activated protein kinase

Received: 23 August 2016  DOI: 10.1111/cbdd.12897

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  Revised: 11 October 2016 

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  Accepted: 23 October 2016

REVIEW ARTICLE

Structural and biochemical insights into the allosteric activation mechanism of AMP-­activated protein kinase Jin Li  |  Shuying Li  |  Fengzhong Wang  |  Fengjiao Xin Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China Correspondence Fengzhong Wang and Fengjiao Xin, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China. Emails: [email protected]; [email protected] Funding information National Natural Science Foundation of China, Grant/Award Number: 31571963

The AMP-­activated protein kinase (AMPK), a complicated αβγ heterotrimer, can sense cellular energy status and maintain energy homeostasis via switching catabolic and anabolic pathways. AMPK also participates in the regulation of many other life activities, including the cell cycle, cell polarity, autophagy, and life span. Therefore, AMPK is widely studied as a potential drug target for treatment of type 2 diabetes and some other metabolic diseases, cancers, and cardiovascular diseases. Similar to other kinases, the phosphorylation of α-­Thr172 in the activation loop by upstream kinases is crucial for the activation of AMPK. In addition, the binding of AMP and its analogues to the γ subunit causes further allosteric activation, which makes AMPK distinctive from other kinases. Here, we give a brief introduction to the domain constitutions of mammalian AMPK and then systematically review its allosteric activation mechanism from a structural and biochemical view. KEYWORDS adenine nucleotides, allosteric activation, AMP-activated protein kinase, energy homeostasis, phosphorylation

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|   IN T RO D U C T ION

AMP-­activated protein kinase (AMPK) is a conserved Ser/ Thr protein kinase, which belongs to the Ca2+/calmodulin-­ dependent protein kinase (CAMK) group of the human kinome. Mammalian AMPK was first discovered independently in 1973 to phosphorylate and negatively regulate two key enzymes in the cholesterol and fatty acid synthesis: HMG-­CoA reductase (3-­hydroxy-­3-­methylglutaryl coenzyme A reductase, HMGCR)[1a,1b] and acetyl-­CoA carboxylase (ACC).[2] Therefore, it got its name as HMGCR kinase or ACC kinase-­3, respectively. It was not until 1988 the name AMPK was finally adopted by its allosteric regulator AMP.[3] As research continues, AMPK is found to be implicated in the regulation of a diverse range of metabolic signaling pathways in the whole body: It activates the catabolic pathways and inhibits the anabolic pathways through phosphorylation of different substrates. Moreover, it also participates in the

regulation of autophagy, mitophagy, mitochondrial biogenesis, cell proliferation, cell polarity, viral infection, and cancer.[4a,4b] Mammalian AMPK is sensitive to the cellular energy status, which is reflected by the AMP/ATP ratio. Once the AMP/ ATP ratio is increased, the AMPK becomes activated to accelerate ATP generation, and once the ratio is decreased, the AMPK will be inactivated to relieve inhibition of ATP consumption. Therefore, AMPK maintains the energy homeostasis.[5] The activity of AMPK is controlled by upstream kinases; it is activated over 100-­fold by upstream kinases (LKB1,[6a,6b] CaMKKβ,[7a,7b,7c] TAK1[8]) via phosphorylation of the conserved α-­Thr172 in the activation loop.[9a,9b,9c] In addition, it is sensitive to the concentration changes in nucleotides (AXPs) which makes it distinguished from other kinases.[10a,10b].The AXPs regulate the activity of AMPK via three mechanisms: (i) AMP allosterically activates AMPK through direct binding to the γ subunit,[11a,11b,11c] (ii) both AMP and

