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July 1st, 2025
Version: 1
The University of Texas at Austin
biochemistry
biorxiv

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

Bohanon, A. L.Open in Google Scholar•Browning, L. S.Open in Google Scholar•Sammons, R. M.Open in Google Scholar•Piserchio, A.Open in Google Scholar•Tavares, C. D. J.Open in Google Scholar•Cho, E. J.Open in Google Scholar•Ghose, R.Open in Google Scholar•DALBY, K. N.Open in Google Scholar

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 Calcium/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 Calcium/CaM, suggesting a mechanism for maintaining basal activity and priming the kinase for rapid reactivation in response to Calcium transients. These findings support a model in which phosphorylation on T348 and S500 synergize to stabilize the active conformation of eEF-2K.

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