosis. They would therefore have been a major source of nutrients and appear to have developed into hubs for nutrient sensing, as discussed below. As these processes were evolving, early eukaryotes would have needed signaling pathways that could monitor the function of their new internal organelles and regulate cell growth and proliferation accordingly. For example, there would have been a need to monitor the output of ATP by mitochondria and to upregulate their ATP-generating capacity if or when the supply of ATP was insufficient; this is now a major function of the AMPK (AMP-activated protein kinase) signaling pathway. In addition, there would have been a requirement to monitor the supply of nutrients, such as amino acids and glucose, produced at the lysosome by phagocytosis, pinocytosis, or autophagy and to upregulate cell growth when these nutrients were available; this is now a key function of the TOR (target-of-rapamycin) pathway. We propose that these two opposing pathways, which are present in almost all present-day eukaryotes, are the descendants of ancient nutrient sensing and signaling pathways that arose very early during eukaryotic evolution. AMPK represents the Yin (“dark” or “passive”) side that signals lack of nutrients or insufficient ATP and inhibits cell growth, whereas TOR represents the Yang (“bright” or “active”) side that signals availability of nutrients and promotes cell growth. Just as in the Chinese philosophy of Taoism from which the Yin-Yang concept is derived, an appropriate balance between these two opposing elements ensures homeostasis and thus a healthy cell or organism. In present-day unicellular eukaryotes, including fungi like Saccharomyces cerevisiae, growth and proliferation are regulated almost entirely by nutrient availability, and the orthologs of AMPK and TOR play crucial roles in this. However, during the development of multicellular organisms, the uptake (and hence the intracellular availability) of nutrients has become modulated by an additional tier of cell-extrinsic regulation mediated by growth factors and cytokines (Palm and Thompson, 2017Palm W. Thompson C.B. Nutrient acquisition strategies of mammalian cells.Nature. 2017; 546: 234-242Crossref PubMed Scopus (71) Google Scholar). It can be argued that these cell-extrinsic factors “license” or allow cells to take up nutrients but that the AMPK and TOR pathways, which sense intracellular nutrient availability, remain the primary internal regulators of cell growth and proliferation. Interestingly, most of the mutations that cause cancer in multicellular organisms appear to affect the higher-level, cell-extrinsic regulation of cell growth. Such mutations allow cancer cells to become “rebels” that have partially reverted to their unicellular origins and that switch over to using cell-intrinsic growth control, based on nutrient availability and controlled by the AMPK and TOR pathways. AMPK appears to occur universally as heterotrimeric complexes comprising catalytic α ubunits and regulatory β and γ subunits (Ross et al., 2016bRoss F.A. MacKintosh C. Hardie D.G. AMP-activated protein kinase: a cellular energy sensor that comes in 12 flavours.FEBS J. 2016; 283: 2987-3001Crossref PubMed Google Scholar). Genes encoding all three subunits are readily found within the genomes of almost all eukaryotes (Table 1 and Figure 1). However, the orthologs in budding yeast (S. cerevisiae) and plants are not allosterically activated by AMP and were discovered independently of mammalian AMPK by genetic approaches (Alderson et al., 1991Alderson A. Sabelli P.A. Dickinson J.R. Cole D. Richardson M. Kreis M. Shewry P.R. Halford N.G. Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA.Proc. Natl. Acad. Sci. USA. 1991; 88: 8602-8605Crossref PubMed Scopus (0) Google Scholar, Celenza and Carlson, 1986Celenza J.L. Carlson M. A yeast gene that is essential for release from glucose repression encodes a protein kinase.Science. 