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ADP can enhance its net phosphorylation by upstream kinase LKB1,[11a-c,12a-b] and (iii) simultaneously, AMP and ADP can inhibit dephosphorylation from the phosphatases.[13a-d] However, it seems that how AMP and ADP regulate AMPK activity is dependent on differing γ isoforms.[14] Recent studies indicate that AMP (also its mimetic compound C2) and a small-­molecule drug A769662, which interacts with the allosteric drug and metabolite-­binding site (ADaM),[15] formed between the small lobe of α subunit and the carbohydrate-­ binding module (CBM) within β subunit, can synergistically activate unphosphorylated α1 containing AMPK, independent of upstream kinases.[16a-c] Although the synergistic activation mechanism is still unclear, the novel C2-­binding sites provide a new route for drug design. AMP-­activated protein kinase is a potential drug target due to its key role in the regulation of type 2 diabetes and other metabolic diseases. A great many of AMPK-­activating agents have been identified. The direct activators are compounds, or their metabolites, which have direct interactions with AMPK, such as AICAR (5-­aminoimidazole-­4-­carboxamide ribose)/ ZMP (the metabolite of AICAR),[17] A-­769662,[18] salicylate,[19a,19b] 991[20] and C2/C13 (cell-­permeable pro-­drug of C2).[16a,16c] The indirect activators activate AMPK by affecting other signaling pathways. For instance, metformin is the most widely used clinical drug in treating type 2 diabetes. It affects activation of AMPK through inhibition of complex I in the respiratory chain, which indicates that it functions indirectly. Moreover, its sister drug phenformin is also an indirect activator, but it was withdrawn in USA for its severe side-­effects.[21a,21b] Furthermore, several natural products can activate AMPK as well, such as resveratrol,[22] berberine,[23] galegine,[24] arctigenin,[25] and so on. Their regulatory mechanism can also be attributed to suppressing mitochondrial ATP production either by inhibiting ATP synthase or by reducing the activity of complex I in respiratory chain.[26] However, many other natural products, such as leuteolin, epigallocatechin gallate (EGCG),[27] and honokiol, were reported as AMPK agonists, but how they activate AMPK is not yet clear.

2  |   D O M A IN CON ST IT U T IO N S OF AMPK AMPK is composed of a catalytic α subunit and regulatory β and γ subunits (Figure 1a,b). In mammals, two isoforms of α and β subunits and three isoforms of γ subunits are found. These isoforms distribute in different tissues in 12 possible αβγ combinations.[28a,28b] α subunit has a conserved kinase domain, followed by the auto-­ inhibitory ­domain (AID). Between the AID and the C-­terminal domain (α-­CTD) is the flexible linker region, which contains two short conserved motifs: the regulatory subunit-­interacting motifs

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F I G U R E   1   Overall structure of mammalian AMPK. (a) Schematic representation of mammalian AMPK. The color scheme is as follows: kinase domain (light blue), AID (blue), α-­linker (light pink), α-­RIM1/ α-­RIM12 (magenta), α-­CTD (brown), GBD (green), β-­CTD (pale green), β-­loop (forest), γ subunit (bright orange). (b) Graphical representations of the phosphorylated human α1β2γ1 (PDB: 4RER). Unless stated, the color scheme represents the same with (a). The AMP, staurosporine, and beta-­cyclodextrin (BCD) are highlighted as marine, green, and blue sticks [Colour figure can be viewed at wileyonlinelibrary.com]

(RIMs) RIM1 and RIM2, which directly bind to the surface of nucleotide-­binding site 2 and site 3.[29a-b] The myristoylation of the β subunit (at Gly2) may contribute to its membrane localization and further activation by upstream kinases.[12a] The C-­terminal domain of β subunit (β-­CTD) works as a scaffold to interact with the α-­CTD and the N-­terminal of γ subunit to stabilize the heterotrimer, and two strands from β and one from γ form an internal β-­sheet.[30a-b] The glycogen-­ binding domain (GBD) locates in the middle of β subunit, but its physiological role has not been clarified.[31a,31b] There are four tandem cystathionine β synthase (CBS) motifs (CBS1-­4)

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in the γ subunit, which fold into two Bateman domains. The γ subunit can bind nucleotides (ATP, ADP, or AMP) and NADH molecules in response to different stimuli.[13d,32] α-­CTD, β-­CTD, and the full length of γ subunit constitute the core domain, which plays a significant role in the regulation of AMPK[30a-b,33] (Figure 2a).

3  |   A L LO ST E R IC AC T IVAT ION ME CH A N IS M S OF A MP K BY A MP AMP-­activated protein kinase works like a delicate instrument. α-­Thr172 is just like a start button; once it is phosphorylated, it achieves the initial activity. The γ subunit works like a sensor which senses the allosteric signal of AMP and induces the long-­range allosteric activation. The allosteric signal will be transduced to the engine, the α subunit, and fully activate the holoenzyme. When ATP and other molecules replace AMP, the allosteric activation is disturbed.

