Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP.

www.biochemj.org Biochem. J. (2010) 426, 109–118 (Printed in Great Britain) 109 doi:10.1042/BJ20091372 Calmodulin-dependent protein kinase kinase-β activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP Sarah FOGARTY, Simon A. HAWLEY, Kevin A. GREEN, Nazan SANER, Kirsty J. MUSTARD and D. Grahame HARDIE1 Activation of AMPK (AMP-activated protein kinase) by phosphorylation at Thr172 is catalysed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and CaMKKβ (Ca2+ /calmodulin-dependent protein kinase kinase-β). The sequence around Thr172 is highly conserved between the two catalytic subunit isoforms of AMPK and the 12 AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. In the present paper we report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKKβ, although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKKα. Despite recent reports, we could not find any evidence that the α and/or β subunits of AMPK formed a stable INTRODUCTION AMPK (AMP-activated protein kinase) is an energy-sensing system involved in regulating energy balance at both the cellular and the whole-body levels [1,2]. The kinase occurs as heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits, with each subunit existing in mammals as isoforms encoded by multiple genes (α1, α2; β1, β2; γ 1, γ 2, γ 3). Metabolic stresses that inhibit ATP synthesis (e.g. hypoxia, hypoglycaemia) or that stimulate ATP consumption (e.g. muscle contraction) cause an increase in the cellular ADP/ATP ratio, which is amplified by adenylate kinase into an even larger increase in the AMP/ATP ratio. AMP and ATP bind antagonistically to two sites formed by the four tandem CBS (cystathionine β-synthase) motifs on the γ subunit [3,4]. The kinase is only active after phosphorylation of a critical threonine residue within the activation loop of the kinase domain (Thr172 in rat α1/α2) by upstream kinases. The major upstream kinase in most cells was identified to be a complex between the tumour suppressor LKB1 and two accessory subunits, STRAD (Ste20-related adaptor) and MO25 (mouse protein 25) [5,6]. LKB1 appears to be constitutively active [7,8] and may therefore phosphorylate AMPK continually, but under basal conditions the phosphate appears to be immediately removed by protein phosphatases. However, binding of AMP to the AMPK γ subunits inhibits dephosphorylation of Thr172 , an effect that is antagonized by high concentrations of ATP [9–11]. In addition, binding of AMP (but not ATP) triggers a further allosteric activation of the phosphorylated kinase by up to 10-fold, with the combination of these two effects producing >1000-fold activation [11]. Both stimulatory effects appear to occur because AMP binding relieves complex with CaMKKβ. We also showed that increasing AMP concentrations in HeLa cells (which lack LKB1) had no effect on basal AMPK phosphorylation, but enhanced the ability of agents that increase intracellular Ca2+ to activate AMPK. This is consistent with the effect of AMP on phosphorylation of Thr172 being due to inhibition of dephosphorylation, and confirms that the effect of AMP is independent of the upstream kinase utilized. Key words: AMP-activated protein kinase (AMPK), AMPactivated protein kinase-related kinase (ARK), Ca2+ /calmodulindependent protein kinase (CaMK), Ca2+ /calmodulin-dependent protein kinase kinase (CaMKK). the inhibitory effects of an auto-inhibitory domain on the α subunit, which binds to the kinase domain on the opposite side to the substrate-binding site [12]. This represents a sensitive switch mechanism that produces a large activation of AMPK in response to a small increase in the cellular AMP/ATP ratio. Some human tumour cells (e.g. HeLa cells) do not express LKB1, but AMPK can still be phosphorylated at Thr172 and activated in such cells using Ca2+ ionophores. This led to the discovery that the CaMKKs [CaMK (Ca2+ /calmodulin-dependent protein kinase) kinases], especially CaMKKβ, could act as alternative upstream kinases that can phosphorylate Thr172 on AMPK [13–15]. CaMKKα and CaMKKβ [16] were originally discovered as CaMKs that acted upstream of CaMKI and CaMKIV. The Ca2+ →CaMKK→AMPK pathway is triggered by a rise in cytosolic Ca2+ without any requirement for an increase in AMP, and is responsible for AMPK activation in response to K+ -induced depolarization in neurons [13], muscarinic activation in neuroblastoma cells [17], thrombin activation of endothelial cells [18], treatment of smooth muscle cells with vasoconstrictors [19] and stimulation of antigen receptors in T-cells [20]. The sequence around Thr172 on the AMPK α subunits is highly conserved (see Figure 1) in the kinase domain sequences of kinases [termed ARKs (AMPK-related kinases)], that lie on the same branch of the human kinome [21]. By studying phosphorylation in cell-free assays and in cells lacking LKB1, including LKB1−/− mouse embryo fibroblasts and HeLa cells, it was shown that at least 12 of these were also dependent on LKB1 for basal phosphorylation of the threonine residue equivalent to Thr172 , and hence for basal activity. These include the brain-specific kinases BRSK1 and BRSK2 (also known as SAD-B and SAD-A), SIK (salt-inducible kinase) 1, Abbreviations used: ACC, acetyl-CoA carboxylase; AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated protein kinase; ARK, AMPK-related kinase; BRSK, brain-specific kinase; CaMK, Ca2+ /calmodulin-dependent protein kinase; CaMKK, Ca2+ /calmodulin-dependent protein kinase kinase; FBS, foetal bovine serum; GFP, green fluorescent protein; GST, glutathione transferase; HEK, human embryonic kidney; MARK, microtubule affinity-regulating kinase; MO25, mouse protein 25; NUAK, SNF1 (sucrose-non-fermenting kinase-1)-like kinase; SIK, salt-inducible kinase; STRAD, Ste20related adaptor; TBS, Tris-buffered saline; UBA, ubiquitin-associated. 1 To whom correspondence should be addressed (email ). © 2010 The Author(s)  c The Authors Journal compilation  c 2010 Biochemical Society The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. Biochemical Journal Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K. 110 S. Fogarty and others SIK2 and SIK3 (also known as SIK, QIK and QSK respectively), NUAK [SNF1 (sucrose-non-fermenting kinase-1)-like kinase] 1 and NUAK2 (also known as ARK5 and SNARK), MARK (microtubule affinity-regulating kinase) 1, MARK2, MARK3 and MARK4, and the testis-specific kinase SNRK (SNF1-relate (...truncated)