1986; 233: 1175-1180Crossref PubMed Google Scholar). They are therefore not usually referred to as AMPK but instead in yeast as Snf1 complexes (SNF1 being the gene encoding the catalytic subunit) and in plants as Snf1-related kinase-1 (SnRK1) complexes.Table 1Gene and Protein Names for AMPK, TORC1, and TORC2 Subunit Orthologs in Humans and Different Model Organisms (Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe)Signaling complexH. sapiensA. thalianaD. melanogasterC. elegansS. cerevisiaeS. pombeGeneProteinLocusProteinGeneProteinGeneProteinGeneProteinGeneProteinAMPKPRKAA1PRKAA2α1α2AT3G01090AT3G29160KIN10KIN11snfAAMPKαaak-1aak-2AAK-1AAK-2SNF1Snf1ssp2ppk9Ssp2Ppk9PRKAB1PRKAB2β1β2AT5G21170AT4G16360AT2G28060KINβ1KINβ2KINβ3aThis is an unusual plant-specific β subunit that contains the C-terminal domain but lacks a CBM (carbohydrate-binding module).alcAMPKβaakb-1aakb-2AAKB-1AAKB-2SIP1SIP2GAL83Sip1Sip2Gal83amk2Amk2PRKAG1PRKAG2PRKAG3γ1γ2γ3AT3G48530AT1G09020KINγbBy sequence, this appears to be an ortholog of mammalian γ subunits, but it does not appear to form functional heterotrimers (Zhao, 2019).KINβγcThis is an unusual plant-specific γ subunit that contains a CBM (carbohydrate-binding module) fused to the four CBS motifs found in other AMPK-γ subunits.SNF4AγAMPKγaakg-1aakg-2aakg-3aakg-4aakg-5AAKG-1AAKG-2AAKG-3AAKG-4AAKG-5SNF4Snf4cbs2Cbs2TORC1MTORmTORAT1G50030TORtorTORlet-363TORTOR1/TOR2TOR1/TOR2tor2Tor2MLST8mLST8AT3G18140AT2G22040LST8-1LST8-2dThis is probably a non-functional protein (Moreau et al., 2012).lst8LST8mlst-8LST-8LST8Lst8pop3Pop3RPTORRAPTORAT5G01770AT3G08850RAPTOR1ARAPTOR1BraptorRAPTORdaf-15DAF-15KOG1Kog1mip1Mip1TORC2MTORmTORAT1G50030TORtorTORlet-363TORTOR2TOR2tor1Tor1MLST8mLST8AT3G18140AT2G2204LST8-1LST8-2dThis is probably a non-functional protein (Moreau et al., 2012).lst8LST8mlst-8LST-8LST8Lst8pop3Pop3RICTORRICTOR--rictorRICTORrict-1RICTORAVO3Avo3ste20Ste20MSIN1mSIN1--sin1SIN1sinh-1SIN1AVO1Avo1sin1Sin1a This is an unusual plant-specific β subunit that contains the C-terminal domain but lacks a CBM (carbohydrate-binding module).b By sequence, this appears to be an ortholog of mammalian γ subunits, but it does not appear to form functional heterotrimers (Zhao, 2019Zhao R.Q. Expression, purification and characterization of the plant Snf1-related protein kinase 1 from Escherichia coli.Protein Expr. Purif. 2019; 162: 24-31Crossref PubMed Scopus (0) Google Scholar).c This is an unusual plant-specific γ subunit that contains a CBM (carbohydrate-binding module) fused to the four CBS motifs found in other AMPK-γ subunits.d This is probably a non-functional protein (Moreau et al., 2012Moreau M. Azzopardi M. Clement G. Dobrenel T. Marchive C. Renne C. Martin-Magniette M.L. Taconnat L. Renou J.P. Robaglia C. Meyer C. Mutations in the Arabidopsis homolog of LST8/GbetaL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days.Plant Cell. 2012; 24: 463-481Crossref PubMed Scopus (0) Google Scholar). Open table in a new tab Interestingly, the only eukaryotes known to lack AMPK subunit orthologs are parasites that spend all or most of their life cycle living inside other eukaryotic cells, including Encephalitozoon cuniculi and Plasmodium falciparum, the latter being the causative agent of human malaria (Figure 1). These parasitic eukaryotes appear to have undergone stringent selection for small genome size, with E. cuniculi having one of the smallest known genomes of any eukaryote, encoding only 29 conventional and 3 atypical protein kinases (compared with >500 in humans) (Miranda-Saavedra et al., 2007Miranda-Saavedra D. Stark M.J. Packer J.C. Vivares C.P. Doerig C. Barton G.J. The complement of protein kinases of the microsporidium Encephalitozoon cuniculi in relation to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe.BMC Genomics. 2007; 8: 309Crossref PubMed Scopus (0) Google Scholar). Ancestors of these organisms most likely did have AMPK genes, but the modern-day descendants may have been able to dispense with them because the host cell would provide AMPK that regulates cellular energy balance on their behalf. Consistent with this, species closely related to P. falciparum that cause malaria in birds (P. gallinaceum and P. relictum) do still have conventional AMPK genes (Böhme et al., 2018Böhme U. Otto T.D. Cotton J.A. Steinbiss S. Sanders M. Oyola S.O. Nicot A. Gandon S. Patra K.P. Herd C. et al.Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals.Genome Res. 2018; 28: 547-560Crossref PubMed Scopus (19) Google Scholar). Interestingly, TOR genes are missing in E. cuniculi and P. falciparum (Figure 1) but are also absent in P. gallinaceum and P. relictum. Mammals, including humans, have two genes encoding isoforms of AMPK-α (α1 and α2), two encoding AMPK-β (β1 and β2), and three encoding AMPK-γ (γ1, γ2, and γ3) (Table 1). These multiple isoforms appear to have arisen during the two rounds of whole-genome duplication that occurred during the early evolution of vertebrates (Ross et al., 2016bRoss F.A. MacKintosh C. Hardie D.G. AMP-activated protein kinase: a cellular energy sensor that comes in 12 flavours.FEBS J. 2016; 283: 2987-3001Crossref PubMed Google Scholar). All twelve combinations of these subunit isoforms are able to form heterotrimeric complexes, although it is not certain that all combinations exist in vivo. Structures for several almost-complete human AMPK heterotrimers, i.e., α2β1γ1 (Xiao et al., 2013Xiao B. Sanders M.J. Carmena D. Bright N.J. Haire L.F. Underwood E. Patel B.R. Heath R.B. Walker P.A. Hallen S. et al.Structural basis of AMPK regulation by small molecule activators.Nat. Commun. 2013; 4: 3017Crossref PubMed Scopus (237) Google Scholar), α1β1γ1 (Calabrese et al., 2014Calabrese M.F. Rajamohan F. Harris M.S. Caspers N.L. Magyar R. Withka J.M. Wang H. Borzilleri K.A. Sahasrabudhe P.V. Hoth L.R. et al.Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms.Structure. 2014; 22: 1161-1172Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), α1β2γ1 (Li et al., 2015Li X. Wang L. Zhou X.E. Ke J. de Waal P.W. Gu X. Tan M.H. Wang D. Wu D. Xu H.E. Melcher K. Structural basis of AMPK regulation by adenine nucleotides and glycogen.Cell Res. 2015; 25: 50-66Crossref PubMed Scopus (69) Google Scholar), and α2β2γ1 (Ngoei et al., 2018Ngoei K.R.W. Langendorf C.G. Ling N.X.Y. Hoque A. Varghese S. Camerino M.A. Walker S.R. Bozikis Y.E. Dite T.A. Ovens A.J. et al.Structural determinants for small-molecule activation of skeletal muscle AMPK alpha2beta2gamma1 by the glucose importagog SC4.Cell Chem. Biol. 2018; 25: 728-737.e9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), have been obtained via X-ray crystallography. The complexes were all crystallized in active conformations, and their structures are very similar; a schematic representation of a generalized AMPK heterotrimer based on these structures is shown in Figure 2. Although the main theme of this review is nutrient sensing, we will first discuss the classical or “canonical” mechanism by which AMPK responds to the changing energy status of cells. The catalytic α subunits of AMPK contain, at their N-termini, conventional serine/threonine kinase domains with a small N-lobe and larger C-lobe and the catalytic site in the cleft between them. As with many other members of the ePK (eukaryotic protein kinase) family, AMPK complexes are only significantly active when phosphorylated at a critical residue within the activation loop, a stretch of ≈20 amino acids in the C-lobe between the highly conserved DFG and APE motifs. In AMPK, the critical phosphorylation site is a threonine, usually referred to as Thr172 after its position in the rat α2 sequence where originally mapped (Hawley et al., 1996Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.J. Biol. Chem. 1996; 271: 27879-27887Crossref PubMed Scopus (866) Google Scholar). Thr172 is not phosphorylated by AMPK itself but by upstream kinases, principally by LKB1 (liver kinase B1) (Hawley et al., 2003Hawley S.A. Boudeau J. Reid J.L. Mustard K.J. Udd L. Mäkelä T.P. 