Direct and indirect activation of eukaryotic elongation factor 2 kinase by AMP-activated protein kinase

Highlights

• AMPK activation increases eEF2 phosphorylation by direct eEF2K activation via multi-site phosphorylation.

• Pharmacological AMPK activators can cause increased eEF2 phosphorylation by increasing intracellular Ca²⁺.

• Increased eEF2 phosphorylation by 991 in MEFs is mainly mTORC1-independent, but dependent on AMPK and eEF2K.

• Ser491/Ser492 eEF2K phosphorylation by AMPK in vitro increases the V_max of eEF2K without affecting Ca²⁺/CaM sensitivity.

• Ser491/Ser492 eEF2K phosphorylation by AMPK in MEFs is important for mTOR-independent protein synthesis inhibition.

Abstract

Background

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a key regulator of protein synthesis in mammalian cells. It phosphorylates and inhibits eEF2, the translation factor necessary for peptide translocation during the elongation phase of protein synthesis. When cellular energy demand outweighs energy supply, AMP-activated protein kinase (AMPK) and eEF2K become activated, leading to eEF2 phosphorylation, which reduces the rate of protein synthesis, a process that consumes a large proportion of cellular energy under optimal conditions.

Aim

The goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis.

Methods

Using genetically modified mouse embryo fibroblasts (MEFs), effects of treatments with commonly used AMPK activators to increase eEF2 phosphorylation were compared with that of the novel compound 991. Bacterially expressed recombinant eEF2K was phosphorylated in vitro by recombinant activated AMPK for phosphorylation site-identification by mass spectrometry followed by site-directed mutagenesis of the identified sites to alanine residues to study effects on the kinetic properties of eEF2K. Wild-type eEF2K and a Ser491/Ser492 mutant were retrovirally re-introduced in eEF2K-deficient MEFs and effects of 991 treatment on eEF2 phosphorylation and protein synthesis rates were studied in these cells.

Results & conclusions

AMPK activation leads to increased eEF2 phosphorylation in MEFs mainly by direct activation of eEF2K and partly by inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment of MEFs with AMPK activators can also lead to eEF2K activation independently of AMPK probably via a rise in intracellular Ca²⁺. AMPK activates eEF2K by multi-site phosphorylation and the newly identified Ser491/Ser492 is important for activation, leading to mTOR-independent inhibition of protein synthesis. Our study provides new insights into the control of eEF2K by AMPK, with implications for linking metabolic stress to decreased protein synthesis to conserve energy reserves, a pathway that is of major importance in cancer cell survival.

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.