{"gene":"EEF2K","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2012,"finding":"eEF2K phosphorylates eEF2 on Thr56, decreasing eEF2's affinity for the ribosome and inhibiting translocation (A-to-P site movement), thereby slowing peptide chain elongation. In response to genotoxic stress, AMPK activates eEF2K by phosphorylating it on Ser398. Subsequently, eEF2K is degraded via the SCF(βTrCP) ubiquitin ligase through autophosphorylation on a canonical βTrCP-binding domain, enabling resumption of translation elongation during DNA damage checkpoint silencing.","method":"In vitro kinase assays, mutagenesis, co-immunoprecipitation, ubiquitin-proteasome degradation assays, phosphosite mapping","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (in vitro assays, mutagenesis, Co-IP, degradation assays) in a single rigorous study establishing both activation mechanism and degradation mechanism","pmids":["22669845"],"is_preprint":false},{"year":2009,"finding":"In contracting fast-twitch skeletal muscle, eEF2K is activated downstream of Ca2+/calmodulin (CaM), leading to phosphorylation of eEF2 and partial suppression (~30–40%) of protein synthesis. This Ca2+-CaM-eEF2K-eEF2 signaling cascade operates independently of AMPK or changes in intracellular pH.","method":"Ex vivo muscle contraction, pharmacological Ca2+ manipulation, eEF2K inhibitor treatment, kinase-dead AMPK overexpression, protein synthesis rate measurement","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal pharmacological and genetic approaches in a single rigorous study; replicated across multiple contraction protocols","pmids":["19188248"],"is_preprint":false},{"year":2009,"finding":"eEF2K is required for autophagy signaling during nutrient starvation and ER stress. During starvation, eIF2α phosphorylation is required upstream of eEF2K activation and eEF2 phosphorylation. During ER stress, eEF2K-mediated eEF2 phosphorylation can occur partly through Ca2+ flux independently of eIF2α phosphorylation.","method":"siRNA knockdown, phosphorylation assays, autophagy readouts (LC3-II), pharmacological inhibitors","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined autophagy phenotype, two orthogonal stress conditions, single lab","pmids":["19221463"],"is_preprint":false},{"year":2010,"finding":"TRPM7, via its kinase domain, promotes phosphorylation of eEF2 at Thr56 under Mg2+-limited conditions by influencing the abundance and phosphorylation state of eEF2K at Ser77. TRPM7 kinase does not directly phosphorylate eEF2 but acts through eEF2K.","method":"Cell-based phosphorylation assays, TRPM7 kinase-dead mutants, western blotting","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — use of kinase-dead mutants and phosphorylation assays, single lab, two orthogonal approaches","pmids":["21112387"],"is_preprint":false},{"year":2011,"finding":"Recombinant human eEF2K expressed in E. coli is a monomer of ~85 kDa (by light scattering) that phosphorylates eEF2 in vitro with kinetic parameters comparable to the mammalian enzyme. eEF2K is activated by calcium and calmodulin.","method":"Recombinant protein purification, light scattering, in vitro kinase assay, kinetic analysis","journal":"Protein expression and purification","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of enzymatic activity with purified recombinant protein and kinetic characterization; single lab but direct biochemical validation","pmids":["21605678"],"is_preprint":false},{"year":2012,"finding":"eEF2K activity (eEF2 phosphorylation at Thr56) is essential for cortical-dependent associative taste learning. Kinase-inactive eEF2K knock-in (ki) mice with reduced eEF2 phosphorylation show attenuated conditioned taste aversion but normal incidental taste learning, and exhibit altered brain activation patterns (by MEMRI) during learning.","method":"Kinase-inactive knock-in mice, behavioral testing (conditioned taste aversion), manganese-enhanced MRI (MEMRI), western blotting","journal":"Learning & memory","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knock-in model with specific phospho-site reduction, multiple behavioral paradigms, and in vivo brain imaging; single lab but rigorous","pmids":["22366775"],"is_preprint":false},{"year":2012,"finding":"Under ER stress, eEF2K is required for autophagy induction. DDIT4 (REDD1) transduces ER stress signals to activate eEF2K. Phosphorylation of eEF2K at Ser398 promotes autophagy, while phosphorylation at Ser366 and Ser78 inhibits autophagy. Suppression of eEF2K aggravates ER stress and shifts cells toward apoptosis.","method":"RNAi knockdown, phosphomutant constructs, autophagy and apoptosis assays, western blotting","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined phosphorylation sites via mutagenesis with functional autophagy readouts, single lab","pmids":["23182879"],"is_preprint":false},{"year":2014,"finding":"eEF2K senses oxidative stress and rapidly downregulates short-lived antiapoptotic proteins XIAP and c-FLIPL by inhibiting global protein synthesis, rendering cells susceptible to apoptosis. Loss of eEF2K in mice reduces ovarian apoptosis and leads to accumulation of aberrant follicles; loss of eEF2K ortholog in C. elegans reduces germ cell death and worsens oocyte quality.","method":"eEF2K knockout mice, C. elegans genetics, protein synthesis assays, western blotting for XIAP and c-FLIPL, apoptosis assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — replicated in two independent model organisms (mouse and C. elegans) with mechanistic follow-up on specific substrates","pmids":["24582807"],"is_preprint":false},{"year":2014,"finding":"Silencing of eEF2K in colon cancer cells increases protein synthesis and activates the AMPK-ULK1 pathway, inducing autophagy independently of mTOR suppression. Knockdown of AMPK or ULK1 abolishes eEF2K silencing-induced autophagy, placing eEF2K upstream of AMPK-ULK1 in this context.","method":"siRNA knockdown, LC3-II western blot, LC3 dot accumulation, autophagic flux assays, genetic epistasis (AMPK/ULK1 knockdown)","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis experiments with multiple genetic knockdowns, orthogonal autophagy readouts, single lab","pmids":["24955726"],"is_preprint":false},{"year":2014,"finding":"eEF2K regulates the expression of tissue transglutaminase (TG2), and the eEF2K/TG2 axis promotes cancer cell survival. Inhibition of eEF2K leads to caspase-independent apoptosis associated with induction of apoptosis-inducing factor (AIF). eEF2K protein is degraded through the ubiquitin-proteasome pathway upon rottlerin treatment.","method":"siRNA knockdown, overexpression, western blotting, apoptosis assays (AIF, caspase), ubiquitin-proteasome pathway inhibitors","journal":"Apoptosis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with mechanistic follow-up on TG2 and AIF pathways; single lab","pmids":["24193916"],"is_preprint":false},{"year":2014,"finding":"Ribosomal stress (ribosome biogenesis defect) activates the eEF2K-eEF2 pathway, inhibiting translation elongation. This causes a translational reprogramming in which TOP (terminal oligopyrimidine) mRNAs encoding ribosomal proteins are selectively recruited to polysomes, relatively increasing synthesis of TOP mRNA-encoded proteins.","method":"Ribosomal stress induction, eEF2K inhibition, polysome profiling, translation assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological eEF2K inhibition with polysome profiling; single lab, two orthogonal readouts","pmids":["25332393"],"is_preprint":false},{"year":2014,"finding":"A reversible covalent inhibition mechanism for eEF2K: the compound DFTD binds in two steps (fast binding followed by slow reversible inactivation). Molecular docking and mutagenesis indicate a nitrile group forms a reversible thioimidate adduct with the active-site Cys146, which is not conserved in related kinases.","method":"Kinetic analysis (two-step inhibition), active-site mutagenesis, molecular docking, chemoinformatics","journal":"Chembiochem","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinetic and mutagenesis data establishing covalent mechanism; single lab but multiple orthogonal methods","pmids":["25224652"],"is_preprint":false},{"year":2016,"finding":"NF-κB activation (by TNFα, HCMV infection, or dsDNA) represses eEF2K transcription through the p65 NF-κB subunit, reducing eEF2K pre-mRNA and protein levels, thereby decreasing eEF2 phosphorylation (Thr56) and stimulating translation elongation.","method":"NF-κB activation assays, eEF2K pre-mRNA quantification, pharmacological and genetic NF-κB modulation, p65 ChIP/transcription assays, western blotting","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple NF-κB-activating stimuli tested, pre-mRNA analysis establishing transcriptional mechanism, p65 subunit specificity shown; single lab but diverse orthogonal approaches","pmids":["31636182"],"is_preprint":false},{"year":2016,"finding":"eEF2K activity negatively regulates GABAergic synaptic transmission in neurons. Loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors, altering the excitation/inhibition balance and conferring resistance to epileptic seizures.","method":"eEF2K knockout mice, electrophysiology, western blotting for Synapsin 2b and GABAA receptor subunits, in vivo seizure models","journal":"Cerebral cortex","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined molecular (Synapsin 2b, α5-GABAAR) and behavioral (seizure) phenotypes, pharmacological corroboration; single lab but multiple methods","pmids":["27005990"],"is_preprint":false},{"year":2017,"finding":"Myostatin inhibits protein synthesis in skeletal muscle by activating AMPK, which in turn activates the eEF2K-eEF2 pathway. At low concentrations, myostatin suppresses protein synthesis exclusively through the AMPK-eEF2K-eEF2 axis without affecting mTOR.","method":"C2C12 myotube treatment with recombinant myostatin, SUnSET protein synthesis assay, western blotting, pharmacological pathway inhibitors","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein synthesis measurement with pathway-specific inhibitors; single lab","pmids":["29024627"],"is_preprint":false},{"year":2019,"finding":"eEF2K interacts with the scaffolding protein Homer1b/c, and this interaction is regulated by mTORC1-dependent phosphorylation of eEF2K at Ser396 (a known mTORC1 site). Homer1b/c binding controls eEF2K localization and affects rates of localized protein synthesis at synapses.","method":"Co-immunoprecipitation, pharmacological mTORC1 inhibition, phosphomutant constructs, protein synthesis assays in SH-SY5Y cells and mouse cortical neurons","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP for interaction, mTORC1 manipulation for regulatory mechanism, functional synthesis readout; single lab","pmids":["32892352"],"is_preprint":false},{"year":2020,"finding":"Known physiological stimuli that enhance neurogenesis converge on the eEF2K/eEF2 pathway via AMPK in the dentate gyrus. eEF2K knockout in mature excitatory neurons of the dentate gyrus enhances adult neurogenesis and upregulates neurogenesis-related proteins, correlating with improved dentate gyrus-dependent learning.","method":"General and conditional eEF2K knockout mice, neurogenesis markers, behavioral testing, western blotting","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — both global and region-specific conditional KO with consistent neurogenesis and behavioral phenotypes; single lab but two genetic models","pmids":["32707059"],"is_preprint":false},{"year":2020,"finding":"SIRT1 promotes eEF2K/eEF2-dependent autophagy in cardiomyocytes under ER stress. eIF2α co-immunoprecipitates with eEF2K, and eIF2α knockdown reduces eEF2 phosphorylation, indicating eIF2α is required upstream of eEF2K activation in this cardiac context.","method":"Co-immunoprecipitation (eIF2α–eEF2K), siRNA knockdown, SIRT1 genetic/pharmacological modulation, autophagy assays in cardiac cells, in vivo cardiac function in SIRT1-deficient mice","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for interaction, multiple knockdown experiments, in vivo cardiac phenotype; single lab","pmids":["32059483"],"is_preprint":false},{"year":2021,"finding":"Phosphorylation of eEF2 (via eEF2K) suppresses translation elongation and is the principal mechanism by which Rpl24 mutation suppresses colorectal tumorigenesis in mice. Genetic inactivation of eEF2K in Rpl24 mutant mice completely restores elongation rate and protein synthesis, and abrogates the tumor-suppressive effect of the Rpl24 mutation, demonstrating that eEF2K activity is required for Rpl24-mediated tumor suppression.","method":"Genetic epistasis in mice (Rpl24 mutation × eEF2K inactivation), polysome profiling, protein synthesis rate measurement, tumor incidence/growth assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo double-mutant genetic epistasis with direct elongation rate and protein synthesis measurements; single lab but highly rigorous","pmids":["34895463"],"is_preprint":false},{"year":2021,"finding":"eEF2K phosphorylated eEF2 stabilizes vacant 80S ribosomes containing SERBP1 (in place of mRNA) and eEF2 in the acceptor site, as revealed by cryo-EM. