{"gene":"TBK1","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2000,"finding":"NAK (TBK1) directly phosphorylates IKKβ in vitro to activate the IKK complex, leading to IκB degradation and NF-κB activation; endogenous NAK is activated by phorbol esters and growth factors, and catalytically inactive NAK specifically inhibits NF-κB activation by PKCε.","method":"In vitro kinase assay, dominant-negative overexpression, IκB degradation assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay demonstrating direct phosphorylation, dominant-negative functional validation, replicated across multiple stimuli","pmids":["10783893"],"is_preprint":false},{"year":2003,"finding":"TBK1 and IKKε are essential components of the IRF3 signaling pathway downstream of TLR3 and viral dsRNA; both kinases phosphorylate IRF3 to drive type I interferon induction.","method":"Dominant-negative and siRNA knockdown, reporter assays, phosphorylation assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function and dominant-negative approaches with IRF3 phosphorylation readout, widely replicated","pmids":["12692549"],"is_preprint":false},{"year":2009,"finding":"TBK1 is selectively essential in KRAS-mutant cancer cells; TBK1 activates NF-κB anti-apoptotic signals involving c-Rel and BCL-XL that are required for survival in these cells.","method":"Systematic RNAi screen, shRNA knockdown with apoptosis readout, NF-κB pathway analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic RNAi screen with mechanistic follow-up across multiple cell lines, widely replicated","pmids":["19847196"],"is_preprint":false},{"year":2012,"finding":"STING functions as a scaffold protein that binds both TBK1 and IRF3; the C-terminal region of STING is necessary and sufficient to activate TBK1 and promote IRF3 phosphorylation; STING mutations that selectively disrupt IRF3 binding abrogate IRF3 phosphorylation without impairing TBK1 activation.","method":"In vitro reconstitution system, mutagenesis, co-immunoprecipitation","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis validating interaction mode and functional consequences","pmids":["22394562"],"is_preprint":false},{"year":2015,"finding":"Loss-of-function mutations in TBK1 cause familial ALS and frontotemporal dementia via haploinsufficiency; C-terminal coiled-coil domain (CCD2) mutations disrupt TBK1 interaction with the adaptor protein optineurin.","method":"Exome sequencing of ALS/FTD patients, in vitro expression and binding assays, linkage analysis","journal":"Nature neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic and in vitro functional validation across multiple families and mutation types","pmids":["25803835"],"is_preprint":false},{"year":2018,"finding":"TBK1 is an endogenous inhibitor of RIPK1 kinase; embryonic lethality of Tbk1-/- mice is rescued by inactivating RIPK1 kinase activity, establishing TBK1 as a suppressor of RIPK1-driven apoptosis and inflammation.","method":"Genetic epistasis (Tbk1-/- × RIPK1 kinase-dead mice), embryonic lethality rescue assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis with complete rescue, multiple phenotypic readouts","pmids":["30146158"],"is_preprint":false},{"year":2018,"finding":"TBK1 and IKKε are recruited to the TNFR1 signaling complex via LUBAC-dependent linear ubiquitin; they phosphorylate RIPK1 in the TNFR1-SC to prevent TNF-induced cell death. NEMO mediates recruitment via adaptors TANK and NAP1.","method":"Co-immunoprecipitation of signaling complex, genetic knockout, in vivo TNF lethal shock model","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple KO cell lines, in vivo rescue experiment","pmids":["30420664"],"is_preprint":false},{"year":2018,"finding":"TBK1 directly phosphorylates RAB7A at Ser72 upon mitochondrial depolarization to promote mitophagy; this modification reduces RAB GDI association and increases FLCN-FNIP1 recruitment, and non-phosphorylatable RAB7A impairs mitophagy and ATG9A vesicle recruitment to damaged mitochondria.","method":"Quantitative phosphoproteomics, in vitro kinase assay, interaction proteomics with phosphomutants, mitophagy imaging","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, phosphoproteomics, and functional mutant analysis in single study","pmids":["30627666"],"is_preprint":false},{"year":2018,"finding":"TBK1 directly inhibits AMPK to repress cellular respiration and