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A summary of the canonical and non-canonical mechanisms that activate AMPK, and selected downstream targets involved in its promotion of catabolic processes, inhibition of anabolic processes, and effects on DNA replication, are shown in Figure 3. In the canonical mechanism that is enshrined in its name, AMPK is activated by binding of 5′-AMP, with activation occurring not by one but three mechanisms: (1) allosteric activation of AMPK already phosphorylated on Thr172 (Carling et al., 1987Carling D. Zammit V.A. Hardie D.G. A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis.FEBS Lett. 1987; 223: 217-222Crossref PubMed Scopus (364) Google Scholar, Ferrer et al., 1985Ferrer A. Caelles C. Massot N. Hegardt F.G. Activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by adenosine 5′-monophosphate.Biochem. Biophys. Res. 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Hardie D.G. 5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC.FEBS Lett. 1995; 377: 421-425Crossref PubMed Scopus (435) Google Scholar). All three effects are due to binding of AMP to AMPK, not to the upstream kinase or phosphatase, and this tripartite mechanism ensures that the system responds to small increases in AMP in a very sensitive manner. Although there is general agreement that only AMP binding causes effect #1 above, ADP binding similarly triggers effects #2 and #3 (Oakhill et al., 2011Oakhill J.S. Steel R. Chen Z.P. Scott J.W. Ling N. Tam S. Kemp B.E. AMPK is a direct adenylate charge-regulated protein kinase.Science. 2011; 332: 1433-1435Crossref PubMed Scopus (324) Google Scholar, Xiao et al., 2011Xiao B. Sanders M.J. Underwood E. Heath R. Mayer F.V. Carmena D. Jing C. Walker P.A. Eccleston J.F. Haire L.F. et al.Structure of mammalian AMPK and its regulation by ADP.Nature. 2011; 472: 230-233Crossref PubMed Scopus (480) Google Scholar). However, most AMPK complexes (apart from those containing the γ2 isoform) are about 10-fold more sensitive to AMP than ADP, suggesting that increases in AMP are the primary activating signal, although increases in ADP may contribute (Ross et al., 2016aRoss F.A. Jensen T.E. Hardie D.G. Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms.Biochem. J. 2016; 473: 189-199Crossref PubMed Scopus (56) Google Scholar). All of the activating effects of AMP and ADP are antagonized by binding of ATP so that the AMPK system effectively monitors cellular AMP:ATP and ADP:ATP ratios. Where are the regulatory binding sites where these adenine nucleotides are sensed? The γ subunits contain four tandem repeats of a sequence termed a CBS (cystathionine β-synthase) motif (Bateman, 1997Bateman A. The structure of a domain common to archaebacteria and the homocystinuria disease protein.Trends Biochem. Sci. 1997; 22: 12-13Abstract Full Text PDF PubMed Scopus (402) Google Scholar). These occur, usually as just two tandem repeats, in about 75 proteins in humans, and they are also found in archaea and bacteria. Single pairs of tandem CBS repeats associate into pseudodimers (termed Bateman modules), potentially creating two pseudo-symmetrical ligand-binding sites in the intervening cleft, although in many cases, only one is utilized. These sites usually bind ligands containing adenosine or (less often) guanosine (Anashkin et al., 2017Anashkin V.A. Baykov A.A. Lahti R. Enzymes regulated via cystathionine beta-synthase domains.Biochemistry (Mosc.). 2017; 82: 1079-1087Crossref PubMed Scopus (5) Google Scholar, Scott et al., 2004Scott J.W. Hawley S.A. Green K.A. Anis M. Stewart G. Scullion G.A. Norman D.G. Hardie D.G. CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations.J. Clin. Invest. 2004; 113: 274-284Crossref PubMed Google Scholar). The two Bateman modules in each AMPK-γ subunit associate head-to-head to form a flattened disk with four potential binding sites for adenine nucleotides in the center (Figure 2). However, only three are utilized, i.e., CBS3, which is accessible from one face of the γ subunit, and CBS1 and CBS4, accessible from the other. The critical site appears to be CBS3; the α-linker, a flexible region of the α subunit that connects the α-AID (α-auto-inhibitory domain) and α-CTD (α-C-terminal domain), wraps around the face of the γ subunit containing CBS3, contacting its bound AMP (Figure 2). This interaction is not thought to occur when ATP is bound at CBS3 instead of AMP, and the consequent release of the α-linker from the γ subunit is proposed to allow the α-AID to rotate back into its inhibitory position behind the kinase domain (Chen et al., 2009Chen L. Jiao Z.H. Zheng L.S. Zhang Y.Y. Xie S.T. Wang Z.X. Wu J.W. Structural insight into the autoinhibition mechanism of AMP-activated protein kinase.Nature. 