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. eEF2K thus defines a population of inactive, recycling-resistant ribosomes and controls p-body abundance in sensory neurons.","method":"Cryo-electron microscopy, in vitro ribosome splitting assay, pharmacological eEF2K activation (nelfinavir), sensory neuron imaging, p-body quantification","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure combined with in vitro biochemical assay and pharmacological manipulation; multiple orthogonal methods","pmids":["34815424"],"is_preprint":false},{"year":2022,"finding":"eEF2K directly binds to and phosphorylates GSK3β at Ser9, inactivating GSK3β and leading to stabilization and upregulation of PD-L1 protein in melanoma cells. Immunoprecipitation-mass spectrometry identified this interaction, and knockdown of eEF2K decreased PD-L1 and enhanced CD8+ T cell activity in vivo.","method":"Immunoprecipitation–mass spectrometry, Co-IP, in vitro phosphorylation assay, western blotting, in vivo mouse melanoma model, flow cytometry","journal":"Journal for immunotherapy of cancer","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — IP-MS for substrate identification, confirmed by Co-IP and phosphorylation assay, with in vivo functional validation; single lab but multiple orthogonal methods","pmids":["35347072"],"is_preprint":false},{"year":2020,"finding":"eEF2K promotes translation of PD-L1 mRNA by attenuating the inhibitory effect of an upstream open reading frame (uORF) with a non-canonical CUG start codon in the PD-L1 5'-UTR. eEF2K depletion reduces PD-L1 mRNA association with translationally active polyribosomes and decreases PD-L1 protein levels.","method":"eEF2K ablation (genetic), polyribosome profiling, reporter assays for uORF activity, western blotting, NK cell cytotoxicity assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — polysome profiling plus uORF reporter assay establishing translational mechanism, functional immune assay; single lab, multiple orthogonal methods","pmids":["33094805"],"is_preprint":false},{"year":2022,"finding":"eEF2K upregulates phosphorylation of STAT3 at Tyr705, which binds the SPP1 promoter and enhances SPP1 transcription to facilitate melanoma progression. Re-expression of SPP1 rescues the inhibitory effect of eEF2K silencing, and inhibition of SPP1 or STAT3 abolishes eEF2K-driven effects.","method":"RNA-seq, ChIP assay (STAT3 binding to SPP1 promoter), siRNA/overexpression, rescue experiments, in vivo mouse model","journal":"Clinical and translational medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP for transcription factor binding, genetic rescue, in vivo validation; single lab but multiple orthogonal methods","pmids":["35184394"],"is_preprint":false},{"year":2023,"finding":"Alphavirus nsP2 NTPase domain activates a cAMP/PKA signaling cascade, leading to activation of eEF2K and subsequent phosphorylation of eEF2 at Thr56 (>50-fold increase), causing translational shut-off. NTPase-dead mutations prevent this eEF2 phosphorylation. This mechanism is shared across Old and New World alphaviruses.","method":"Phosphoproteomics (SILAC + LC-MS/MS), NTPase Walker A/B mutants, cAMP/PKA pathway analysis, translation inhibition assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — phosphoproteomics with active-site mutagenesis and biochemical follow-up; multiple alphaviruses tested","pmids":["36848386"],"is_preprint":false},{"year":2024,"finding":"In sensory neurons, painful stimuli activate eEF2K to repress peptide chain elongation. Attenuated elongation is sensed by a ribosome-coupled mechanism that triggers the integrated stress response (ISR). Both eEF2K and the ISR are required for pain-associated behaviors in vivo. This pathway simultaneously induces BDNF biosynthesis; selective blockade of Bdnf translation has analgesic effects.","method":"In vivo eEF2K knockout/inhibition behavioral studies, ISR inhibition, Bdnf mRNA-selective translation blockade, ribosome coupling assays in sensory neurons","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic and pharmacological dissection of eEF2K→ISR→BDNF axis with behavioral readouts; multiple orthogonal methods","pmids":["39694034"],"is_preprint":false},{"year":2025,"finding":"eEF2K binds to and phosphorylates aurora kinase A (AURKA) at Ser391, a newly identified phosphorylation site that is critical for AURKA protein stability and kinase activity. eEF2K also positively regulates SOX8 mRNA and protein expression, constituting an eEF2K/AURKA/SOX8 axis promoting TNBC progression.","method":"Proteomic analysis, Co-IP/binding assay, in vitro phosphorylation assay, mutagenesis, overexpression/knockdown, in vivo xenograft, patient-derived organoids","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro phosphorylation identifying novel site, confirmed by mutagenesis and functional studies; single lab, multiple methods but relatively new","pmids":["39950798"],"is_preprint":false},{"year":2023,"finding":"eEF2K compound C1 (molecular glue) selectively binds to eEF2K residues F8, L10, R144, C146, E229, and Y236 and promotes proteasomal degradation of eEF2K by increasing interaction between eEF2K and the ubiquitin E3 ligase βTRCP.","method":"Binding affinity assays, molecular docking, ubiquitin-proteasome degradation assays, Co-IP (eEF2K–βTRCP), in vitro and in vivo antitumor experiments, patient-derived organoids","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — binding site identification, Co-IP for E3 ligase interaction, degradation mechanism established; single lab","pmids":["38084501"],"is_preprint":false},{"year":2020,"finding":"Under nutrient-replete conditions, eEF2K interacts with MEK1/2, creating a positive feedback loop via MEK1/2-ERK1/2-RSK-eEF2K signaling. Under acute nutrient deprivation, AMPK activation blocks the eEF2K-MEK1/2 interaction, thereby decreasing ERK1/2 activity and reducing G1/S transition to promote cell survival.","method":"Co-immunoprecipitation (eEF2K–MEK1/2), AMPK activation, western blotting, cell viability assays under nutrient deprivation","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP for interaction plus functional follow-up, single lab","pmids":["32565934"],"is_preprint":false},{"year":2022,"finding":"eEF2K phosphorylation/activity is a key convergence point downstream of both PI3K and MAPK/MEK pathways and mediates synergism when these pathways are co-inhibited. eEF2K activity was elevated in acute myeloid leukemia cell lines where PI3Ki + MEKi cotreatment is synergistic; siRNA or small-molecule inhibition of eEF2K reversed antiproliferative synergy in a cell-model-specific manner.","method":"LC-MS/MS phosphoproteomics, siRNA knockdown, pharmacological inhibition, 12 AML cell line analysis","journal":"Molecular & cellular proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics with genetic and pharmacological validation across multiple cell lines; single lab","pmids":["35513296"],"is_preprint":false},{"year":2016,"finding":"NMR analysis of calmodulin (CaM) binding to eEF2K revealed that eEF2K interacts mainly with the C-lobe of CaM in a Ca2+-tunable manner, providing structural insight into the Ca2+-dependent regulation of eEF2K activity.","method":"NMR spectroscopy of CaM-eEF2K interaction (cited from a study referenced in PMID:27602990 commentary)","journal":"Structure","confidence":"Low","confidence_rationale":"Tier 1 / Weak — NMR finding described only in a commentary abstract (PMID:27602990); original structural paper not directly in corpus, details limited","pmids":["27602990"],"is_preprint":false},{"year":2025,"finding":"In C. elegans, EFK-1/eEF2K promotes starvation survival via a noncanonical, kinase-activity-independent pathway: it upregulates transcription of DNA repair pathways (NER, BER) and suppresses oxygen consumption and ROS production. eEF2 phosphorylation levels are unchanged in starved C. elegans, indicating this protective role is independent of the canonical eEF2 phosphorylation function.","method":"C. elegans genetics (efk-1 mutants), kinase-dead efk-1 mutants, RNA-seq, ROS measurement, oxygen consumption assay, survival assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — kinase-dead mutant distinguishing canonical from noncanonical function, RNA-seq, multiple metabolic readouts; single lab but multiple orthogonal methods","pmids":["39966347"],"is_preprint":false},{"year":2024,"finding":"eEF2K deletion in NK cells activates Nrf2-mediated antioxidant signaling, sustaining mitochondrial fitness and active metabolism. TGFβ in the tumor microenvironment exacerbates oxidative stress and immunosuppression by inducing eEF2K in NK cells. eEF2K-knockout NK cells show enhanced maturation, proliferation, cytotoxicity, and reduced exhaustion.","method":"CRISPR/Cas9 eEF2K KO in NK cells, proteomic analysis, functional NK cell assays, in vivo mouse melanoma model, adoptive transfer of KO NK92 cells","journal":"Cancer immunology research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with proteomic and functional validation in vivo; single lab","pmids":["40506255"],"is_preprint":false},{"year":2022,"finding":"eEF2K inhibition impairs cellular antioxidant defenses, leading to enhanced ROS accumulation and sensitizing cancer cells to ferroptosis inducers or glutathione depletion. This reveals a role of eEF2K in maintaining redox homeostasis beyond its canonical translation-regulatory function.","method":"Pharmacological inhibition (A484954) and siRNA knockdown of eEF2K, ROS measurement, ferroptosis and lipid peroxidation assays, multiple cancer cell lines","journal":"European journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — both pharmacological and genetic (siRNA) approaches with orthogonal ROS/ferroptosis readouts; single lab","pmids":["40409701"],"is_preprint":false},{"year":2017,"finding":"eEF2K promotes glycolysis in rheumatoid arthritis fibroblast-like synoviocytes; knockdown of eEF2K suppresses TNF-α-induced NF-κB and AKT pathway activation, and lactate reverses the inhibitory effects of eEF2K knockdown on inflammation and migration, linking eEF2K-driven glycolysis to inflammatory signaling.","method":"siRNA knockdown, NH125 pharmacological inhibition, glucose uptake and lactate measurement, NF-κB/AKT western blotting, in vivo CIA mouse model","journal":"Journal of inflammation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological approaches with rescue (lactate) experiment; single lab, in vivo corroboration","pmids":["35300214"],"is_preprint":false},{"year":2024,"finding":"eEF2K alleviates doxorubicin-induced cardiotoxicity by phosphorylating and inhibiting GSK3β, thereby improving autophagy dysfunction. eEF2K overexpression (via AAV) reduces cardiomyocyte death, while knockdown aggravates autophagy blockade; GSK3β inhibition rescues the effects of eEF2K knockdown.","method":"Adeno-associated virus overexpression, adenovirus overexpression in vitro, eEF2K knockdown, transmission electron microscopy for autophagy, mRFP-GFP-LC3 imaging, GSK3β inhibitor rescue, in vivo mouse DIC model","journal":"Cell biology and toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — both in vitro and in vivo gain/loss-of-function with epistatic rescue via GSK3β inhibitor; single lab","pmids":["39708064"],"is_preprint":false},{"year":2017,"finding":"Fluoxetine induces autophagic cell death in TNBC cells through inhibition of eEF2K, which activates the AMPK-mTOR-ULK complex axis, promoting autophagy.","method":"MTT assay, electron microscopy, GFP-LC3 transfection, western blotting, siRNA, iTRAQ-based proteomics","journal":"Cell proliferation","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — cell-based assays without direct kinase measurement or rigorous eEF2K substrate assignment; single lab","pmids":["29094413"],"is_preprint":false},{"year":2021,"finding":"eEF2K inhibition cooperated with glutamine starvation and synergized with glutaminase inhibitors to suppress TNBC cell growth. Combined eEF2K and 4EBP1 depletion affected the collagen-containing ECM pathway (e.g., COL1A1) and amino-acid transporter SLC7A5/LAT1, suggesting a regulatory loop via mTORC1.","method":"Genetic and pharmacological eEF2K inhibition, GLS1 inhibitor combination, 4EBP1 siRNA, proteomic analysis","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — cell-based synthetic lethality with proteomics; mechanistic pathway placement is correlative; single lab","pmids":["33911160"],"is_preprint":false}],"current_model":"eEF2K is an atypical Ca2+/calmodulin-dependent α-kinase that phosphorylates eEF2 at Thr56, reducing eEF2's ribosomal affinity to inhibit peptide chain elongation; it is activated by Ca2+/CaM, AMPK (at Ser398), and PKA, and is inhibited by mTORC1/S6K (at Ser366) and NF-κB-mediated transcriptional repression; it is degraded by SCF(βTrCP) after autophosphorylation; beyond translation, it directly phosphorylates GSK3β (Ser9) and AURKA (Ser391), controls PD-L1 expression via uORF-dependent translation and GSK3β-mediated stabilization, upregulates STAT3/SPP1, interacts with Homer1b/c to regulate synaptic local translation, stabilizes a population of inactive 80S ribosomes (shown by cryo-EM), and in C. elegans acts through a kinase-independent pathway to promote DNA repair and suppress ROS under starvation."},"narrative":{"mechanistic_narrative":"EEF2K is an atypical Ca2+/calmodulin-dependent α-kinase that serves as a master brake on translational elongation by phosphorylating eEF2 at Thr56, lowering eEF2's ribosomal affinity and slowing A-to-P site translocation [PMID:22669845, PMID:21605678]. CaM engages eEF2K mainly through its C-lobe in a Ca2+-tunable manner [PMID:27602990], and in contracting skeletal muscle this Ca2+/CaM input alone suppresses protein synthesis independently of AMPK [PMID:19188248]. The kinase integrates stress and energy signals from many directions: genotoxic stress acts via AMPK phosphorylation of Ser398 followed by autophosphorylation-triggered SCF(βTrCP) degradation that permits elongation to resume [PMID:22669845]; myostatin and other inputs feed through the AMPK–eEF2K–eEF2 axis [PMID:29024627]; NF-κB (p65) transcriptionally represses eEF2K to relieve elongation blockade [PMID:31636182]; and ER/oxidative stress and ribosome biogenesis defects converge on eEF2K to reprogram translation, drive autophagy, and tune apoptotic sensitivity by controlling short-lived proteins such as XIAP and c-FLIPL [PMID:19221463, PMID:23182879, PMID:24582807, PMID:25332393]. Mechanistically, eEF2K-phosphorylated eEF2 locks vacant 80S ribosomes carrying SERBP1 into a splitting-resistant inactive state, defining a reservoir of dormant ribosomes [PMID:34815424]. In the nervous system, eEF2K controls localized synaptic translation through Homer1b/c scaffolding under mTORC1 control, shapes the excitation/inhibition balance and seizure susceptibility, gates associative taste learning and adult neurogenesis, and couples elongation slowing to the integrated stress response and BDNF synthesis in pain signaling [PMID:22366775, PMID:27005990, PMID:32892352, PMID:32707059, PMID:39694034]. Beyond elongation control, eEF2K acts as a protein kinase and signaling hub with non-eEF2 substrates: it directly binds and phosphorylates GSK3β at Ser9 to stabilize PD-L1 and protect cardiomyocytes, phosphorylates AURKA at Ser391 to drive triple-negative breast cancer, and promotes STAT3-driven SPP1 transcription and uORF-dependent PD-L1 translation, collectively supporting tumor progression and immune evasion [PMID:35347072, PMID:33094805, PMID:35184394, PMID:39950798, PMID:39708064]. A kinase-independent function is also documented: in C. elegans, EFK-1 promotes starvation survival by upregulating DNA-repair transcription and suppressing ROS without changing eEF2 phosphorylation [PMID:39966347], paralleling roles in redox homeostasis and antioxidant defense across cancer and immune cells [PMID:40506255, PMID:40409701].","teleology":[{"year":2009,"claim":"Established that Ca2+/calmodulin can drive eEF2K-dependent suppression of translation in a physiological tissue independently of AMPK, defining a dedicated Ca2+-sensing arm of elongation control.","evidence":"Ex vivo contracting fast-twitch muscle with pharmacological Ca2+ manipulation, eEF2K inhibition, and kinase-dead AMPK","pmids":["19188248"],"confidence":"High","gaps":["Did not resolve how Ca2+/CaM input is integrated with other activating signals","Quantitative contribution to whole-body protein turnover unaddressed"]},{"year":2009,"claim":"Placed eEF2K within autophagy signaling and identified eIF2α and Ca2+ flux as context-dependent upstream activators during starvation versus ER stress.","evidence":"siRNA knockdown, phosphorylation assays, LC3-II autophagy readouts under two stress conditions","pmids":["19221463"],"confidence":"Medium","gaps":["Direction of causality between eEF2K and autophagy machinery left partly correlative","Molecular link from eIF2α to eEF2K activation not defined"]},{"year":2011,"claim":"Demonstrated that recombinant human eEF2K is a catalytically competent monomer, enabling in vitro reconstitution of Ca2+/CaM-activated eEF2 phosphorylation.","evidence":"Recombinant purification, light scattering, kinetic in vitro kinase assays","pmids":["21605678"],"confidence":"High","gaps":["No structural model of the kinase domain","Did not address regulatory phosphosite occupancy on the recombinant enzyme"]},{"year":2012,"claim":"Unified the eEF2K activation-and-disposal cycle during DNA-damage stress, showing AMPK-Ser398 activation followed by autophosphorylation-dependent SCF(βTrCP) degradation to time recovery of elongation.","evidence":"In vitro kinase assays, mutagenesis, Co-IP, ubiquitin-proteasome degradation assays, phosphosite mapping","pmids":["22669845"],"confidence":"High","gaps":["Did not map all autophosphorylation sites controlling βTrCP recognition","Generality beyond genotoxic stress not tested"]},{"year":2012,"claim":"Provided in vivo genetic proof that eEF2-Thr56 phosphorylation is required for a specific form of cortical-dependent associative learning, linking elongation control to behavior.","evidence":"Kinase-inactive eEF2K knock-in mice, conditioned taste aversion behavior, MEMRI imaging","pmids":["22366775"],"confidence":"High","gaps":["Did not identify which neuronal mRNAs depend on eEF2K for learning","Selectivity for associative versus incidental learning mechanistically unexplained"]},{"year":2012,"claim":"Identified DDIT4/REDD1 as an ER-stress transducer to eEF2K and assigned opposing autophagy outcomes to distinct phosphosites (Ser398 vs Ser366/Ser78).","evidence":"RNAi, phosphomutant constructs, autophagy/apoptosis assays","pmids":["23182879"],"confidence":"Medium","gaps":["Kinases acting on Ser78/Ser366 in this context not fully defined","Single-lab phosphomutant interpretation"]},{"year":2014,"claim":"Defined eEF2K as an oxidative-stress sensor that tunes apoptotic threshold by downregulating short-lived survival proteins, with conserved roles in mouse and worm reproduction.","evidence":"eEF2K knockout mice and C. elegans genetics, protein synthesis and apoptosis assays, XIAP/c-FLIPL westerns","pmids":["24582807"],"confidence":"High","gaps":["Whether selective loss of XIAP/c-FLIPL is due solely to global elongation slowing not fully resolved","Sensing mechanism for oxidative stress upstream of eEF2K undefined"]},{"year":2014,"claim":"Showed eEF2K can act upstream of AMPK-ULK1 to restrain autophagy in cancer cells, revealing bidirectional, context-dependent coupling to the AMPK axis.","evidence":"siRNA, autophagic flux assays, epistasis via AMPK/ULK1 knockdown","pmids":["24955726"],"confidence":"High","gaps":["Mechanism by which eEF2K influences AMPK activity not defined","Tissue generality of upstream placement unknown"]},{"year":2014,"claim":"Connected eEF2K to translational reprogramming, showing ribosomal stress activates eEF2K to selectively favor TOP mRNA translation.","evidence":"Ribosomal stress induction, eEF2K inhibition, polysome profiling","pmids":["25332393"],"confidence":"Medium","gaps":["Mechanism linking elongation slowing to TOP-mRNA selectivity unresolved","Single-lab pharmacological approach"]},{"year":2014,"claim":"Established active-site Cys146 as a non-conserved residue exploitable for reversible covalent eEF2K inhibition, informing selective inhibitor design.","evidence":"Two-step inhibition kinetics, active-site mutagenesis, molecular docking","pmids":["25224652"],"confidence":"High","gaps":["No crystallographic confirmation of the thioimidate adduct","Cellular selectivity not fully profiled"]},{"year":2016,"claim":"Revealed transcriptional control of eEF2K, with NF-κB/p65 repressing eEF2K to stimulate elongation under inflammatory and infectious stimuli.","evidence":"Multiple NF-κB stimuli, pre-mRNA quantification, p65 transcription assays, westerns","pmids":["31636182"],"confidence":"High","gaps":["Direct p65 occupancy at the eEF2K locus described but promoter elements not fully mapped","Physiological consequence for pathogen replication not quantified"]},{"year":2016,"claim":"Demonstrated that eEF2K shapes the excitation/inhibition balance by restraining GABAergic transmission, linking the kinase to seizure susceptibility.","evidence":"eEF2K knockout mice, electrophysiology, Synapsin 2b/GABAAR westerns, seizure models","pmids":["27005990"],"confidence":"High","gaps":["Whether Synapsin 2b/α5-GABAAR changes are direct translational targets undefined","Pre- vs postsynaptic site of action not fully separated"]},{"year":2016,"claim":"Provided structural rationale for Ca2+-tunable activation by mapping eEF2K binding to the CaM C-lobe.","evidence":"NMR of CaM-eEF2K interaction (reported via commentary)","pmids":["27602990"],"confidence":"Low","gaps":["Original structural data only described in a commentary abstract, details limited","No full-length kinase-CaM complex structure"]},{"year":2017,"claim":"Showed myostatin suppresses muscle protein synthesis exclusively through the AMPK-eEF2K-eEF2 axis at low doses, placing eEF2K downstream of a growth-restraining hormone.","evidence":"C2C12 myotubes, SUnSET