increase energy storage in adipocytes; conversely, AMPK can increase TBK1 activity via its downstream target ULK1; TBK1 also suppresses inflammation by phosphorylating and inducing degradation of the IKK kinase NIK.","method":"Adipocyte-specific TBK1 knockout mice, in vitro kinase assays, phosphorylation and degradation assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with metabolic phenotype plus in vitro kinase assays for multiple substrates","pmids":["29425491"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structure of TBK1 in complex with cGAMP-bound STING reveals that the STING C-terminal tail inserts as a β-strand into a groove between the TBK1 kinase domain and SDD of the second subunit; phosphorylation site Ser366 cannot reach the active site of bound TBK1, indicating that STING phosphorylation requires oligomerization of both proteins.","method":"Cryo-electron microscopy structure determination, mutational analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with mutational validation, mechanistic model experimentally confirmed","pmids":["30842653"],"is_preprint":false},{"year":2019,"finding":"NDP52 initiates selective autophagy by localizing and activating the ULK1 complex in a TBK1-dependent manner; ectopic placement of NDP52 on organelles is sufficient to induce selective autophagy; tethering ULK1 directly to cargo bypasses both autophagy receptors and TBK1 requirement.","method":"Chemically inducible dimerization (CID) assays, CRISPR KO lines, live-cell imaging","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic CID approach with CRISPR KO controls and rescue experiments","pmids":["30853401"],"is_preprint":false},{"year":2019,"finding":"TBK1 phosphorylates Syntaxin 17 at Ser202; phospho-Stx17 translocates from the Golgi to ATG13+FIP200+ pre-autophagosomal structures to control autophagy initiation; TBK1 or Stx17 KO blocks ATG13/FIP200 puncta formation and reduces ULK1 complex assembly.","method":"Phosphorylation mapping, in vitro kinase assay, CRISPR KO, live-cell imaging, co-immunoprecipitation","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay identifying substrate site, KO with defined phenotypic rescue, localization experiments","pmids":["30827897"],"is_preprint":false},{"year":2019,"finding":"TBK1 is activated by VHL loss or hypoxia in cancer cells; VHL-mediated hydroxylation of TBK1 at Pro48 promotes PPM1B phosphatase binding and TBK1 dephosphorylation; TBK1 phosphorylates p62/SQSTM1 at Ser366, which is essential for p62 stability and cancer cell proliferation.","method":"Genetic ablation, pharmacological inhibition, PROTAC degradation, phosphorylation assays, xenograft model","journal":"Cancer discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic and pharmacological tools, mechanistic pathway mapping, in vivo validation","pmids":["31810986"],"is_preprint":false},{"year":2019,"finding":"TBKBP1 recruits TBK1 to PKCθ via scaffold protein CARD10; PKCθ phosphorylates TBK1 at Ser716, a step required for TBK1 activation by growth factors but not innate immune stimuli; the TBK1-TBKBP1 axis mediates mTORC1 activation, oncogenesis, and PD-L1 induction.","method":"Co-immunoprecipitation, phosphorylation assays, conditional knockout mice, lung cancer model","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, phosphosite identification, conditional KO with tumor phenotype, multiple orthogonal approaches","pmids":["31792381"],"is_preprint":false},{"year":2019,"finding":"PINK1/Parkin-mediated TBK1 activation at damaged mitochondria during mitophagy sequesters TBK1 from its physiological role at centrosomes, causing a block in mitosis.","method":"Genetic interaction screen, live-cell imaging, mitosis assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic interaction with mitosis readout and TBK1 localization data, single lab","pmids":["31577952"],"is_preprint":false},{"year":2019,"finding":"Axl receptor tyrosine kinase induces TBK1 activity through a Ras-RalB-dependent mechanism to drive epithelial-mesenchymal transition in KRAS-mutant pancreatic cancer.","method":"Genetic knockdown, pharmacological inhibition, EMT marker analysis, invasion/metastasis assays","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with defined pathway placement and functional EMT readout, single lab","pmids":["30938713"],"is_preprint":false},{"year":2019,"finding":"TBK1 promotes