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3.1  |  γ subunit directly senses the allosteric signal of AMP As early as 1987, Hegardt and his colleagues used affinity-­ labeling assay and obtained the evidence for separated catalytic and allosteric sties, but not until 2000 year, AMP binding to the γ subunit was proved.[34a,34b] However, it takes a very long time and much effort to get the direct structural evidence. There was a large breakthrough in 2007, three groups separately solved the structures of core domain of yeast and rat AMPK, and they all shared a similar three-­dimensional (3D) architecture. The γ subunit consists of four cystathionine β-­synthase (CBS) motifs and folds a globular domain, shaped as a flattened disk. It folds to generate two Bateman domains: Bateman domain 1 is composed of CBS1 and CBS2, and Bateman domain 2 is composed of the other two. The α and β subunits sit atop the γ subunit, and two strands from β subunit and one from γ subunit form an intersubunit β-­sheet, which enforces the rigidity of

F I G U R E   2   Allosteric activation mechanism of mammalian AMPK. (a) γ subunit directly senses the allosteric signal of AMP. Schematic representation of the core domain which contains the α-­CTD, β-­CTD, and the full length of γ subunit, and AMP directly binds to the γ subunit at sites 1, 3, and 4. (b) α-­RIM1, α-­RIM2, and β-­loop transduce the allosteric activation signal of AMP. α-­RIM1 and α-­RIM2 are set apart in the two sides of β-­loop. The α-­RIM1 directly binds to the non-­bound γ-­site 2, α-­RIM2 is sandwiched between γ-­site 3 and β-­loop, and they form extensive interactions. (c) AID directly binds to the backside of KD and keeps the KD in a relatively open, inactive state. Schematic representation of unphosphorylated KD–AID from Schizosaccharomyces pombe (PDB: 3H4J); KD and AID are colored in blue and purple blue, respectively. (d) A model for the allosteric regulation mechanism of mammalian AMPK. In the resting state, the concentration of ATP is high and AMPK is saturated with ATP at γ-­sites 1 and 4; the holoenzyme adopts relatively a loose conformation. The AID stably binds to the backside of KD and keeps it in an inactive state, and the α-­linker is flexible and disordered in the structure (dotted line). Once the AMP/ATP ratio increases, the AMP will competitively bind to the γ-­sites 1, 3, and 4 and induce a lot of conformational changes and then recruit α-­RIM1/2 and the induced β-­loop. The constrained α-­linker forces the disassociation of AID from KD and therefore releases its inhibition effect to KD, and the AID binds to the N-­terminus of γ subunit. The holoenzyme becomes more compact and is allosterically activated [Colour figure can be viewed at wileyonlinelibrary.com]

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the backbone of the three subunits.[30a-b,33] The mammalian AMPK (PDB: 2V8Q) is in complex with three AMP molecules, which occupy nucleotide-­binding sites 1, 3, and 4. Site 2 is empty as predicted, due to the replacement of the conserved Asp with Arg. The adenine moiety of AMP nestles into the hydrophobic pocket, which is formed by the two adjacent CBS domains within the same Bateman domain. The phosphate group interacts with surrounding residues, mainly through polar interactions[30b] (Figure 2a). There are three types of free adenine nucleotides in mammals: ATP, ADP, and AMP. They possess similar structures and properties but differ in the number of the phosphate groups (it is 3, 2, and 1, respectively, just as their name mentioned). Structural and biochemical studies indicate that their binding patterns and biological functions are different from each other. According to the crystal soaking experiments, the γ-­site 4 seems to bind AMP permanently, which is recognized as a “non-­exchangeable” site.[30b] However, soaking crystallization method is based on the preformed crystal ­lattice, and the original structure cannot be changed even though ATP molecules replace other two AMPs (γ-­sites 1 and 3). So the “non-­exchangeable” site 4 may be an artifact of binding ­ligands to preformed crystals. In 2011, Lei Chen and his colleagues used the method of cocrystallization to solve the core structure of AMPK in complex with ATP, which represents a very different conformation in comparison with the previously solved AMP/ATP-­bound structures. ATP binds to γ-­sites 1 and 4, which induces many conformational changes. The two Bateman domains rotated with respect to each other, and key amino acids and secondary structural elements are shifted a lot. Particularly, the γ-­site 3 becomes malformed without binding any nucleotide. Therefore, the γ-­site 4 is no longer “non-­exchangeable” and it can bind ATP at some circumstances.[35] It seems that the γ-­site 4 has much higher affinity for AMP than other small molecules and may play the dominant role in the discrimination of AXPs. However, the exact dissociation constant is difficult to determine. While the fluorescence competition assays prove that both γ-­site 1 and γ-­site 3 bind AXPs with similar affinities, γ-­site 1 shows at least 30-­fold tighter binding affinity than γ-­site 3, so γ-­site 1 is defined as the tighter site and γ-­site 3 is the weaker site.[13d] Among the three functional sites, the γ-­site 3 and γ-­site 4 are responsible for the allosteric activation of AMPK.[35,36]