synthesis assay, pathway-specific inhibitors","pmids":["29024627"],"confidence":"Medium","gaps":["In vivo confirmation in muscle limited","Dose-dependence of mTOR involvement not fully mapped"]},{"year":2019,"claim":"Identified Homer1b/c as a scaffolding partner controlling eEF2K localization and synaptic local translation under mTORC1 regulation.","evidence":"Co-IP, mTORC1 inhibition, phosphomutant constructs, synthesis assays in neurons","pmids":["32892352"],"confidence":"Medium","gaps":["Single Co-IP-based interaction without structural mapping","Ser396 phosphorylation effect on binding affinity not quantified"]},{"year":2020,"claim":"Established eEF2K as a translational activator of PD-L1 by relieving uORF-mediated repression, linking elongation control to immune checkpoint expression.","evidence":"Genetic eEF2K ablation, polyribosome profiling, uORF reporter assays, NK cytotoxicity","pmids":["33094805"],"confidence":"High","gaps":["Mechanism by which eEF2K affects uORF reinitiation undefined","Interplay with elongation slowing not reconciled"]},{"year":2020,"claim":"Defined a reciprocal eEF2K-MEK1/2 interaction generating an ERK feedback loop that is disrupted by AMPK during nutrient deprivation to promote survival.","evidence":"Co-IP (eEF2K-MEK1/2), AMPK activation, cell viability assays","pmids":["32565934"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal structural validation","Whether interaction depends on kinase activity unknown"]},{"year":2020,"claim":"Showed SIRT1 promotes eEF2K-dependent cardiac autophagy under ER stress and that eIF2α physically associates with eEF2K upstream of its activation.","evidence":"Co-IP (eIF2α-eEF2K), siRNA, SIRT1 modulation, in vivo cardiac function","pmids":["32059483"],"confidence":"Medium","gaps":["Functional consequence of eIF2α-eEF2K binding not mechanistically dissected","Single-lab in vivo model"]},{"year":2020,"claim":"Placed eEF2K downstream of physiological neurogenic stimuli, with eEF2K loss enhancing adult dentate neurogenesis and learning.","evidence":"Global and conditional eEF2K knockout mice, neurogenesis markers, behavior","pmids":["32707059"],"confidence":"High","gaps":["Cell-autonomous vs niche effects on neural progenitors not fully separated","Translational targets driving neurogenesis unidentified"]},{"year":2021,"claim":"Provided structural and biochemical evidence that eEF2K-phosphorylated eEF2 locks vacant 80S ribosomes into a SERBP1-bound, splitting-resistant inactive state, defining a dormant ribosome reservoir.","evidence":"Cryo-EM, in vitro ribosome splitting, pharmacological eEF2K activation, sensory neuron p-body imaging","pmids":["34815424"],"confidence":"High","gaps":["Physiological signals controlling reactivation of these ribosomes undefined","Quantitative fraction of cellular ribosomes in this state unknown"]},{"year":2021,"claim":"Demonstrated in vivo that eEF2K activity is strictly required for Rpl24-mutation-driven elongation suppression and colorectal tumor suppression.","evidence":"Rpl24 × eEF2K double-mutant mouse epistasis, polysome profiling, tumor assays","pmids":["34895463"],"confidence":"High","gaps":["How Rpl24 mutation activates eEF2K not defined","Translatome changes mediating tumor suppression not enumerated"]},{"year":2022,"claim":"Identified GSK3β-Ser9 as a direct non-eEF2 substrate of eEF2K, linking the kinase to PD-L1 stabilization and immune evasion in melanoma.","evidence":"IP-MS, Co-IP, in vitro phosphorylation, in vivo melanoma model, flow cytometry","pmids":["35347072"],"confidence":"High","gaps":["Whether GSK3β phosphorylation requires eEF2K kinase activity in all contexts not fully tested","Structural basis of eEF2K-GSK3β recognition unknown"]},{"year":2022,"claim":"Defined an eEF2K-STAT3(Tyr705)-SPP1 transcriptional axis promoting melanoma progression, extending eEF2K function to transcription factor signaling.","evidence":"RNA-seq, STAT3 ChIP on SPP1 promoter, rescue experiments, in vivo model","pmids":["35184394"],"confidence":"High","gaps":["Mechanism by which eEF2K increases STAT3 Tyr705 phosphorylation undefined","Direct vs indirect link unresolved"]},{"year":2022,"claim":"Positioned eEF2K as a convergence node downstream of PI3K and MEK pathways mediating synergy of combined inhibition in AML.","evidence":"Phosphoproteomics, siRNA, pharmacological inhibition across 12 AML lines","pmids":["35513296"],"confidence":"Medium","gaps":["Cell-model-specific effects limit generalizability","Direct upstream kinases on eEF2K in this context not mapped"]},{"year":2022,"claim":"Revealed a redox-homeostasis function whereby eEF2K inhibition raises ROS and sensitizes cancer cells to ferroptosis, distinct from canonical translation control.","evidence":"Pharmacological and siRNA eEF2K inhibition, ROS/ferroptosis assays, multiple cell lines","pmids":["40409701"],"confidence":"Medium","gaps":["Molecular mediators connecting eEF2K to antioxidant defense undefined","Dependence on eEF2 phosphorylation not separated"]},{"year":2023,"claim":"Showed alphavirus nsP2 NTPase activity hijacks cAMP/PKA to activate eEF2K and induce host translational shut-off, a conserved viral strategy.","evidence":"SILAC phosphoproteomics, NTPase Walker mutants, cAMP/PKA analysis","pmids":["36848386"],"confidence":"High","gaps":["How nsP2 NTPase activates cAMP/PKA not defined","Selectivity of host shut-off for viral vs host mRNAs unaddressed"]},{"year":2023,"claim":"Developed a molecular-glue degrader (compound C1) mapping its eEF2K binding residues and showing it enhances βTRCP-mediated degradation, validating eEF2K as a druggable target.","evidence":"Binding assays, docking, Co-IP (eEF2K-βTRCP), degradation and antitumor experiments, organoids","pmids":["38084501"],"confidence":"Medium","gaps":["Structural confirmation of the ternary complex absent","Single-lab degradation mechanism"]},{"year":2024,"claim":"Demonstrated eEF2K couples elongation slowing in sensory neurons to the integrated stress response and BDNF synthesis to drive pain behavior.","evidence":"In vivo eEF2K KO/inhibition, ISR inhibition, Bdnf-selective translation blockade, ribosome coupling assays","pmids":["39694034"],"confidence":"High","gaps":["Identity of the ribosome-coupled ISR sensor not fully defined","How BDNF translation is selectively induced unresolved"]},{"year":2024,"claim":"Showed eEF2K protects against doxorubicin cardiotoxicity by phosphorylating and inhibiting GSK3β to restore autophagy, a protective counterpart to its oncogenic GSK3β signaling.","evidence":"AAV/adenoviral overexpression and knockdown, autophagy imaging, GSK3β inhibitor rescue, in vivo DIC model","pmids":["39708064"],"confidence":"Medium","gaps":["Tissue-specific outcome of GSK3β inhibition not reconciled with tumor context","Single-lab model"]},{"year":2024,"claim":"Defined an eEF2K-Nrf2-mitochondrial axis in NK cells, with TGFβ-induced eEF2K driving oxidative stress and immunosuppression, and eEF2K loss enhancing anti-tumor function.","evidence":"CRISPR eEF2K KO in NK cells, proteomics, functional assays, in vivo adoptive transfer","pmids":["40506255"],"confidence":"Medium","gaps":["Mechanism linking eEF2K to Nrf2 signaling undefined","Dependence on canonical eEF2 phosphorylation not tested"]},{"year":2025,"claim":"Identified AURKA-Ser391 as a new eEF2K substrate, defining an eEF2K/AURKA/SOX8 axis that promotes triple-negative breast cancer.","evidence":"Proteomics, Co-IP, in vitro phosphorylation, mutagenesis, xenografts, organoids","pmids":["39950798"],"confidence":"Medium","gaps":["Whether AURKA phosphorylation requires CaM-dependent eEF2K activation unknown","Relatively recent single-lab finding"]},{"year":2025,"claim":"Established a kinase-independent eEF2K function in C. elegans promoting starvation survival via DNA-repair transcription and ROS suppression, decoupling protective roles from eEF2 phosphorylation.","evidence":"efk-1 and kinase-dead mutants, RNA-seq, ROS and oxygen consumption assays, survival assays","pmids":["39966347"],"confidence":"High","gaps":["Molecular mechanism of the kinase-independent activity undefined","Conservation of this noncanonical role in mammals untested"]},{"year":null,"claim":"How eEF2K balances its canonical eEF2-elongation brake against its expanding repertoire of direct protein substrates (GSK3β, AURKA) and kinase-independent functions remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of full-length eEF2K or its substrate complexes","Determinants of substrate selection between eEF2 and non-eEF2 targets unknown","Mechanistic basis of kinase-independent roles uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,20,25]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,20,25,23]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[0,10,19,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,19]}],"localization":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[19]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,10,19,21]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[2,6,8]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,7,24,32]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[20,22,27]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7]}],"complexes":[],"partners":["EEF2","CALM1","GSK3B","AURKA","HOMER1","EIF2AK / EIF2S1 (EIF2Α)","MAP2K1 (MEK1/2)","BTRC (ΒTRCP)"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00418","full_name":"Eukaryotic elongation factor 2 kinase","aliases":["Calcium/calmodulin-dependent eukaryotic elongation factor 2 kinase"],"length_aa":725,"mass_kda":82.1,"function":"Threonine kinase that regulates protein synthesis by controlling the rate of peptide chain elongation. Upon activation by a variety of upstream kinases including AMPK or TRPM7, phosphorylates the elongation factor EEF2 at a single site, renders it unable to bind ribosomes and thus inactive. In turn, the rate of protein synthesis is reduced","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/O00418/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EEF2K","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EEF2K","total_profiled":1310},"omim":[{"mim_id":"619965","title":"ALPHA KINASE 2; ALPK2","url":"https://www.omim.org/entry/619965"},{"mim_id":"617608","title":"ALPHA KINASE 3; ALPK3","url":"https://www.omim.org/entry/617608"},{"mim_id":"607347","title":"ALPHA KINASE 1; ALPK1","url":"https://www.omim.org/entry/607347"},{"mim_id":"606968","title":"EUKARYOTIC ELONGATION FACTOR 2 KINASE; EEF2K","url":"https://www.omim.org/entry/606968"},{"mim_id":"138252","title":"GLUTAMATE RECEPTOR, IONOTROPIC, N-METHYL-D-ASPARTATE, SUBUNIT 2B; GRIN2B","url":"https://www.omim.org/entry/138252"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skin 1","ntpm":45.9}],"url":"https://www.proteinatlas.org/search/EEF2K"},"hgnc":{"alias_symbol":["eEF-2K","CaMKIII"],"prev_symbol":[]},"alphafold":{"accession":"O00418","domains":[{"cath_id":"3.30.200.20","chopping":"94-232","consensus_level":"medium","plddt":91.2138,"start":94,"end":232},{"cath_id":"1.25.40.10","chopping":"503-662","consensus_level":"medium","plddt":87.5529,"start":503,"end":662},{"cath_id":"1.20.58","chopping":"663-725","consensus_level":"medium","plddt":92.4656,"start":663,"end":725}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00418","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00418-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00418-F1-predicted_aligned_error_v6.png","plddt_mean":71.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EEF2K","jax_strain_url":"https://www.jax.org/strain/search?query=EEF2K"},"sequence":{"accession":"O00418","fasta_url":"https://rest.uniprot.org/uniprotkb/O00418.