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

• Published in: Cellular signalling Vol. 36; pp. 212 - 221
• Main Authors: Johanns, M., Pyr dit Ruys, S., Houddane, A., Vertommen, D., Herinckx, G., Hue, L., Proud, C.G., Rider, M.H.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01-08-2017
• Subjects: AMP-Activated Protein Kinases - metabolism; AMPK; Animals; Calcium - pharmacology; Compound 991; eEF2; eEF2K; Elongation Factor 2 Kinase - metabolism; Enzyme Activation - drug effects; Enzyme Activators - pharmacology; Humans; Mechanistic Target of Rapamycin Complex 1 - metabolism; Mice; mTORC1; Phosphorylation - drug effects; Phosphoserine - metabolism; Protein Biosynthesis - drug effects; Translation; eEF2K; AMPK; eEF2; Compound 991; Translation; mTORC1
• DOI: 10.1016/j.cellsig.2017.05.010
• EISSN: 1873-3913
• ISSN: 0898-6568

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.
The goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis.
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.
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.
Mechanism of AMPK-induced inhibition of protein synthesis via eEF2K activation. [Display omitted]
•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 Vmax of eEF2K without affecting Ca2+/CaM sensitivity.•Ser491/Ser492 eEF2K phosphorylation by AMPK in MEFs is important for mTOR-independent protein synthesis inhibition.