3.2  |  AID responses for the allosteric activation signal of AMP The kinase domain of AMPK is the central functional domain. Phosphorylation of the conserved Thr172 in the activation loop is a prerequisite to get basic catalytic activity, and then, the kinase can phosphorylate a large set of substrates. Several crystal structures of the isolated kinase domain (KD) have been solved, such as the unphosphorylated KD of

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Saccharomyces cerevisiae SNF1 (PDB: 3HYH, 2FH9),[37a,37b] the unphosphorylated KD of human α2 (PDB: 2H6D), and its phosphor-­mimetic (T172D) form (PDB: 2YZA).[38a,38b] The KD of different species adopts the classical bilobal kinase fold, a small β-­stranded N-­lobe, and a larger α-­helical C-­lobe, and the two lobes are connected through a short hinge region. The relatively “open” and “closed” conformations represent its “inactive” and “active” states, respectively. The ATP-­ binding pocket is formed by a deep cleft and the hinge region, with flexible activation loop sitting in and behaving like a gate to dynamically regulate ATP binding. The two elements are crucial for the kinase activity of AMPK.[39a,39b] The auto-­inhibition regulation mechanism of AMPK has been well studied. In 1998, Crute et al. analyzed the functional domains of the catalytic α subunit and identified a conserved, auto-­inhibitory sequence that negatively regulated the AMPK’s activity, which was later defined as the auto-­ inhibitory domain (AID).[40a,40b] In 2009, Lei Chen et al. reported the crystal structure of unphosphorylated KD–AID from Schizosaccharomyces pombe (PDB: 3H4J, Figure 2c) and phosphorylated KD from Saccharomyces cerevisiae SNF1 (PDB: 3DAE), confirmed the existence of AID, and revealed the auto-­ inhibition mechanism at the molecular level. The three α-­helical (α1–α3) AID binds to the KD from the backside. This interaction constrains the mobility of the N and C lobes, especially the helix αC, which maintains the KD in a relatively “open” inactive state. The mutations of the key amino acids in the KD–AID interface disrupt the inhibition effect to varying degrees, and most of the mutated holoenzymes have a higher initial activity and behave independently of AMP change. The enzyme kinetic studies also proved that AMP allosterically activates AMPK by releasing the auto-­inhibition effect of AID to KD.[41] In 2015, Xiaodan Li et al. reported the structure of human α1 KD–AID (PDB: 4RED), and their structural and biochemical studies are in agreement with Lei Chen’s previous results.[36] The crystal structure of isolated rat α1 AID (PDB: 4F2L) and NMR solution structure of human α2 AID (PDB: 2LTU) also indicate the stable existence and its regulatory role in the trimeric protein.[29a] Therefore, AID is the central response element in the allosteric regulation of AMPK by AMP.

