Direct and indirect activation of eukaryotic elongation factor 2 kinase by AMP-activated protein kinase
Graphical abstract
Mechanism of AMPK-induced inhibition of protein synthesis via eEF2K activation.
Introduction
eEF2K is a highly conserved Ser/Thr kinase and member of the atypical alpha kinase family [1], [2], [3]. eEF2K is a highly regulated protein kinase and its activity is almost entirely dependent on Ca2+/calmodulin (CaM), which binds to an amino-terminal regulatory region [4], [5]. eEF2K activity is also controlled by multi-site phosphorylation, which modulates CaM-binding, kinetic properties and proteasomal degradation [6], [7], [8], [9]. Besides control by protein kinases from various signaling pathways, such as inactivation by p70 ribosomal S6 kinase (p70S6K) downstream of mammalian target of rapamycin complex-1 (mTORC1) [10], [11] and activation by AMPK [12] or cAMP-dependent protein kinase (PKA) [13], [14], eEF2K extensively autophosphorylates at several sites in the presence of Ca2+/CaM [15], [16] to acquire maximal activity. Once activated, eEF2K phosphorylates eEF2 on Thr56 [17], [18], preventing its binding to the ribosome, which leads to ribosomal stalling and reversibly halts protein synthesis elongation. It is noteworthy that eEF2K is the only protein kinase known to phosphorylate eEF2 at Thr56.
AMPK is a key regulator of cellular energy homeostasis becoming activated during metabolic stress via a rise in AMP:ATP ratio. AMPK phosphorylates eEF2K in vitro and phosphorylation at Ser398 was proposed to cause eEF2K activation [12]. The inhibition of protein synthesis by AMPK at peptide elongation is crucial for survival under energy-depleting conditions and logical, since this step of protein synthesis is the most costly in terms of ATP equivalents consumed [19]. It is therefore not surprising that AMPK activation leads to the phosphorylation of eEF2 [20], thereby decreasing the rate of protein synthesis. AMPK activation also inhibits protein synthesis initiation by decreasing PKB/mTORC1 signaling at different levels [21], [22], [23].
Several cellular stresses, most of which activate AMPK, have been shown to increase eEF2 Thr56 phosphorylation, namely skeletal muscle contraction [24], [25], [26], ischemia [27], hypoxia [28], increasing cell density [29], nutrient deprivation [30], growth factor retrieval [31], genotoxic agents [32], endoplasmic reticulum stress [33], [34], ribosomal stress [35], oxidative stress [29], [36], osmotic stress [37], chemical stress [29], alcohol [38], and changes in pH [39], [40] or temperature [41]. Also, treatment of cells with AMPK activators such as 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) [12], 2-deoxy-d-glucose (2DG) [12], the “Abbott compound” A-769662 [42], metformin/phenformin [43] or oligomycin [20] leads to increased eEF2 phosphorylation. Most of these chemical treatments activate AMPK indirectly by causing ATP depletion (2DG [44], metformin [45], oligomycin [46]) and thus lack specificity. On the other hand, A-769622 and a small-molecule benzimidazole derivative called “991” activate AMPK by binding directly to the AMPKβ subunit [47]. Compound 991 is the more potent of the two direct AMPK activators and A-769622 seems only to target AMPKβ1. Incubation of skeletal muscles with 991 led to activation of both AMPKβ1- and AMPKβ2-containing complexes to increase glucose-uptake [48], [49] and 991 treatment of hepatocytes antagonized glucagon signaling [50], both in an AMPK-dependent manner.
In the present study, we used 991 to activate AMPK in genetically modified mouse embryonic fibroblast (MEF) cell lines deficient either for the two AMPK catalytic subunits, for tuberin of the tuberous sclerosis complex (TSC2), a negative regulator of mTORC1 signaling, or for eEF2K to monitor eEF2 phosphorylation. In parallel, we identified Ser491/Ser492 as a new key in vitro phosphorylation site for AMPK in eEF2K and studied eEF2 phosphorylation by 991 treatment and effects on protein synthesis in eEF2K-null MEFs in which wild-type eEF2K or a S491A/S492A mutant had been re-introduced by viral transfection. Our data provide new insights into the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation with implications for protein synthesis inhibition in response to cellular stresses.
Section snippets
Reagents and materials
Compound 991 (previously referred to as ex229 [47] from patent application WO2010036613, Merck Sharp & Dohme Corp., Metabasis Therapeutics, Inc. Novel cyclic benzimidazole derivatives useful anti-diabetic agents, 2010) was kindly provided by AstraZeneca, Mölndal, Sweden. All other reagents were from Sigma Aldrich. Cell culture reagents were from Life Technologies. Oligonucleotides were from Integrated DNA Technologies (IDT). [γ−32P] ATP was from Perkin Elmer. Anti-total ACC (Merck Millipore,
Increased eEF2 phosphorylation by AMPK activators requires Ca2+ and is partly dependent on AMPK
Several studies have reported that both physiological and pharmacological AMPK activation leads to increased eEF2 phosphorylation, most likely via eEF2K activation. Accordingly, in immortalized MEF cells lacking eEF2K, no eEF2 Thr56 phosphorylation was seen under conditions that led to AMPK activation monitored by AMPKα Thr172 phosphorylation (Supplemental Fig. S1). We therefore investigated whether eEF2 phosphorylation was also strictly dependent on AMPK. In wild-type MEF cells incubated under
Discussion
eEF2K is a complicated multi-modulated protein kinase and the control of its activity integrates inputs from many cellular signaling pathways. In addition, eEF2K activity is controlled by [Ca2+]i and pH, such that an increase in [Ca2+]i via CaM leads to increased eEF2K autophosphorylation/activity and eEF2K displays optimal activity at pH 6.4. Multi-site phosphorylation by protein kinases of different signaling pathways can lead to eEF2K activation or inactivation, change CaM affinity/Ca2+
Conclusions
Activation of AMPK using pharmacological activators leads to increased phosphorylation of eEF2 through different mechanisms, including increased cytosolic [Ca2+]i, inhibition of mTORC1 by AMPK and direct activation of eEF2K by AMPK-mediated multi-site phosphorylation. Direct activation of eEF2K is the major mechanism, increasing the Vmax of the kinase and implicating phosphorylation of the newly identified Ser491/Ser492 in human eEF2K.
Acknowledgements
We thank Benoît Viollet and Marc Foretz (INSERM and Cochin Institute, Paris) for kindly providing AMPK- and LKB1-deficient MEFs, Nicolas Tajeddine for help with intracellular [Ca2+]i measurements and Melissa Drappier for help with production of retroviruses and infection of cells. M.J. was supported by the Fund for Scientific Research in Industry and Agriculture (FRIA). Funding was from the Interuniversity Poles of Attraction Belgian Science Policy (P7/13), the Directorate General Higher
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These authors contributed equally to this work.