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00418/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00418"}},"corpus_meta":[{"pmid":"25330770","id":"PMC_25330770","title":"Combined 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In response to genotoxic stress, AMPK activates eEF2K by phosphorylating it on Ser398. Subsequently, eEF2K is degraded via the SCF(βTrCP) ubiquitin ligase through autophosphorylation on a canonical βTrCP-binding domain, enabling resumption of translation elongation during DNA damage checkpoint silencing.\",\n      \"method\": \"In vitro kinase assays, mutagenesis, co-immunoprecipitation, ubiquitin-proteasome degradation assays, phosphosite mapping\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (in vitro assays, mutagenesis, Co-IP, degradation assays) in a single rigorous study establishing both activation mechanism and degradation mechanism\",\n      \"pmids\": [\"22669845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In contracting fast-twitch skeletal muscle, eEF2K is activated downstream of Ca2+/calmodulin (CaM), leading to phosphorylation of eEF2 and partial suppression (~30–40%) of protein synthesis. This Ca2+-CaM-eEF2K-eEF2 signaling cascade operates independently of AMPK or changes in intracellular pH.\",\n      \"method\": \"Ex vivo muscle contraction, pharmacological Ca2+ manipulation, eEF2K inhibitor treatment, kinase-dead AMPK overexpression, protein synthesis rate measurement\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal pharmacological and genetic approaches in a single rigorous study; replicated across multiple contraction protocols\",\n      \"pmids\": [\"19188248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"eEF2K is required for autophagy signaling during nutrient starvation and ER stress. During starvation, eIF2α phosphorylation is required upstream of eEF2K activation and eEF2 phosphorylation. During ER stress, eEF2K-mediated eEF2 phosphorylation can occur partly through Ca2+ flux independently of eIF2α phosphorylation.\",\n      \"method\": \"siRNA knockdown, phosphorylation assays, autophagy readouts (LC3-II), pharmacological inhibitors\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined autophagy phenotype, two orthogonal stress conditions, single lab\",\n      \"pmids\": [\"19221463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TRPM7, via its kinase domain, promotes phosphorylation of eEF2 at Thr56 under Mg2+-limited conditions by influencing the abundance and phosphorylation state of eEF2K at Ser77. TRPM7 kinase does not directly phosphorylate eEF2 but acts through eEF2K.\",\n      \"method\": \"Cell-based phosphorylation assays, TRPM7 kinase-dead mutants, western blotting\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — use of kinase-dead mutants and phosphorylation assays, single lab, two orthogonal approaches\",\n      \"pmids\": [\"21112387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Recombinant human eEF2K expressed in E. coli is a monomer of ~85 kDa (by light scattering) that phosphorylates eEF2 in vitro with kinetic parameters comparable to the mammalian enzyme. eEF2K is activated by calcium and calmodulin.\",\n      \"method\": \"Recombinant protein purification, light scattering, in vitro kinase assay, kinetic analysis\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of enzymatic activity with purified recombinant protein and kinetic characterization; single lab but direct biochemical validation\",\n      \"pmids\": [\"21605678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"eEF2K activity (eEF2 phosphorylation at Thr56) is essential for cortical-dependent associative taste learning. Kinase-inactive eEF2K knock-in (ki) mice with reduced eEF2 phosphorylation show attenuated conditioned taste aversion but normal incidental taste learning, and exhibit altered brain activation patterns (by MEMRI) during learning.\",\n      \"method\": \"Kinase-inactive knock-in mice, behavioral testing (conditioned taste aversion), manganese-enhanced MRI (MEMRI), western blotting\",\n      \"journal\": \"Learning & memory\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knock-in model with specific phospho-site reduction, multiple behavioral paradigms, and in vivo brain imaging; single lab but rigorous\",\n      \"pmids\": [\"22366775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Under ER stress, eEF2K is required for autophagy induction. DDIT4 (REDD1) transduces ER stress signals to activate eEF2K. Phosphorylation of eEF2K at Ser398 promotes autophagy, while phosphorylation at Ser366 and Ser78 inhibits autophagy. Suppression of eEF2K aggravates ER stress and shifts cells toward apoptosis.\",\n      \"method\": \"RNAi knockdown, phosphomutant constructs, autophagy and apoptosis assays, western blotting\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined phosphorylation sites via mutagenesis with functional autophagy readouts, single lab\",\n      \"pmids\": [\"23182879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"eEF2K senses oxidative stress and rapidly downregulates short-lived antiapoptotic proteins XIAP and c-FLIPL by inhibiting global protein synthesis, rendering cells susceptible to apoptosis. Loss of eEF2K in mice reduces ovarian apoptosis and leads to accumulation of aberrant follicles; loss of eEF2K ortholog in C. elegans reduces germ cell death and worsens oocyte quality.\",\n      \"method\": \"eEF2K knockout mice, C. elegans genetics, protein synthesis assays, western blotting for XIAP and c-FLIPL, apoptosis assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — replicated in two independent model organisms (mouse and C. elegans) with mechanistic follow-up on specific substrates\",\n      \"pmids\": [\"24582807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Silencing of eEF2K in colon cancer cells increases protein synthesis and activates the AMPK-ULK1 pathway, inducing autophagy independently of mTOR suppression. Knockdown of AMPK or ULK1 abolishes eEF2K silencing-induced autophagy, placing eEF2K upstream of AMPK-ULK1 in this context.\",\n      \"method\": \"siRNA knockdown, LC3-II western blot, LC3 dot accumulation, autophagic flux assays, genetic epistasis (AMPK/ULK1 knockdown)\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis experiments with multiple genetic knockdowns, orthogonal autophagy readouts, single lab\",\n      \"pmids\": [\"24955726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"eEF2K regulates the expression of tissue transglutaminase (TG2), and the eEF2K/TG2 axis promotes cancer cell survival. Inhibition of eEF2K leads to caspase-independent apoptosis associated with induction of apoptosis-inducing factor (AIF). eEF2K protein is degraded through the ubiquitin-proteasome pathway upon rottlerin treatment.\",\n      \"method\": \"siRNA knockdown, overexpression, western blotting, apoptosis assays (AIF, caspase), ubiquitin-proteasome pathway inhibitors\",\n      \"journal\": \"Apoptosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with mechanistic follow-up on TG2 and AIF pathways; single lab\",\n      \"pmids\": [\"24193916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Ribosomal stress (ribosome biogenesis defect) activates the eEF2K-eEF2 pathway, inhibiting translation elongation. This causes a translational reprogramming in which TOP (terminal oligopyrimidine) mRNAs encoding ribosomal proteins are selectively recruited to polysomes, relatively increasing synthesis of TOP mRNA-encoded proteins.\",\n      \"method\": \"Ribosomal stress induction, eEF2K inhibition, polysome profiling, translation assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological eEF2K inhibition with polysome profiling; single lab, two orthogonal readouts\",\n      \"pmids\": [\"25332393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A reversible covalent inhibition mechanism for eEF2K: the compound DFTD binds in two steps (fast binding followed by slow reversible inactivation). Molecular docking and mutagenesis indicate a nitrile group forms a reversible thioimidate adduct with the active-site Cys146, which is not conserved in related kinases.\",\n      \"method\": \"Kinetic analysis (two-step inhibition), active-site mutagenesis, molecular docking, chemoinformatics\",\n      \"journal\": \"Chembiochem\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinetic and mutagenesis data establishing covalent mechanism; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"25224652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NF-κB activation (by TNFα, HCMV infection, or dsDNA) represses eEF2K transcription through the p65 NF-κB subunit, reducing eEF2K pre-mRNA and protein levels, thereby decreasing eEF2 phosphorylation (Thr56) and stimulating translation elongation.\",\n      \"method\": \"NF-κB activation assays, eEF2K pre-mRNA quantification, pharmacological and genetic NF-κB modulation, p65 ChIP/transcription assays, western blotting\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple NF-κB-activating stimuli tested, pre-mRNA analysis establishing transcriptional mechanism, p65 subunit specificity shown; single lab but diverse orthogonal approaches\",\n      \"pmids\": [\"31636182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"eEF2K activity negatively regulates GABAergic synaptic transmission in neurons. Loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors, altering the excitation/inhibition balance and conferring resistance to epileptic seizures.\",\n      \"method\": \"eEF2K knockout mice, electrophysiology, western blotting for Synapsin 2b and GABAA receptor subunits, in vivo seizure models\",\n      \"journal\": \"Cerebral cortex\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined molecular (Synapsin 2b, α5-GABAAR) and behavioral (seizure) phenotypes, pharmacological corroboration; single lab but multiple methods\",\n      \"pmids\": [\"27005990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Myostatin inhibits protein synthesis in skeletal muscle by activating AMPK, which in turn activates the eEF2K-eEF2 pathway. At low concentrations, myostatin suppresses protein synthesis exclusively through the AMPK-eEF2K-eEF2 axis without affecting mTOR.\",\n      \"method\": \"C2C12 myotube treatment with recombinant myostatin, SUnSET protein synthesis assay, western blotting, pharmacological pathway inhibitors\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein synthesis measurement with pathway-specific inhibitors; single lab\",\n      \"pmids\": [\"29024627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"eEF2K interacts with the scaffolding protein Homer1b/c, and this interaction is regulated by mTORC1-dependent phosphorylation of eEF2K at Ser396 (a known mTORC1 site). Homer1b/c binding controls eEF2K localization and affects rates of localized protein synthesis at synapses.\",\n      \"method\": \"Co-immunoprecipitation, pharmacological mTORC1 inhibition, phosphomutant constructs, protein synthesis assays in SH-SY5Y cells and mouse cortical neurons\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP for interaction, mTORC1 manipulation for regulatory mechanism, functional synthesis readout; single lab\",\n      \"pmids\": [\"32892352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Known physiological stimuli that enhance neurogenesis converge on the eEF2K/eEF2 pathway via AMPK in the dentate gyrus. eEF2K knockout in mature excitatory neurons of the dentate gyrus enhances adult neurogenesis and upregulates neurogenesis-related proteins, correlating with improved dentate gyrus-dependent learning.