AMP-activated protein kinase: also regulated by ADP? D. Grahame Hardie 1 , David Carling 2 and Steven J. Gamblin 3 1 College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK 2 MRC Clinical Sciences Center, Imperial College, London W12 0NN, UK 3 MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK AMPK is a ubiquitous sensor of cellular energy status in eukaryotic cells. It is activated by stresses causing ATP depletion and, once activated, maintains energy homeo- stasis by phosphorylating targets that activate catabo- lism and inhibit energy-consuming processes. Evidence derived from non-mammalian orthologs suggests that its ancestral role was in the response to starvation for a carbon source. We review recent findings showing that AMPK is activated by ADP as well as AMP, and discuss the mechanism by which binding of these nucleotides prevent its dephosphorylation and inactivation. We also discuss the role of the carbohydrate-binding module on the b subunit and the mechanisms by which it is activat- ed by drugs and xenobiotics such as metformin and resveratrol. Historical background In 1987, evidence was provided that two apparently dis- tinct protein kinase activities that inactivated enzymes of lipid biosynthesis (acetyl-CoA carboxylase and HMG-CoA reductase) were functions of a single entity [1]. Both activ- ities had previously been shown to be activated by AMP [2,3] and it was therefore renamed AMP-activated protein kinase (AMPK) [4]. There are now around 1000 papers published on the system every year, so, here, we cannot provide a comprehensive coverage; readers should consult other reviews for discussion of identification of down- stream targets and roles in regulation of cell growth and metabolism [5,6]. In this article, we focus on the role of non- mammalian orthologs, on identification of upstream kinases and phosphatases, on recent findings concerning regulation of AMPK by adenine nucleotides and binding to glycogen, and on the mechanisms by which it is activated by drugs and xenobiotics. AMPK: roles of non-mammalian orthologs AMPK is expressed ubiquitously in eukaryotic cells as heterotrimeric complexes comprising a catalytic a subunit and regulatory b and g subunits. Genes encoding orthologs of these subunits are found in all eukaryotes including protists, fungi, plants and animals. One interesting excep- tion that ‘proves the rule’ is the microsporidian parasite Encephalitozoon cuniculi, which (unlike apicomplexan parasites such as Plasmodium falciparum) lacks an AMPK ortholog [7]. E. cuniculi has the smallest known eukaryotic genome, encoding only approximately 2000 proteins and 32 protein kinases (compared with >20 000 and >500 in humans), including two orthologs of the AMPK-related kinases MARK1–MARK4 [7]. E. cuniculi lives as an obli- gate intracellular parasite inside mammalian cells and lacks metabolically active mitochondria. However, it has genes related to the ATP/ADP translocases found in chlor- oplasts, and it seems probable that it uses these to ex- change ADP for ATP made by the host cell [8]. Thus, this parasite, with its stripped-down genome, might be able to exist without AMPK because the host cell provides it anyway. Genetic studies in non-mammalian eukaryotes provide compelling evidence that the ancestral role of AMPK was in the response to starvation for a carbon source. In the yeast Saccharomyces cerevisiae, the AMPK ortholog (the SNF1 complex) is required for most responses to glucose starvation, including expression of enzymes required for the switch from fermentative to oxidative metabolism when glucose runs low, and for catabolism of other fer- mentable carbon sources [9]. The expression of over 400 mRNAs was >2-fold different between wild-type and SNF1-deficient yeast strains grown in low glucose [10]. In Caenorhabditis elegans, the AMPK orthologs are re- quired for the extension of lifespan and inhibition of germ cell development that occurs in response to caloric restric- tion [11,12]. In the moss Physcomitrella patens, AMPK orthologs are not required for growth in continuous light, but are essential for growth in alternate light/dark cycles [13] (darkness is, of course, the equivalent of starvation for a plant). A role in transcriptional responses to starvation (and other stresses such as hypoxia) in higher plants was also supported by studies in which the orthologs in Arabi- dopsis thaliana were either overexpressed or silenced. In this case, the growth defects in kinase-deficient plants (unlike in the moss) were not rescued by continuous illu- mination, although starch mobilization during the dark phase was impaired [14]. Identification of upstream kinases and phosphatases AMPK and its orthologs are only significantly active after phosphorylation of a conserved threonine residue within the activation loop of the kinase domain (Thr172 in rat [15] and Thr210 in S. cerevisiae [16]). Identification of the upstream kinases and phosphatases that modify these sites proved challenging. The breakthrough occurred in S. cerevisiae where three upstream kinases, Pak1/Sak1, Review Corresponding author: Hardie, D.G. (d.g.hardie@dundee.ac.uk). 470 0968-0004/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2011.06.004 Trends in Biochemical Sciences, September 2011, Vol. 36, No. 9