3.3  |  Conserved regulatory elements (α-­RIM1, α-­RIM2, and β-­loop) transduce the allosteric activation signal of AMP The molecular regulation mechanism of AMPK has been interpreted a lot from extensive studies of isolated domain structures, for example, KD, KD–AID, core domain. However, the allosteric activation is a kind of long-­range regulation mechanism, so it is difficult to determine how the signal of AMP binding transfers to the active site of α subunit. Hence, a structure of holoenzyme is required. However,

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the constitution of the 145-­kDa holoenzyme is very complicated, and the existence of the flexible loops restricts its crystallization. After a prolonged endeavor, Xiao et al.[13d] solved the crystal structure of a partial αβγ-­trimer (PDB: 2Y94) in 2011. Combined with the studies of Lei Chen and his colleagues, a more reasonable model (PDB: 4CFH) was proposed. This structure shows the position of KD in the holoenzyme for the first time. Two years later, Xiao et al.[20] solved the crystal structure of the human AMPK α2β1γ1 complexed with small-­molecule activators (A769662/991, PDB: 4CFF and 4CFE) in the active state. Then, two papers published in 2014 presented the structures of the phosphorylated (PDB: 4RER, Figure 1b) and unphosphorylated mammalian holoenzymes (PDB: 4REW and 4QFG, for human and rat, respectively), and the nearly complete 3-­dimensional (3D) structure gave more detailed information about the complicated allosteric activation.[36,42] All of the existing structures adopt a similar 3D architectural arrangement: The core domain performs as the backbone, the interformed β-­sheet formed by β-­CTD and the N-­terminus of γ subunit sits atop one shoulder of the γ subunit, and the kinase domain sits atop the other shoulder of γ subunit. The GBD of the β subunit sits atop the N-­lobe of the KD. It is worth mentioning that all of the holoenzyme structures, even the non-­phosphorylated structure (PDB: 4REW), are in complex with AMPs (or one AMP and two ADPs). AMP occupies at least two of the nucleotide-­binding sites, site 3 and site 4, which is in agreement with Lei Chen’s studies. There is a long flexible region between AID and α-­CTD, which is not included in the previous structures, but is well traceable in the partial or entire holoenzyme structures. This flexible region forms a loop and extends to the γ subunit and contacts the nucleotide-­binding sites 2 and 3 directly through two short conserved motifs (regulatory subunit-­interacting motifs 1 and 2, α-­RIM1 and α-­RIM2, Figure 2b). An induced β-­loop in the β-­CTD also participates in the regulation. These conserved regulatory elements play very important roles in mediating the transduction of the allosteric activation signal. The structures of AMPK holoenzymes published until now are in one of the three states: the non-­phosphorylated, AMP-­ bound inactive state; the phosphorylated, AMP-­bound active state; and the phosphorylated, AMP-­and A769662/991/BCD (beta-­cyclodextrin)-­bound active state. As the function of GBD is not clear yet and beyond the scope of this review, the third state will not be discussed here. • The non-phosphorylated, AMP-bound inactive state. In the human AMPK (PDB: 4REW),[36] although the γ subunit binds AMP at all of the functional sites, and its conformation is comparable to the isolated AMP-bound core domains, it is still in an inactive state. This structure contains almost all of the functional domains except the GBD,