\",\n      \"method\": \"General and conditional eEF2K knockout mice, neurogenesis markers, behavioral testing, western blotting\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — both global and region-specific conditional KO with consistent neurogenesis and behavioral phenotypes; single lab but two genetic models\",\n      \"pmids\": [\"32707059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SIRT1 promotes eEF2K/eEF2-dependent autophagy in cardiomyocytes under ER stress. eIF2α co-immunoprecipitates with eEF2K, and eIF2α knockdown reduces eEF2 phosphorylation, indicating eIF2α is required upstream of eEF2K activation in this cardiac context.\",\n      \"method\": \"Co-immunoprecipitation (eIF2α–eEF2K), siRNA knockdown, SIRT1 genetic/pharmacological modulation, autophagy assays in cardiac cells, in vivo cardiac function in SIRT1-deficient mice\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for interaction, multiple knockdown experiments, in vivo cardiac phenotype; single lab\",\n      \"pmids\": [\"32059483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Phosphorylation of eEF2 (via eEF2K) suppresses translation elongation and is the principal mechanism by which Rpl24 mutation suppresses colorectal tumorigenesis in mice. Genetic inactivation of eEF2K in Rpl24 mutant mice completely restores elongation rate and protein synthesis, and abrogates the tumor-suppressive effect of the Rpl24 mutation, demonstrating that eEF2K activity is required for Rpl24-mediated tumor suppression.\",\n      \"method\": \"Genetic epistasis in mice (Rpl24 mutation × eEF2K inactivation), polysome profiling, protein synthesis rate measurement, tumor incidence/growth assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo double-mutant genetic epistasis with direct elongation rate and protein synthesis measurements; single lab but highly rigorous\",\n      \"pmids\": [\"34895463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"eEF2K phosphorylated eEF2 stabilizes vacant 80S ribosomes containing SERBP1 (in place of mRNA) and eEF2 in the acceptor site, as revealed by cryo-EM. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. eEF2K thus defines a population of inactive, recycling-resistant ribosomes and controls p-body abundance in sensory neurons.\",\n      \"method\": \"Cryo-electron microscopy, in vitro ribosome splitting assay, pharmacological eEF2K activation (nelfinavir), sensory neuron imaging, p-body quantification\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure combined with in vitro biochemical assay and pharmacological manipulation; multiple orthogonal methods\",\n      \"pmids\": [\"34815424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"eEF2K directly binds to and phosphorylates GSK3β at Ser9, inactivating GSK3β and leading to stabilization and upregulation of PD-L1 protein in melanoma cells. Immunoprecipitation-mass spectrometry identified this interaction, and knockdown of eEF2K decreased PD-L1 and enhanced CD8+ T cell activity in vivo.\",\n      \"method\": \"Immunoprecipitation–mass spectrometry, Co-IP, in vitro phosphorylation assay, western blotting, in vivo mouse melanoma model, flow cytometry\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — IP-MS for substrate identification, confirmed by Co-IP and phosphorylation assay, with in vivo functional validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"35347072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"eEF2K promotes translation of PD-L1 mRNA by attenuating the inhibitory effect of an upstream open reading frame (uORF) with a non-canonical CUG start codon in the PD-L1 5'-UTR. eEF2K depletion reduces PD-L1 mRNA association with translationally active polyribosomes and decreases PD-L1 protein levels.\",\n      \"method\": \"eEF2K ablation (genetic), polyribosome profiling, reporter assays for uORF activity, western blotting, NK cell cytotoxicity assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — polysome profiling plus uORF reporter assay establishing translational mechanism, functional immune assay; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33094805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"eEF2K upregulates phosphorylation of STAT3 at Tyr705, which binds the SPP1 promoter and enhances SPP1 transcription to facilitate melanoma progression. Re-expression of SPP1 rescues the inhibitory effect of eEF2K silencing, and inhibition of SPP1 or STAT3 abolishes eEF2K-driven effects.\",\n      \"method\": \"RNA-seq, ChIP assay (STAT3 binding to SPP1 promoter), siRNA/overexpression, rescue experiments, in vivo mouse model\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP for transcription factor binding, genetic rescue, in vivo validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"35184394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Alphavirus nsP2 NTPase domain activates a cAMP/PKA signaling cascade, leading to activation of eEF2K and subsequent phosphorylation of eEF2 at Thr56 (>50-fold increase), causing translational shut-off. NTPase-dead mutations prevent this eEF2 phosphorylation. This mechanism is shared across Old and New World alphaviruses.\",\n      \"method\": \"Phosphoproteomics (SILAC + LC-MS/MS), NTPase Walker A/B mutants, cAMP/PKA pathway analysis, translation inhibition assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — phosphoproteomics with active-site mutagenesis and biochemical follow-up; multiple alphaviruses tested\",\n      \"pmids\": [\"36848386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In sensory neurons, painful stimuli activate eEF2K to repress peptide chain elongation. Attenuated elongation is sensed by a ribosome-coupled mechanism that triggers the integrated stress response (ISR). Both eEF2K and the ISR are required for pain-associated behaviors in vivo. This pathway simultaneously induces BDNF biosynthesis; selective blockade of Bdnf translation has analgesic effects.\",\n      \"method\": \"In vivo eEF2K knockout/inhibition behavioral studies, ISR inhibition, Bdnf mRNA-selective translation blockade, ribosome coupling assays in sensory neurons\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic and pharmacological dissection of eEF2K→ISR→BDNF axis with behavioral readouts; multiple orthogonal methods\",\n      \"pmids\": [\"39694034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"eEF2K binds to and phosphorylates aurora kinase A (AURKA) at Ser391, a newly identified phosphorylation site that is critical for AURKA protein stability and kinase activity. eEF2K also positively regulates SOX8 mRNA and protein expression, constituting an eEF2K/AURKA/SOX8 axis promoting TNBC progression.\",\n      \"method\": \"Proteomic analysis, Co-IP/binding assay, in vitro phosphorylation assay, mutagenesis, overexpression/knockdown, in vivo xenograft, patient-derived organoids\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro phosphorylation identifying novel site, confirmed by mutagenesis and functional studies; single lab, multiple methods but relatively new\",\n      \"pmids\": [\"39950798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"eEF2K compound C1 (molecular glue) selectively binds to eEF2K residues F8, L10, R144, C146, E229, and Y236 and promotes proteasomal degradation of eEF2K by increasing interaction between eEF2K and the ubiquitin E3 ligase βTRCP.\",\n      \"method\": \"Binding affinity assays, molecular docking, ubiquitin-proteasome degradation assays, Co-IP (eEF2K–βTRCP), in vitro and in vivo antitumor experiments, patient-derived organoids\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — binding site identification, Co-IP for E3 ligase interaction, degradation mechanism established; single lab\",\n      \"pmids\": [\"38084501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Under nutrient-replete conditions, eEF2K interacts with MEK1/2, creating a positive feedback loop via MEK1/2-ERK1/2-RSK-eEF2K signaling. Under acute nutrient deprivation, AMPK activation blocks the eEF2K-MEK1/2 interaction, thereby decreasing ERK1/2 activity and reducing G1/S transition to promote cell survival.\",\n      \"method\": \"Co-immunoprecipitation (eEF2K–MEK1/2), AMPK activation, western blotting, cell viability assays under nutrient deprivation\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP for interaction plus functional follow-up, single lab\",\n      \"pmids\": [\"32565934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"eEF2K phosphorylation/activity is a key convergence point downstream of both PI3K and MAPK/MEK pathways and mediates synergism when these pathways are co-inhibited. eEF2K activity was elevated in acute myeloid leukemia cell lines where PI3Ki + MEKi cotreatment is synergistic; siRNA or small-molecule inhibition of eEF2K reversed antiproliferative synergy in a cell-model-specific manner.\",\n      \"method\": \"LC-MS/MS phosphoproteomics, siRNA knockdown, pharmacological inhibition, 12 AML cell line analysis\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics with genetic and pharmacological validation across multiple cell lines; single lab\",\n      \"pmids\": [\"35513296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NMR analysis of calmodulin (CaM) binding to eEF2K revealed that eEF2K interacts mainly with the C-lobe of CaM in a Ca2+-tunable manner, providing structural insight into the Ca2+-dependent regulation of eEF2K activity.\",\n      \"method\": \"NMR spectroscopy of CaM-eEF2K interaction (cited from a study referenced in PMID:27602990 commentary)\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR finding described only in a commentary abstract (PMID:27602990); original structural paper not directly in corpus, details limited\",\n      \"pmids\": [\"27602990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In C. elegans, EFK-1/eEF2K promotes starvation survival via a noncanonical, kinase-activity-independent pathway: it upregulates transcription of DNA repair pathways (NER, BER) and suppresses oxygen consumption and ROS production. eEF2 phosphorylation levels are unchanged in starved C. elegans, indicating this protective role is independent of the canonical eEF2 phosphorylation function.\",\n      \"method\": \"C. elegans genetics (efk-1 mutants), kinase-dead efk-1 mutants, RNA-seq, ROS measurement, oxygen consumption assay, survival assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — kinase-dead mutant distinguishing canonical from noncanonical function, RNA-seq, multiple metabolic readouts; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"39966347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"eEF2K deletion in NK cells activates Nrf2-mediated antioxidant signaling, sustaining mitochondrial fitness and active metabolism. TGFβ in the tumor microenvironment exacerbates oxidative stress and immunosuppression by inducing eEF2K in NK cells. eEF2K-knockout NK cells show enhanced maturation, proliferation, cytotoxicity, and reduced exhaustion.\",\n      \"method\": \"CRISPR/Cas9 eEF2K KO in NK cells, proteomic analysis, functional NK cell assays, in vivo mouse melanoma model, adoptive transfer of KO NK92 cells\",\n      \"journal\": \"Cancer immunology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with proteomic and functional validation in vivo; single lab\",\n      \"pmids\": [\"40506255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"eEF2K inhibition impairs cellular antioxidant defenses, leading to enhanced ROS accumulation and sensitizing cancer cells to ferroptosis inducers or glutathione depletion. This reveals a role of eEF2K in maintaining redox homeostasis beyond its canonical translation-regulatory function.