2009; 459: 1146-1149Crossref PubMed Scopus (145) Google Scholar, Chen et al., 2013Chen L. Xin F.J. Wang J. Hu J. Zhang Y.Y. Wan S. Cao L.S. Lu C. Li P. Yan S.F. et al.Conserved regulatory elements in AMPK.Nature. 2013; 498: E8-E10Crossref PubMed Scopus (58) Google Scholar, Li et al., 2015Li X. Wang L. Zhou X.E. Ke J. de Waal P.W. Gu X. Tan M.H. Wang D. Wu D. Xu H.E. Melcher K. Structural basis of AMPK regulation by adenine nucleotides and glycogen.Cell Res. 2015; 25: 50-66Crossref PubMed Scopus (69) Google Scholar, Xiao et al., 2011Xiao B. Sanders M.J. Underwood E. Heath R. Mayer F.V. Carmena D. Jing C. Walker P.A. Eccleston J.F. Haire L.F. et al.Structure of mammalian AMPK and its regulation by ADP.Nature. 2011; 472: 230-233Crossref PubMed Scopus (480) Google Scholar, Xin et al., 2013Xin F.J. Wang J. Zhao R.Q. Wang Z.X. Wu J.W. Coordinated regulation of AMPK activity by multiple elements in the α-subunit.Cell Res. 2013; 23: 1237-1240Crossref PubMed Scopus (31) Google Scholar); this model thus explains allosteric activation by AMP as well as its antagonism by ATP. At the same time, the resulting conformational changes may alter the accessibility of Thr172 for phosphorylation and/or dephosphorylation, although those aspects of the mechanism are less well understood. The functions of the CBS1 and CBS4 sites are less clear, although they are close to the CBS3 site in the center of the CBS repeats, where the three sites interact. One proposal is that CBS1 binds ATP permanently, while CBS4 binds AMP permanently, and that these constitutive binding events alter the conformation of the CBS3 site such that it has a higher affinity for AMP than ADP or ATP (Gu et al., 2017bGu X. Yan Y. Novick S.J. Kovach A. Goswami D. Ke J. Tan M.H.E. Wang L. Li X. de Waal P.W. et al.Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing.J. Biol. Chem. 2017; 292: 12653-12666Crossref PubMed Scopus (0) Google Scholar). This helps to explain how AMPK achieves the difficult task of sensing changes in AMP in the 30–300 μM range despite the presence of mM concentrations of ATP (Gowans et al., 2013Gowans G.J. Hawley S.A. Ross F.A. Hardie D.G. AMP is a true physiological regulator of AMP-activated protein kinase by both allosteric activation and enhancing net phosphorylation.Cell Metab. 2013; 18: 556-566Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). An additional explanation is that only Mg2+-free ATP competes with AMP at the CBS3 site (Pelosse et al., 2019Pelosse M. Cottet-Rousselle C. Bidan C.M. Dupont A. Gupta K. Berger I. Schlattner U. Synthetic energy sensor AMPfret deciphers adenylate-dependent AMPK activation mechanism.Nat. Commun. 2019; 10: 1038Crossref PubMed Scopus (4) Google Scholar), although 90% of intracellular ATP is thought to be present at the Mg.ATP2- complex. According to this model, the ATP and AMP constitutively bound at the CBS1 and CBS4 sites, respectively, act essentially as regulatory co-factors. This explains why a functional CBS4 site is required for activation even when overall AMP levels remain at the basal level (Zong et al., 2019Zong Y. Zhang C.S. Li M. Wang W. Wang Z. Hawley S.A. Ma T. Feng J.W. Tian X. Qi Q. et al.Hierarchical activation of compartmentalized pools of AMPK depends on severity of nutrient or energy stress.Cell Res. 2019; 29: 460-473Crossref PubMed Scopus (3) Google Scholar). Although the sequences of the α, β, and γ subunits are well conserved, the regulation by adenine nucleotides of AMPK orthologs from eukaryotes other than mammals is much less well studied. As mentioned earlier, neither Snf1 complexes from S. cerevisiae (Wilson et al., 1996Wilson W.A. Hawley S.A. Hardie D.G. Gluc