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whose electron density cannot be detected. Combining with the biochemical studies, the authors concluded that the GBD is disassociated from the KD in the non-phosphorylated AMPK. The GBD is traceable in the structure of non-phosphorylated rat AMPK holoenzyme (PDB: 4QFG),[42] and it is also disassociated from KD, which is in accordance with the studies of human AMPK. Moreover, the α-RIMs, AID, and the induced β-loop collectively move toward, but are still not suitable to interact with the γ subunit. Furthermore, the KD keeps an inactive conformation and the “swung-out” helix αC conformation restricts the formation of the crucial Lys-Glu salt bridge, which is a hallmark of a kinase in an inactive state. • The phosphorylated, AMP-bound active state. In the first fully active AMPK structure (PDB: 4CFH),[13d] GBD is truncated in the construct for better crystallization. In the subsequent solved structures, the GBD is clearly visible with the help of GBD-binding molecules: the small-molecule compounds A769662/911 or glycogen-mimic cyclodextrin (BCD). The core domain is similar to the previously reported AMP-bound structures, but the holoenzyme behaves more compact enabling the protection of the conserved p-Thr172 from the phosphatases. The holoenzyme undergoes evident conformational changes upon AMP binding to the γ subunit. α-RIM1 and α-RIM2 are recruited to γ-site 2 and γ-site 3, and the β-loop is sandwiched between them. The α-RIM1 partially overlaps with its preceding AID and interacts with non-bound γ-site 2. In particular, γArg170, which is the main determinant of non-bound γ-site 2, contributes a lot to the αγ interactions. The α-RIM2 is located in the middle of β-loop and γ-site 3 and interacts with both of them extensively. In the low energy state, AMPK is saturated with AMP and induced a series of conformational changes, and then recruited the α-RIM1, α-RIM2, β-loop, γ-site 2, and γ-site 3 to form a “zipper”-like interaction mode, and the AID acts as a response node. Any disturbance that affects the interactions will trigger a chain reaction to stretch the response element AID, which then collectively moves toward and directly binds to the γ subunit with the integrity of its stable 3-helix conformation. Thereby, the auto-inhibitory effect is relieved and the AMPK is allosterically activated.[29b] AMP and ATP dynamically regulate the allosteric activation of AMPK. Once the concentration of ATP is high enough, ATP will competitively bind to the γ subunit. Although the structure of the trimeric AMPK in complex with ATP has not been solved until now, the cocrystallized core structure, which is bound with ATP, will give some information. As mentioned above, the binding of ATP to the γ-­site 1 and γ-­site 4 induces many conformational changes, and the intersubunit β-­ sheet collectively moves upward, which disrupts mounts of hydrogen bonds between α-­RIM1 and γ N-­terminus. Moreover, the

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γ-­site 2 and γ-­site 3 become distorted, which further abolishes their interactions with α-­RIMs. Therefore, ATP weakens the αγ interaction and negatively affects the release of AID from KD, which inhibits the allosteric activation. Biochemical analysis also confirms this conclusion.[29b,35,36]

3.4  |  A proposed allosteric activation model The energy status holds a dynamic equilibrium in eukaryotic cells. In the resting state, AMPK stays in the ATP-­bound form and ATP occupies γ-­site 1 and γ-­site 4. The conformational changes in the γ subunit and the collective upward movement of the intersubunit β-­sheet make the holoenzyme less compact and also restrict the interaction between α-­RIMs and the γ-­sites. The α-­linker is flexible, and the AID stably binds to the kinase domain and maintains it in a relatively open, inactive conformation. When the energy level decreases, the concentration of AMP will increase and AMP competitively binds to the AMPK at γ-­site 1, γ-­site 3, and γ-­site 4. This induces substantial conformational changes that enable α-­RIM 1 and α-­RIM 2 to be recruited to the γ-­site 2 and γ-­site 3, respectively. Then, the constrained α-­linker forces the disassociation of the adjacent AID from the KD; then, the AID moves toward and directly binds to the γ subunit. Thus, the KD is in a more closed, active conformation and the AMPK is allosterically activated (Figure 2d).

4  |   CO NC LU SION S A N D FU TU R E P E R SP E C T IV E S AMP-­ activated protein kinase is an important regulatory center and participates in almost every aspect of cellular metabolism. Scientists around the world never stop exploring its secrets. During the past 5 years, scientists solved the structures of partial and full holoenzymes in different states. Combined with the biological studies, they gradually elucidated the allosteric activation mechanism of AMPK from the molecular perspective. However, there are still a lot of problems that need further studies. Recently, ADP has been shown to regulate the activity of AMPK,[12b,13d,32,43] and it behaves differently from AMP and ATP.[11c] Although scientists have solved the ADP-­bound AMPK structures, they still cannot explain the underlying mechanism. NADH is another important molecular regulator in eukaryotic cells that binds to AMPK at γ-­site 1, but its physical function and regulatory mechanism remain elusive.[13d,32] There are at least 12 combinations of αβγ heterotrimers, and their mechanisms of allosteric activation are distinct. Although the structures of the holoenzyme containing α1 and α2 have both been solved, they still cannot give any detailed evidence. The binding of AMP and ADP can increase the phosphorylation of α-­Thr172 by upstream kinase LKB1. Yalin Zhang et al. proved that AMP, but not

