Regulation of elongation factor 2 kinase (eEF-2K) by acidity

Eukaryotic elongation factor 2 kinase (eEF-2K) is a calmodulin (CaM)/calcium regulated kinase whose activity disrupts translation elongation resulting in the global decrease of protein synthesis¹. Translation is one of the most energy intensive processes in a cell; eEF-2K is critical in maintaining cellular homeostasis. The dysregulation of eEF-2K is associated with an assortment of diseases including several types of cancer², depression [superscript 3,4], and Alzheimer’s disease⁵, which has prompted closer examination across multiple studies of the enzyme in recent decades. Notably, the kinetic mechanism of eEF-2K has been detailed as a two-step process: 1) The binding of Ca²⁺-CaM enables the autophosphorylation of Thr-348 and 2) The resulting conformational change enables eEF-2K to bind and phosphorylate its substrate. We have uncovered many surprising layers of eEF-2K regulation in past years, from kinetic mechanism to upstream affecters to models of CaM binding. This study focuses on the role of pH in regulating eEF-2K. pH homeostasis is also vital for cellular function. Even a slight disruption between the intra- and extracellular pH ratio can affect an array of processes such as ATP synthesis, enzyme function, as well as the proliferation, migration, and invasion of tumor cells⁶. Acidosis is a common effect of high intensity exercise, diabetic ketoacidosis, ischemia, and solid tumors [superscript 7–9]. It has been demonstrated that eEF-2K activity is increased and protein translation decreased in cells under acidic conditions, suggesting the enzyme’s potential cytoprotective effects [superscript 10–12]. This work reports the following: 1) A novel mechanism where H⁺ instead of Ca²⁺ activates eEF-2K; 2) A novel mechanism where H⁺ can replace Ca²⁺ in promoting eEF-2K substrate phosphorylation; 3) H⁺ promotes the binding of CaM to eEF-2K in a Ca²⁺-independent manner; 4) There are five histidine residues on eEF-2K that may contribute to the stabilized activity of eEF-2K under acidic conditions.Cellular and Molecular Biolog