Browning, L. S. | bioRxiv Science

bioRxiv2025

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

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the -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{superscript 2}/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 Ca2+/CaM, suggesting a mechanism for maintaining basal activity and priming the kinase for rapid reactivation in response to Ca2+ transients. These findings support a model in which phosphorylation on T348 and S500 synergize to stabilize the active conformation of eEF-2K.

biochemistry↗

bioRxiv2025

The Critical Role of the C-terminal Lobe of Calmodulin in Activating Eukaryotic Elongation Factor 2 Kinase

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the -kinase family, modulates translational rates by phosphorylating eEF-2, a GTPase that facilitates the translocation of the nascent chain on the ribosome during the elongation phase of protein synthesis. eEF-2K is regulated by diverse cellular cues, many of which sensitize it to the Ca2+-effector protein calmodulin (CaM). CaM, which binds and allosterically activates eEF-2K in the presence of Ca2+, contains two structural "lobes," each with a pair of Ca2+-binding EF-hands. Using kinetic analysis, we demonstrate that the isolated C-terminal lobe of CaM (CaMC) is sufficient to engage and fully activate eEF-2K in a Ca2+-dependent fashion. Genetically fusing CaMC to the N-terminus of eEF-2K, upstream of its critical CaM-targeting motif (CTM) via a flexible 2-glycine linker, results in a chimeric species (C-LiNK) that is constitutively active independent of external CaM and Ca2+. A structure of the C-LiNK functional core reveals no significant deviation in the overall conformations of the interacting modules and orientations of key catalytic-site residues relative to the heterodimeric complex between full-length CaM and eEF-2K. These observations demonstrate that, in contrast to other CaM-regulated kinases, CaMC alone is sufficient to activate eEF-2K fully. The proximity effect of CaMC in the context of C-LiNK removes the requirement for external Ca2+, whose apparent role is to enhance the CaM-affinity of eEF-2K and drive kinase activation. The responsiveness of eEF-2K to regulatory stimuli in cells appears to be lost in C-LiNK, presumably due to its permanently "on" state.

biochemistry↗