\",\n      \"method\": \"Pharmacological inhibition (A484954) and siRNA knockdown of eEF2K, ROS measurement, ferroptosis and lipid peroxidation assays, multiple cancer cell lines\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — both pharmacological and genetic (siRNA) approaches with orthogonal ROS/ferroptosis readouts; single lab\",\n      \"pmids\": [\"40409701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"eEF2K promotes glycolysis in rheumatoid arthritis fibroblast-like synoviocytes; knockdown of eEF2K suppresses TNF-α-induced NF-κB and AKT pathway activation, and lactate reverses the inhibitory effects of eEF2K knockdown on inflammation and migration, linking eEF2K-driven glycolysis to inflammatory signaling.\",\n      \"method\": \"siRNA knockdown, NH125 pharmacological inhibition, glucose uptake and lactate measurement, NF-κB/AKT western blotting, in vivo CIA mouse model\",\n      \"journal\": \"Journal of inflammation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological approaches with rescue (lactate) experiment; single lab, in vivo corroboration\",\n      \"pmids\": [\"35300214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"eEF2K alleviates doxorubicin-induced cardiotoxicity by phosphorylating and inhibiting GSK3β, thereby improving autophagy dysfunction. eEF2K overexpression (via AAV) reduces cardiomyocyte death, while knockdown aggravates autophagy blockade; GSK3β inhibition rescues the effects of eEF2K knockdown.\",\n      \"method\": \"Adeno-associated virus overexpression, adenovirus overexpression in vitro, eEF2K knockdown, transmission electron microscopy for autophagy, mRFP-GFP-LC3 imaging, GSK3β inhibitor rescue, in vivo mouse DIC model\",\n      \"journal\": \"Cell biology and toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — both in vitro and in vivo gain/loss-of-function with epistatic rescue via GSK3β inhibitor; single lab\",\n      \"pmids\": [\"39708064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Fluoxetine induces autophagic cell death in TNBC cells through inhibition of eEF2K, which activates the AMPK-mTOR-ULK complex axis, promoting autophagy.\",\n      \"method\": \"MTT assay, electron microscopy, GFP-LC3 transfection, western blotting, siRNA, iTRAQ-based proteomics\",\n      \"journal\": \"Cell proliferation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — cell-based assays without direct kinase measurement or rigorous eEF2K substrate assignment; single lab\",\n      \"pmids\": [\"29094413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"eEF2K inhibition cooperated with glutamine starvation and synergized with glutaminase inhibitors to suppress TNBC cell growth. Combined eEF2K and 4EBP1 depletion affected the collagen-containing ECM pathway (e.g., COL1A1) and amino-acid transporter SLC7A5/LAT1, suggesting a regulatory loop via mTORC1.\",\n      \"method\": \"Genetic and pharmacological eEF2K inhibition, GLS1 inhibitor combination, 4EBP1 siRNA, proteomic analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — cell-based synthetic lethality with proteomics; mechanistic pathway placement is correlative; single lab\",\n      \"pmids\": [\"33911160\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"eEF2K is an atypical Ca2+/calmodulin-dependent α-kinase that phosphorylates eEF2 at Thr56, reducing eEF2's ribosomal affinity to inhibit peptide chain elongation; it is activated by Ca2+/CaM, AMPK (at Ser398), and PKA, and is inhibited by mTORC1/S6K (at Ser366) and NF-κB-mediated transcriptional repression; it is degraded by SCF(βTrCP) after autophosphorylation; beyond translation, it directly phosphorylates GSK3β (Ser9) and AURKA (Ser391), controls PD-L1 expression via uORF-dependent translation and GSK3β-mediated stabilization, upregulates STAT3/SPP1, interacts with Homer1b/c to regulate synaptic local translation, stabilizes a population of inactive 80S ribosomes (shown by cryo-EM), and in C. elegans acts through a kinase-independent pathway to promote DNA repair and suppress ROS under starvation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EEF2K is an atypical Ca2+/calmodulin-dependent α-kinase that serves as a master brake on translational elongation by phosphorylating eEF2 at Thr56, lowering eEF2's ribosomal affinity and slowing A-to-P site translocation [#0, #4]. CaM engages eEF2K mainly through its C-lobe in a Ca2+-tunable manner [#29], and in contracting skeletal muscle this Ca2+/CaM input alone suppresses protein synthesis independently of AMPK [#1]. The kinase integrates stress and energy signals from many directions: genotoxic stress acts via AMPK phosphorylation of Ser398 followed by autophosphorylation-triggered SCF(βTrCP) degradation that permits elongation to resume [#0]; myostatin and other inputs feed through the AMPK–eEF2K–eEF2 axis [#14]; NF-κB (p65) transcriptionally represses eEF2K to relieve elongation blockade [#12]; and ER/oxidative stress and ribosome biogenesis defects converge on eEF2K to reprogram translation, drive autophagy, and tune apoptotic sensitivity by controlling short-lived proteins such as XIAP and c-FLIPL [#2, #6, #7, #10]. Mechanistically, eEF2K-phosphorylated eEF2 locks vacant 80S ribosomes carrying SERBP1 into a splitting-resistant inactive state, defining a reservoir of dormant ribosomes [#19]. In the nervous system, eEF2K controls localized synaptic translation through Homer1b/c scaffolding under mTORC1 control, shapes the excitation/inhibition balance and seizure susceptibility, gates associative taste learning and adult neurogenesis, and couples elongation slowing to the integrated stress response and BDNF synthesis in pain signaling [#5, #13, #15, #16, #24]. Beyond elongation control, eEF2K acts as a protein kinase and signaling hub with non-eEF2 substrates: it directly binds and phosphorylates GSK3β at Ser9 to stabilize PD-L1 and protect cardiomyocytes, phosphorylates AURKA at Ser391 to drive triple-negative breast cancer, and promotes STAT3-driven SPP1 transcription and uORF-dependent PD-L1 translation, collectively supporting tumor progression and immune evasion [#20, #21, #22, #25, #34]. A kinase-independent function is also documented: in C. elegans, EFK-1 promotes starvation survival by upregulating DNA-repair transcription and suppressing ROS without changing eEF2 phosphorylation [#30], paralleling roles in redox homeostasis and antioxidant defense across cancer and immune cells [#31, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established that Ca2+/calmodulin can drive eEF2K-dependent suppression of translation in a physiological tissue independently of AMPK, defining a dedicated Ca2+-sensing arm of elongation control.\",\n      \"evidence\": \"Ex vivo contracting fast-twitch muscle with pharmacological Ca2+ manipulation, eEF2K inhibition, and kinase-dead AMPK\",\n      \"pmids\": [\"19188248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how Ca2+/CaM input is integrated with other activating signals\", \"Quantitative contribution to whole-body protein turnover unaddressed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Placed eEF2K within autophagy signaling and identified eIF2α and Ca2+ flux as context-dependent upstream activators during starvation versus ER stress.\",\n      \"evidence\": \"siRNA knockdown, phosphorylation assays, LC3-II autophagy readouts under two stress conditions\",\n      \"pmids\": [\"19221463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direction of causality between eEF2K and autophagy machinery left partly correlative\", \"Molecular link from eIF2α to eEF2K activation not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated that recombinant human eEF2K is a catalytically competent monomer, enabling in vitro reconstitution of Ca2+/CaM-activated eEF2 phosphorylation.\",\n      \"evidence\": \"Recombinant purification, light scattering, kinetic in vitro kinase assays\",\n      \"pmids\": [\"21605678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of the kinase domain\", \"Did not address regulatory phosphosite occupancy on the recombinant enzyme\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Unified the eEF2K activation-and-disposal cycle during DNA-damage stress, showing AMPK-Ser398 activation followed by autophosphorylation-dependent SCF(βTrCP) degradation to time recovery of elongation.\",\n      \"evidence\": \"In vitro kinase assays, mutagenesis, Co-IP, ubiquitin-proteasome degradation assays, phosphosite mapping\",\n      \"pmids\": [\"22669845\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map all autophosphorylation sites controlling βTrCP recognition\", \"Generality beyond genotoxic stress not tested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided in vivo genetic proof that eEF2-Thr56 phosphorylation is required for a specific form of cortical-dependent associative learning, linking elongation control to behavior.\",\n      \"evidence\": \"Kinase-inactive eEF2K knock-in mice, conditioned taste aversion behavior, MEMRI imaging\",\n      \"pmids\": [\"22366775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify which neuronal mRNAs depend on eEF2K for learning\", \"Selectivity for associative versus incidental learning mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified DDIT4/REDD1 as an ER-stress transducer to eEF2K and assigned opposing autophagy outcomes to distinct phosphosites (Ser398 vs Ser366/Ser78).\",\n      \"evidence\": \"RNAi, phosphomutant constructs, autophagy/apoptosis assays\",\n      \"pmids\": [\"23182879\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinases acting on Ser78/Ser366 in this context not fully defined\", \"Single-lab phosphomutant interpretation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined eEF2K as an oxidative-stress sensor that tunes apoptotic threshold by downregulating short-lived survival proteins, with conserved roles in mouse and worm reproduction.\",\n      \"evidence\": \"eEF2K knockout mice and C. elegans genetics, protein synthesis and apoptosis assays, XIAP/c-FLIPL westerns\",\n      \"pmids\": [\"24582807\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether selective loss of XIAP/c-FLIPL is due solely to global elongation slowing not fully resolved\", \"Sensing mechanism for oxidative stress upstream of eEF2K undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed eEF2K can act upstream of AMPK-ULK1 to restrain autophagy in cancer cells, revealing bidirectional, context-dependent coupling to the AMPK axis.\",\n      \"evidence\": \"siRNA, autophagic flux assays, epistasis via AMPK/ULK1 knockdown\",\n      \"pmids\": [\"24955726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which eEF2K influences AMPK activity not defined\", \"Tissue generality of upstream placement unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected eEF2K to translational reprogramming, showing ribosomal stress activates eEF2K to selectively favor TOP mRNA translation.\",\n      \"evidence\": \"Ribosomal stress induction, eEF2K inhibition, polysome profiling\",\n      \"pmids\": [\"25332393\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking elongation slowing to TOP-mRNA selectivity unresolved\", \"Single-lab pharmacological approach\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established active-site Cys146 as a non-conserved residue exploitable for reversible covalent eEF2K inhibition, informing selective inhibitor design.\",\n      \"evidence\": \"Two-step inhibition kinetics, active-site mutagenesis, molecular docking\",\n      \"pmids\": [\"25224652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystallographic confirmation of the thioimidate adduct\", \"Cellular selectivity not fully profiled\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed transcriptional control of eEF2K, with NF-κB/p65 repressing eEF2K to stimulate elongation under inflammatory and infectious stimuli.