Stimulation of the AMP-activated Protein Kinase Leads to Activation of Eukaryotic Elongation Factor 2 Kinase and to Its Phosphorylation at a Novel Site, Serine 398

Abstract

Protein synthesis consumes a high proportion of the metabolic energy of mammalian cells, and most of this is used by peptide chain elongation. An important regulator of energy supply and demand in eukaryotic cells is the AMP-activated protein kinase (AMPK). The rate of peptide chain elongation can be modulated through the phosphorylation of eukaryotic elongation factor (eEF) 2, which inhibits its activity and is catalyzed by a specific calcium/calmodulin-dependent protein kinase termed eEF2 kinase. Here we show that AMPK directly phosphorylates eEF2 kinase, and we identify the major site of phosphorylation as Ser-398 in a regulatory domain of eEF2 kinase. AMPK also phosphorylates two other sites (Ser-78 and Ser-366) in eEF2 kinase in vitro. We develop appropriate phosphospecific antisera and show that phosphorylation of Ser-398 in eEF2 kinase is enhanced in intact cells under a range of conditions that activate AMPK and increase the phosphorylation of eEF2. Ser-78 and Ser-366 do not appear to be phosphorylated by AMPK within cells. Although cardiomyocytes appear to contain a distinct isoform of eEF2 kinase, it also contains a site corresponding to Ser-398 that is phosphorylated by AMPK in vitro. Stimuli that activate AMPK and increase eEF2 phosphorylation within cells increase the activity of eEF2 kinase. Thus, AMPK and eEF2 kinase may provide a key link between cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy.Protein synthesis is a process of fundamental importance for all living cells. It also places substantial demands on the cell in terms of requirements for precursors, viz. amino acids and metabolic energy. Recent work (1-3) has shown that amino acids regulate the translational machinery in a number of ways, e.g. through control of the mTOR 1 signaling pathway.Protein synthesis is a major consumer of cellular energy with estimates of 30 -50% of total energy being used in this process. The vast majority of this is consumed by peptide elongation, either directly as GTP (two are hydrolyzed for each amino acid added) or indirectly during aminoacyl-tRNA charging, one ATP being hydrolyzed to AMP for each amino acid charging event.Despite this, little is known of the mechanisms by which cells couple the rate of protein synthesis to the availability of energy, i.e. ATP. In mammalian cells, peptide chain elongation requires two main elongation factors, eEF1A and eEF2. The latter mediates the translocation step of elongation in which the ribosome moves by the equivalent of one codon relative to the mRNA, and the peptidyl-tRNA shifts from the A-into the P-site on the ribosome (4). eEF2 is a phosphoprotein, and phosphorylation at a single site, Thr-56, renders it unable to bind ribosomes, thereby inactivating it (5-7). Phosphorylation is catalyzed by a specific and very unusual kinase, eEF2 kinase (8, 9). Phosphorylation of eEF2 is decreased by insulin and certain other stimuli through signaling events that require the mammalian target of rapamycin, mTOR (reviewed in ...

A pharmacoproteomic approach implicates eukaryotic elongation factor 2 kinase in ER stress-induced cell death

“…Furthermore, pharmacological inducers of ER stress such as Tm and thapsigargin (Tg), an inhibitor of the ER-resident Ca 2 þ -dependent ATPase, induced eEF-2 phosphorylation with kinetics similar to those of sal (Supplementary Figure 3). EEF-2 phosphorylation has been observed in response to several stimuli, [26][27][28][29][30][31][32][33] but never in the context of ER stress. Our observations suggest that eEF-2 might be regulated by phosphorylation during ESR.…”

“…[22][23][24] The phosphorylation of eEF-2 is catalyzed by eEF-2 kinase (eEF-2K), an unusual calcium/calmodulin-dependent enzyme belonging to the alpha kinase family of atypical protein kinases. 25 EEF-2 phosphorylation has been shown to play an important role in coupling protein synthesis to energy metabolism in response to calcium flux, 26 cyclic adenosine monophosphate (AMP)/protein kinase A and AMP-activated protein kinase signaling, [27][28][29][30] amino acid or glucose limitation, 31,32 and cytoplasmic pH changes, 33 but no role for eEF-2 phosphorylation in ER stress has been reported.…”

The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation

“…input signal), this mechanism provides for exquisite control of eEF-2K output. To date, the molecular mechanisms underlying the activation of eEF-2K by PKA (32,54) and AMPK (55), which phosphorylate Ser-500 and Ser-398, respectively, are unknown. We believe this study provides a mechanistic foundation for the elucidation of these mechanisms.…”

“Translating” Tumor Hypoxia: Unfolded Protein Response (UPR)–Dependent and UPR-Independent Pathways

“…Both AMPK and mTOR have been shown to phosphorylate eEF2 kinase, which leads to subsequent phosphorylation of the elongation factor eEF2 and inhibition in translation elongation (76,77). Liu et al showed that activation of AMPK during hypoxia resulted in strong eEF2 phosphorylation.…”