{"doi":"10.1016/j.jbc.2025.111087","title":"Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation","abstract":"Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the α-kinase family of atypical serine/threonine kinases, phosphorylates eEF-2 to slow ribosomal translocation and modulate translational elongation. eEF-2K activation requires Ca 2+ /calmodulin (CaM) and integrates upstream signals through specific sites within an intrinsically disordered regulatory loop (R-loop; ∼321–520) that links the α-kinase core to a C-terminal domain. Unlike canonical CaM-dependent kinases that are activated by displacement of an autoinhibitory segment that occludes the active site, eEF-2K is activated by CaM-driven stabilization of an active state; Ca 2+ /CaM engagement triggers rapid autophosphorylation at T348, which is essential for full activity. Phosphorylation on S500, by eEF-2K or PKA, lowers the CaM requirement (∼20-fold) without increasing maximal catalytic turnover. Here we show that the phosphomimetic S500D markedly enhances CaM binding in the T348-phosphorylated enzyme. S500D also elevates CaM-independent (\"intrinsic\") activity even in the absence of phosphorylation at T348, although maximal activity requires modification at both sites. Hydrogen–deuterium exchange mass spectrometry reveals CaM-dependent conformational changes near S500, consistent with relief of inhibitory constraints. Deletion of residues near S500 mimics S500D, increasing intrinsic activity and CaM binding in vitro and enhancing eEF-2 phosphorylation in cells, supporting an inhibitory role for this segment. Prior studies have linked S500 phosphorylation to eEF-2K degradation, suggesting a dual regulatory role. We demonstrate that phosphorylation at T348 and S500 synergize to stabilize an active-like conformation and increase CaM responsiveness, effectively lowering the Ca 2+ /CaM threshold for eEF-2K activation and enabling the integration of Ca 2+ , cAMP/PKA, and metabolic cues.","journal":"Journal of Biological Chemistry","year":2025,"id":549698,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9513,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1068452,"name":"Luke S. Browning","orcid":"0000-0002-1301-321X","position":1,"is_corresponding":false},{"id":751030,"name":"Andrea Piserchio","orcid":"0000-0001-7865-9065","position":2,"is_corresponding":false},{"id":751031,"name":"Kimberly J. Long","orcid":"0000-0002-3647-705X","position":3,"is_corresponding":false},{"id":1444822,"name":"Sharon Jacob","orcid":null,"position":4,"is_corresponding":false},{"id":1116916,"name":"Rae M. Sammons","orcid":null,"position":5,"is_corresponding":false},{"id":322198,"name":"Clint D.J. Tavares","orcid":null,"position":6,"is_corresponding":false},{"id":517669,"name":"Eun Jeong Cho","orcid":"0000-0002-3298-2462","position":7,"is_corresponding":false},{"id":751032,"name":"Ranajeet Ghose","orcid":"0000-0002-0763-8686","position":8,"is_corresponding":false},{"id":292931,"name":"Kevin N. Dalby","orcid":"0000-0001-9272-5129","position":9,"is_corresponding":false},{"id":804363,"name":"Amanda L. Bohanon","orcid":"0000-0002-3371-3416","position":0,"is_corresponding":true}],"reference_count":42,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:54:12.321988Z","pmid":"41443425","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}