Raptor Ser877 phosphorylation in vitro and in cells; this modification limits mTORC1 activity, as a non-phosphorylatable Raptor S877A mutant increases mTORC1 signaling.","method":"In vitro kinase assay coupled with mass spectrometry, phosphosite mutagenesis, mTORC1 activity assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with phosphosite identification and functional mutagenesis, single lab","pmids":["31530866"],"is_preprint":false},{"year":2020,"finding":"TBK1 is redundant with IKKε for STING-induced NF-κB responses in myeloid cells; IRF3 activation is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition.","method":"Genetic KO (single and double), pharmacological inhibition, reporter assays, in vivo validation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic and pharmacological approaches, human and mouse cells, and in vivo validation distinguishing IRF3 vs NF-κB dependency","pmids":["32268090"],"is_preprint":false},{"year":2020,"finding":"TBK1 directly phosphorylates DRP1/DNM1L upon RLR-MAVS signaling activation; this phosphorylation prevents DRP1 oligomerization and mitochondrial fragmentation, and is required for assembly of large MAVS aggregates and efficient antiviral immunity.","method":"In vitro kinase assay, knockin (KI) strategy mimicking TBK1-DRP1 signaling, co-immunoprecipitation, mitochondrial morphology assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, KI dominant-negative phenotype, KI rescue, multiple orthogonal methods in one study","pmids":["33171123"],"is_preprint":false},{"year":2020,"finding":"TBK1 directly interacts with mTORC2 and phosphorylates mTOR at Ser2159 within mTORC2; this increases mTORC2 intrinsic catalytic activity and Akt phosphorylation; TBK1-mTOR S2159 phosphorylation also promotes mTORC1 signaling.","method":"Co-immunoprecipitation of mTORC2, in vitro kinase assay, Mtor S2159A knock-in MEFs and mice","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted kinase assay, co-IP, and knock-in allele validating the TBK1-mTOR axis","pmids":["34245780"],"is_preprint":false},{"year":2017,"finding":"TBK1 directly phosphorylates the autophagy adaptor optineurin (OPTN) at Ser177 after Salmonella invasion, enhancing OPTN interaction with Atg8 family proteins and recruiting the autophagic machinery to intracellular bacteria for xenophagy.","method":"In vitro kinase assay, co-immunoprecipitation, bacterial clearance assays","journal":"Science signaling (review citing primary data); primary mechanism originally described 2011","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with phosphosite identification, supported by multiple labs","pmids":["21868362"],"is_preprint":false},{"year":2017,"finding":"TRIM23 undergoes K27-linked auto-ubiquitination on its ARF domain, enabling GTP hydrolysis activity and facilitating TBK1 dimerization; active TBK1 then phosphorylates p62 to promote selective autophagy.","method":"RNAi screen, mutagenesis of ubiquitination sites, TBK1 dimerization assay, p62 phosphorylation assay","journal":"Nature microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic dissection with mutagenesis of ubiquitination site and functional readouts, multiple viral pathogens tested","pmids":["28871090"],"is_preprint":false},{"year":2017,"finding":"MAVS recruits TBK1/IKKε via TRAFs; TRAFs interact directly with the SDD domain of TBK1/IKKε via their coiled-coil domain; TRAF E3 ligase activity synthesizes ubiquitin chains to activate NEMO; NEMO-activated IKKα/β then further phosphorylate and activate TBK1/IKKε.","method":"TRAF quadruple knockout cells, co-immunoprecipitation, ubiquitination assays, domain mapping","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — quadruple KO cells with complete loss of viral response, Co-IP domain mapping, multiple orthogonal methods","pmids":["29125880"],"is_preprint":false},{"year":2017,"finding":"USP1-UAF1 deubiquitinase complex binds TBK1, removes K48-linked polyubiquitination, and prevents TBK1 proteasomal degradation, thereby stabilizing TBK1 and enhancing antiviral IFN responses.","method":"Co-immunoprecipitation, ubiquitination assays, USP1-UAF1 inhibitor ML323, in vitro and in vivo viral infection","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, pharmacological inhibitor, single lab","pmids":["29138248"],"is_preprint":false},{"year":2015,"finding":"TBK1 controls AKT ubiquitination and