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ADP, can drive AXIN to tether LKB1 and phosphorylated AMPK to form a complex.[44] However, the difference between AMP and ADP in this regulation process is unclear. Thus, there are still many questions about AMPK that are worth of further study, and we will benefit a lot from fully understanding AMPK in the treatment of different diseases. ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (No. 31571963). CONFLICT OF INTEREST All authors have declared there is no conflict of interest. R E F E R E NC E S [1a]  Z. H. Beg, D. W. Allmann, D. M. Gibson, Biochem. Biophys. Res. Commun. 1973, 54, 1362. [1b] M. S. Brown, G. Y. Brunschede, J. L. Goldstein, J. Biol. Chem. 1975, 250, 2502. [2] C. A. Carlson, K. H. Kim, J. Biol. Chem. 1973, 248, 378. [3] M. R. Munday, D. G. Campbell, D. Carling, D. G. Hardie, Eur. J. Biochem. FEBS 1988, 175, 331. [4a] D. G. Hardie, F. A. Ross, S. A. Hawley, Nat. Rev. Mol. Cell Biol. 2012a, 13, 251. [4b] M. M. Mihaylova, R. J. Shaw, Nat. Cell Biol. 1016, 2011, 13. [5] D. G. Hardie, Nat. Rev. Mol. Cell Biol. 2007a, 8, 774. [6a] A. Woods, S. R. Johnstone, K. Dickerson, F. C. Leiper, L. G. Fryer, D. Neumann, U. Schlattner, T. Wallimann, M. Carlson, D. Carling, Curr. Biol. 2004, 2003, 13. [6b] S. A. Hawley, J. Boudeau, J. L. Reid, K. J. Mustard, L. Udd, T. P. Makela, D. R. Alessi, D. G. Hardie, J. Biol. 2003, 2(4), 28. [7a] S. A. Hawley, D. A. Pan, K. J. Mustard, L. Ross, J. Bain, A. M. Edelman, B. G. Frenguelli, D. G. Hardie, Cell Metab. 2005, 2(1), 9. [7b] A. Woods, K. Dickerson, R. Heath, S. P. Hong, M. Momcilovic, S. R. Johnstone, M. Carlson, D. Carling, Cell Metab. 2005, 2(1), 21. [7c] R. L. Hurley, K. A. Anderson, J. M. Franzone, B. E. Kemp, A. R. Means, L. A. Witters, J. Biol. Chem. 2005, 280, 29060. [8] M. Momcilovic, S. P. Hong, M. Carlson, J. Biol. Chem. 2006, 281, 25336. [9a] S. A. Hawley, M. Davison, A. Woods, S. P. Davies, R. K. Beri, D. Carling, D. G. Hardie, J. Biol. Chem. 1996, 271, 27879. [9b] S. P. Hong, F. C. Leiper, A. Woods, D. Carling, M. Carlson, Proc. Natl Acad. Sci. USA 2003, 100, 8839. [9c] D. G. Hardie, J. Cell Sci. 2004, 117(Pt 23), 5479. [10a] J. S. Oakhill, J. W. Scott, B. E. Kemp, Trends Endocinol. Metab. 2012, 23(3), 125. [10b] D. Carling, F. V. Mayer, M. J. Sanders, S. J. Gamblin, Nat. Chem. Biol. 2011, 7, 512. [11a] L. A. Yeh, K. H. Lee, K. H. Kim, J. Biol. Chem. 1980, 255, 2308. [11b] I. Salt, J. W. Celler, S. A. Hawley, A. Prescott, A. Woods, D. Carling, D. G. Hardie, Biochem. J. 1998, 334(Pt 1), 177. [11c] G. J. Gowans, S. A. Hawley, F. A. Ross, D. G. Hardie, Cell Metab. 2013, 18, 556. [12a] J. S. Oakhill, Z. P. Chen, J. W. Scott, R. Steel, L. A. Castelli, N. Ling, S. L. Macaulay, B. E. Kemp, Proc. Natl Acad. Sci. USA 2010, 107, 19237. [12b] J. S. Oakhill, R. Steel, Z. P. Chen, J. W. Scott, N. Ling, S. Tam, B. E. Kemp, Science 2011, 332, 1433. [13a] S. P. Davies, N. R. Helps, P. T. Cohen, D. G. Hardie, FEBS Lett. 1995, 377, 421.

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