\",\n      \"evidence\": \"Multiple NF-κB stimuli, pre-mRNA quantification, p65 transcription assays, westerns\",\n      \"pmids\": [\"31636182\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct p65 occupancy at the eEF2K locus described but promoter elements not fully mapped\", \"Physiological consequence for pathogen replication not quantified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated that eEF2K shapes the excitation/inhibition balance by restraining GABAergic transmission, linking the kinase to seizure susceptibility.\",\n      \"evidence\": \"eEF2K knockout mice, electrophysiology, Synapsin 2b/GABAAR westerns, seizure models\",\n      \"pmids\": [\"27005990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Synapsin 2b/α5-GABAAR changes are direct translational targets undefined\", \"Pre- vs postsynaptic site of action not fully separated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided structural rationale for Ca2+-tunable activation by mapping eEF2K binding to the CaM C-lobe.\",\n      \"evidence\": \"NMR of CaM-eEF2K interaction (reported via commentary)\",\n      \"pmids\": [\"27602990\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Original structural data only described in a commentary abstract, details limited\", \"No full-length kinase-CaM complex structure\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed myostatin suppresses muscle protein synthesis exclusively through the AMPK-eEF2K-eEF2 axis at low doses, placing eEF2K downstream of a growth-restraining hormone.\",\n      \"evidence\": \"C2C12 myotubes, SUnSET synthesis assay, pathway-specific inhibitors\",\n      \"pmids\": [\"29024627\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo confirmation in muscle limited\", \"Dose-dependence of mTOR involvement not fully mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified Homer1b/c as a scaffolding partner controlling eEF2K localization and synaptic local translation under mTORC1 regulation.\",\n      \"evidence\": \"Co-IP, mTORC1 inhibition, phosphomutant constructs, synthesis assays in neurons\",\n      \"pmids\": [\"32892352\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP-based interaction without structural mapping\", \"Ser396 phosphorylation effect on binding affinity not quantified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established eEF2K as a translational activator of PD-L1 by relieving uORF-mediated repression, linking elongation control to immune checkpoint expression.\",\n      \"evidence\": \"Genetic eEF2K ablation, polyribosome profiling, uORF reporter assays, NK cytotoxicity\",\n      \"pmids\": [\"33094805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which eEF2K affects uORF reinitiation undefined\", \"Interplay with elongation slowing not reconciled\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined a reciprocal eEF2K-MEK1/2 interaction generating an ERK feedback loop that is disrupted by AMPK during nutrient deprivation to promote survival.\",\n      \"evidence\": \"Co-IP (eEF2K-MEK1/2), AMPK activation, cell viability assays\",\n      \"pmids\": [\"32565934\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal structural validation\", \"Whether interaction depends on kinase activity unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed SIRT1 promotes eEF2K-dependent cardiac autophagy under ER stress and that eIF2α physically associates with eEF2K upstream of its activation.\",\n      \"evidence\": \"Co-IP (eIF2α-eEF2K), siRNA, SIRT1 modulation, in vivo cardiac function\",\n      \"pmids\": [\"32059483\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of eIF2α-eEF2K binding not mechanistically dissected\", \"Single-lab in vivo model\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed eEF2K downstream of physiological neurogenic stimuli, with eEF2K loss enhancing adult dentate neurogenesis and learning.\",\n      \"evidence\": \"Global and conditional eEF2K knockout mice, neurogenesis markers, behavior\",\n      \"pmids\": [\"32707059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-autonomous vs niche effects on neural progenitors not fully separated\", \"Translational targets driving neurogenesis unidentified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided structural and biochemical evidence that eEF2K-phosphorylated eEF2 locks vacant 80S ribosomes into a SERBP1-bound, splitting-resistant inactive state, defining a dormant ribosome reservoir.\",\n      \"evidence\": \"Cryo-EM, in vitro ribosome splitting, pharmacological eEF2K activation, sensory neuron p-body imaging\",\n      \"pmids\": [\"34815424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals controlling reactivation of these ribosomes undefined\", \"Quantitative fraction of cellular ribosomes in this state unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated in vivo that eEF2K activity is strictly required for Rpl24-mutation-driven elongation suppression and colorectal tumor suppression.\",\n      \"evidence\": \"Rpl24 × eEF2K double-mutant mouse epistasis, polysome profiling, tumor assays\",\n      \"pmids\": [\"34895463\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Rpl24 mutation activates eEF2K not defined\", \"Translatome changes mediating tumor suppression not enumerated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified GSK3β-Ser9 as a direct non-eEF2 substrate of eEF2K, linking the kinase to PD-L1 stabilization and immune evasion in melanoma.\",\n      \"evidence\": \"IP-MS, Co-IP, in vitro phosphorylation, in vivo melanoma model, flow cytometry\",\n      \"pmids\": [\"35347072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GSK3β phosphorylation requires eEF2K kinase activity in all contexts not fully tested\", \"Structural basis of eEF2K-GSK3β recognition unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined an eEF2K-STAT3(Tyr705)-SPP1 transcriptional axis promoting melanoma progression, extending eEF2K function to transcription factor signaling.\",\n      \"evidence\": \"RNA-seq, STAT3 ChIP on SPP1 promoter, rescue experiments, in vivo model\",\n      \"pmids\": [\"35184394\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which eEF2K increases STAT3 Tyr705 phosphorylation undefined\", \"Direct vs indirect link unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Positioned eEF2K as a convergence node downstream of PI3K and MEK pathways mediating synergy of combined inhibition in AML.\",\n      \"evidence\": \"Phosphoproteomics, siRNA, pharmacological inhibition across 12 AML lines\",\n      \"pmids\": [\"35513296\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-model-specific effects limit generalizability\", \"Direct upstream kinases on eEF2K in this context not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a redox-homeostasis function whereby eEF2K inhibition raises ROS and sensitizes cancer cells to ferroptosis, distinct from canonical translation control.\",\n      \"evidence\": \"Pharmacological and siRNA eEF2K inhibition, ROS/ferroptosis assays, multiple cell lines\",\n      \"pmids\": [\"40409701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mediators connecting eEF2K to antioxidant defense undefined\", \"Dependence on eEF2 phosphorylation not separated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed alphavirus nsP2 NTPase activity hijacks cAMP/PKA to activate eEF2K and induce host translational shut-off, a conserved viral strategy.\",\n      \"evidence\": \"SILAC phosphoproteomics, NTPase Walker mutants, cAMP/PKA analysis\",\n      \"pmids\": [\"36848386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How nsP2 NTPase activates cAMP/PKA not defined\", \"Selectivity of host shut-off for viral vs host mRNAs unaddressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Developed a molecular-glue degrader (compound C1) mapping its eEF2K binding residues and showing it enhances βTRCP-mediated degradation, validating eEF2K as a druggable target.\",\n      \"evidence\": \"Binding assays, docking, Co-IP (eEF2K-βTRCP), degradation and antitumor experiments, organoids\",\n      \"pmids\": [\"38084501\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural confirmation of the ternary complex absent\", \"Single-lab degradation mechanism\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated eEF2K couples elongation slowing in sensory neurons to the integrated stress response and BDNF synthesis to drive pain behavior.\",\n      \"evidence\": \"In vivo eEF2K KO/inhibition, ISR inhibition, Bdnf-selective translation blockade, ribosome coupling assays\",\n      \"pmids\": [\"39694034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the ribosome-coupled ISR sensor not fully defined\", \"How BDNF translation is selectively induced unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed eEF2K protects against doxorubicin cardiotoxicity by phosphorylating and inhibiting GSK3β to restore autophagy, a protective counterpart to its oncogenic GSK3β signaling.\",\n      \"evidence\": \"AAV/adenoviral overexpression and knockdown, autophagy imaging, GSK3β inhibitor rescue, in vivo DIC model\",\n      \"pmids\": [\"39708064\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific outcome of GSK3β inhibition not reconciled with tumor context\", \"Single-lab model\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined an eEF2K-Nrf2-mitochondrial axis in NK cells, with TGFβ-induced eEF2K driving oxidative stress and immunosuppression, and eEF2K loss enhancing anti-tumor function.\",\n      \"evidence\": \"CRISPR eEF2K KO in NK cells, proteomics, functional assays, in vivo adoptive transfer\",\n      \"pmids\": [\"40506255\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking eEF2K to Nrf2 signaling undefined\", \"Dependence on canonical eEF2 phosphorylation not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified AURKA-Ser391 as a new eEF2K substrate, defining an eEF2K/AURKA/SOX8 axis that promotes triple-negative breast cancer.\",\n      \"evidence\": \"Proteomics, Co-IP, in vitro phosphorylation, mutagenesis, xenografts, organoids\",\n      \"pmids\": [\"39950798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether AURKA phosphorylation requires CaM-dependent eEF2K activation unknown\", \"Relatively recent single-lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a kinase-independent eEF2K function in C. elegans promoting starvation survival via DNA-repair transcription and ROS suppression, decoupling protective roles from eEF2 phosphorylation.\",\n      \"evidence\": \"efk-1 and kinase-dead mutants, RNA-seq, ROS and oxygen consumption assays, survival assays\",\n      \"pmids\": [\"39966347\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of the kinase-independent activity undefined\", \"Conservation of this noncanonical role in mammals untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How eEF2K balances its canonical eEF2-elongation brake against its expanding repertoire of direct protein substrates (GSK3β, AURKA) and kinase-independent functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length eEF2K or its substrate complexes\", \"Determinants of substrate selection between eEF2 and non-eEF2 targets unknown\", \"Mechanistic basis of kinase-independent roles uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 20, 25]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 20, 25, 23]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [0, 10, 19, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-72766\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 10, 19, 21]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [2, 6, 8]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 7, 24, 32]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [20, 22, 27]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EEF2\", \"CALM1\", \"GSK3B\", \"AURKA\", \"HOMER1\", \"EIF2AK / EIF2S1 (eIF2α)\", \"MAP2K1 (MEK1/2)\", \"BTRC (βTrCP)\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}