Signal Integration at Elongation Factor 2 Kinase: THE ROLES OF CALCIUM, CALMODULIN, AND SER-500 PHOSPHORYLATION

J Biol Chem. 2017 Feb 3;292(5):2032-2045. doi: 10.1074/jbc.M116.753277. Epub 2016 Dec 12.


Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca2+/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca2+ and CaM to influence eEF-2K activity.

Keywords: CaMK-III; S500D; Ser-500; Thr-348; calcium; calmodulin (CaM); eEF-2K; phosphorylation; translation; translation elongation factor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Calcium / metabolism*
  • Calmodulin / genetics
  • Calmodulin / metabolism*
  • Cell Line, Tumor
  • Elongation Factor 2 Kinase / genetics
  • Elongation Factor 2 Kinase / metabolism*
  • Humans
  • Mutation, Missense
  • Phosphorylation / genetics
  • Serine / genetics
  • Serine / metabolism


  • Calmodulin
  • Serine
  • EEF2K protein, human
  • Elongation Factor 2 Kinase
  • Calcium

Associated data

  • PDB/5J8H