Abstract 2172 Identifying a Novel Mechanism of Translation Regulation: The ADP-dependent Activation of eEF-2K

Protein translation is an energy-intensive process that requires strict regulation by proliferating cells to balance the demands of growth with energy availability. Despite being the most energy-consumptive phase in translation, the control of the elongation phase remains under-investigated. Elongation is, in part, regulated by the phosphorylation of eukaryotic elongation factor 2 (eEF-2), a GTPase that facilitates the translocation step of elongation. The atypical kinase eukaryotic elongation factor 2 kinase (eEF-2K) negatively regulates eEF-2 via phosphorylation. eEF-2K itself is known to be controlled through multiple inputs. Calmodulin (CaM), a calcium modulated protein, binds eEF-2K and is an essential allosteric activator. Additionally, eEF-2K serves as a key point of integration for signal transduction pathways critical for cell proliferation and metabolic regulation. The mechanism by which eEF-2K integrates and prioritizes these disparate signals has yet to be elucidated. Here we identify a novel mechanism of regulation of eEF-2K. To better understand the interaction of eEF-2K and its essential activator CaM, we determined the crystal structure of CaM•eEF-2K. During our investigation, we discovered ADP bound to a pocket in the interface of the two proteins, termed the basic pocket (BP). Biochemical analysis determined that ADP increases the affinity of eEF-2K for CaM in vitro. Further, expression of eEF-2K harboring mutations of residues within the BP into eEF-2K knockout cells, abrogated the ability of the glycolysis inhibitor 2-deoxyglucose to sufficiently induce phosphorylation of eEF-2 compared to wild type (WT) eEF-2K. However, both WT and mutant eEF-2K were activated by the removal of all nutrients and growth factors. To determine why mutant eEF-2K displayed differential activation, the phosphorylation status of eEF-2K at known sites of inhibitory phosphorylations was assessed under both conditions. The removal of nutrients and growth factors, but not the addition of 2-deoxyglucose, reduced the presence of inhibitory phosphorylations of eEF-2K. These findings may identify a novel mechanism of activation of eEF-2K: one in which changes in cellular ADP concentrations modulate the affinity of eEF-2K for CaM and allow eEF-2K to directly sense cellular energy availability to properly regulate elongation rates. Lastly, the ability of ADP binding to activate eEF-2K without the removal of inhibitory phosphorylations provides insight into how eEF-2K prioritizes upstream inputs to properly regulate rates of protein translation. This work is supported by NIH award R01 GM123252 (to K.N.D. and R.G.) and a Welch Foundation award F-1390 (K.N.D.).