degradation to regulate T-cell activation and migration; T-cell-specific TBK1 ablation deregulates AKT-mTORC1 signaling, enhancing T-cell activation and impairing effector T-cell egress from draining lymph nodes.","method":"T-cell-conditional TBK1 knockout mice, AKT ubiquitination assay, mTORC1 activity assays, EAE model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined cellular phenotype and mechanistic follow-up, single lab","pmids":["25606824"],"is_preprint":false},{"year":2021,"finding":"TBK1 is recruited to STING via the STING C-terminal tail; STING S365A mutation (abolishing IRF3 binding) allows TBK1 recruitment and NF-κB activation but not IRF3 activation; disruption of TBK1 binding to STING (L373A or C-terminal truncation) prevents both pathways and rescues autoinflammatory disease in DNase II-/- mice.","method":"Genetic knockin mice (STING S365A, L373A, C-terminal truncation), disease rescue experiments in DNase II-/- mice, cytokine neutralization","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic knockin alleles with in vivo disease rescue, orthogonal experiments dissecting IRF3 vs NF-κB pathway","pmids":["34901991"],"is_preprint":false},{"year":2021,"finding":"TBK1 loss in humans causes autoinflammation driven by TNF-induced RIPK1-dependent cell death (necroptosis); patient-derived fibroblasts with homozygous TBK1 loss show elevated necroptosis in vitro; IFN-I induction via RIG-I/MDA5 is hypomorphic but sufficient in the absence of TBK1.","method":"Patient-derived fibroblasts, necroptosis assays, ex vivo cytokine profiling, anti-TNF treatment","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — human genetic loss-of-function with functional cellular assays and in vivo treatment response","pmids":["34363755"],"is_preprint":false},{"year":2021,"finding":"TBK1 phosphorylates full-length and N-terminal huntingtin at Ser13/Ser16 in vitro and at Ser13 in cells and in vivo; TBK1-mediated phosphorylation inhibits mutant HTT aggregation and increases autophagic clearance, reducing cytotoxicity.","method":"In vitro kinase assay, cell-based phosphorylation assay, aggregation assays, autophagy flux assay, C. elegans model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, in vivo validation, multiple model systems","pmids":["32757223"],"is_preprint":false},{"year":2021,"finding":"TBK1 interacts with tau in human AD brain tissue and phosphorylates tau in vitro at multiple sites; TBK1 activation in Drosophila tauopathy model triggers tau hyperphosphorylation and enhanced neurodegeneration, while TBK1 ortholog knockdown suppresses tau toxicity.","method":"Immunoaffinity enrichment + mass spectrometry from human brain, in vitro kinase assay + MS, Drosophila genetic epistasis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with MS mapping, Drosophila genetic validation, human brain Co-IP, single lab","pmids":["33965374"],"is_preprint":false},{"year":2021,"finding":"NEDD4 E3 ligase catalyzes K27-linked polyubiquitination of TBK1 at K344, targeting TBK1 for NDP52-mediated selective autophagic degradation; this limits TBK1 protein levels at late stages of viral infection to prevent excessive immune response.","method":"Ubiquitination assays (K27 linkage), co-immunoprecipitation, autophagy flux assays, viral infection","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific ubiquitin linkage and lysine site identified, autophagic degradation pathway confirmed, single lab","pmids":["34257412"],"is_preprint":false},{"year":2021,"finding":"PRMT1 directly interacts with TBK1 and catalyzes asymmetric arginine methylation at R54, R134, and R228; this modification enhances TBK1 oligomerization and subsequent Ser172 autophosphorylation and downstream type I IFN production.","method":"Co-immunoprecipitation, in vitro methylation assay, site-directed mutagenesis, oligomerization assay, myeloid-specific Prmt1 KO mice","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with site mutagenesis, functional KO validation in vivo, mechanistic link to oligomerization confirmed","pmids":["34551290"],"is_preprint":false},{"year":2021,"finding":"TBK1 interacts with NLRP3 and acts as a phospho-switch to limit NLRP3 inflammasome activation; TBK1 and IKKε together constitute an OFF switch for NLRP3 via phosphorylation at a site distinct from Ser3; simultaneous deletion of both kinases is required to fully