Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation.

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the α-kinase family of atypical kinases, phosphorylates eukaryotic elongation factor 2 (eEF-2), thereby inhibiting ribosomal translocation and downregulating translational elongation in response to diverse cellular cues. eEF-2K is activated by Ca/calmodulin (CaM) and integrates upstream inputs from diverse signaling pathways, including PKA and mTOR, which target regulatory sites on a disordered regulatory loop. Among these, serine 500 (S500) has been identified as a key phosphorylation site targeted by both eEF-2K and PKA. However, the influence of this post-translational modification on the properties of eEF-2K has remained unclear. Prior studies have shown that S500 phosphorylation accelerates autophosphorylation of eEF-2K at its primary activating site, threonine 348 (T348). Here, we demonstrate that S500 phosphorylation, mimicked by a S500D mutation, works in conjunction with T348 phosphorylation to enhance the intrinsic (CaM-independent) activity of eEF-2K. Hydrogen-deuterium exchange mass spectrometry reveals that CaM binding, and consequent enhancement in eEF-2K activity, is accompanied by conformational changes proximal to S500. Deletion of S500 and surrounding residues mimics the effects of S500D, promoting robust CaM-independent activity. These data suggest that CaM binding or S500 phosphorylation have similar effects, likely relieving an inhibitory constraint to enhance activity. Further, S500 phosphorylation enhances binding to both apo-CaM and Ca/CaM, suggesting a mechanism for maintaining basal activity and priming the kinase for rapid reactivation in response to Ca transients. These findings support a model in which phosphorylation on T348 and S500 synergize to stabilize the active conformation of eEF-2K.