Modulation of eukaryotic elongation factor 2 kinase activity via phosphorylation on Ser-500 (773.1)

Eukaryotic elongation factor 2 kinase (eEF‐2K) is an atypical calcium/calmodulin (Ca2+/CaM)‐dependent kinase known to modulate protein synthesis through the inhibitory phosphorylation of elongation factor 2. Despite its pivotal role in translation regulation and potential role in tumorigenesis, the mechanism of regulation of eEF‐2K activity by phosphorylation is still unclear. Our objective was to analyze the activation of eEF‐2K by phosphorylation at Ser‐500 (phosphorylated by protein kinase A or Ca2+/CaM‐induced autophosphorylation). In vitro analysis indicates that autophosphorylation of eEF‐2K induces Ca2+‐independent activity. Western blotting analysis as well as mutagenesis of Ser‐500 to Ala or Asp suggests that Ser‐500 phosphorylation is responsible for this Ca2+‐independent activity. Interestingly, CaM appears to be essential for this Ca2+‐independent activity of eEF‐2K. Mechanistic studies indicate that Ser‐500 phosphorylation increases eEF‐2K affinity for CaM in the presence or absence of Ca2+. Ser‐500 phosphorylation also increases the rate of Thr‐348 phosphorylation (a key site for the allosteric regulation of eEF‐2K activity). We are currently studying the dynamic nature of Ser‐500 phosphorylation in cells. Phosphorylation of eEF‐2K on Ser‐500 could be a significant mechanism for its regulation, especially upon the influx of Ca2+ during cellular signaling events. Grant Funding Source : NIH (GM059802 and CA167505) to KND