release NLRP3 activation.","method":"Single and double KO macrophages, pharmacological inhibition, NLRP3 mutagenesis, co-immunoprecipitation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological approaches, NLRP3 mutagenesis, mechanistic interaction confirmed by Co-IP, single lab","pmids":["34518217"],"is_preprint":false},{"year":2022,"finding":"TBK1 phosphorylates METTL3 at Ser67 upon viral infection; phospho-METTL3 interacts with the translational complex to enhance protein translation and m6A modification, stabilizing IRF3 mRNA to facilitate antiviral responses.","method":"Co-immunoprecipitation, phosphorylation mapping, METTL3 KO cells and Mettl3 conditional KO mice, viral infection models","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation site identified, functional KO validation with viral phenotype, mechanistic link to translational complex by Co-IP, single lab","pmids":["35172162"],"is_preprint":false},{"year":2020,"finding":"TBC1D9, a Ca2+-binding protein, mediates TBK1 activation during xenophagy and mitophagy; upon bacterial infection, Ca2+ increase promotes TBC1D9 binding to ubiquitin-coated bacteria via its UBR and Ca2+-binding motif, and TBC1D9 KO suppresses TBK1 activation and ULK1 complex recruitment.","method":"Co-immunoprecipitation, TBC1D9 knockout cells, Ca2+ chelation experiments, live-cell imaging","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with defined phenotype, Co-IP, Ca2+ perturbation experiments, single lab","pmids":["32034138"],"is_preprint":false},{"year":2011,"finding":"TBK1 controls an antiapoptotic response downstream of TNF by triggering a specific RelA/p65 phosphorylation event, resulting in PAI-2 expression; PAI-2 limits caspase-3 activation by stabilizing transglutaminase 2, which cross-links and inactivates procaspase-3.","method":"Genetic studies in Tg2-/- mice, RelA phosphorylation mapping, caspase-3 activation assay, TNF liver injury models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO model with defined pathway, mechanistic assays linking TBK1→RelA phosphorylation→PAI-2→TG2→procaspase-3 axis","pmids":["22203995"],"is_preprint":false},{"year":2011,"finding":"IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288, and Ser293, converting it into an active E3 ubiquitin ligase; endogenous Pellino 1 E3 ligase activity is activated in LPS/poly(I:C)-stimulated macrophages in a TBK1/IKKε-dependent manner.","method":"In vitro kinase assay, two-hybrid screen, E3 ligase activity assay, IKKε/TBK1 pharmacological inhibition in macrophages","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with phosphosite mapping, functional E3 ligase activity measured, single lab","pmids":["21204785"],"is_preprint":false},{"year":2018,"finding":"ALS-associated TBK1 missense mutations (G217R, R357X) abolish TBK1 ability to phosphorylate IRF3 and optineurin, impair TBK1 homodimerization (G217R), and reduce optineurin binding; proportion of active (phosphorylated) TBK1 is reduced in lymphoblastoid cell lines from mutation carriers.","method":"In vitro kinase assay with IRF3 and OPTN substrates, dimerization assay, patient-derived cell lines","journal":"Neurobiology of aging","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assays with substrates, patient-derived cells, single lab","pmids":["30033073"],"is_preprint":false},{"year":2021,"finding":"Optineurin provides a unique platform for TBK1 activation at mitophagy contact sites between damaged mitochondria and autophagosome formation sites; both OPTN-ubiquitin and OPTN-PAS interaction axes facilitate OPTN-TBK1 complex assembly, initiating a positive feedback loop of TBK1 hetero-autophosphorylation.","method":"Live-cell imaging, engineered monobodies blocking OPTN, Co-IP, mitophagy flux assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — monobody functional inhibition, live imaging, Co-IP, and defined mechanistic feedback loop, multiple orthogonal approaches","pmids":["38287189"],"is_preprint":false},{"year":2021,"finding":"ALS-linked TBK1 mutations that abolish both dimerization and kinase activity are most disruptive to mitophagy; TBK1 recruitment and OPTN phosphorylation at S177 are each necessary for engulfment of damaged mitochondria; ULK1 activity also contributes to OPTN S177 phosphorylation.","method":"Quantitative imaging of mitophagy in cells expressing ALS mutants, kinase assays, ULK1 inhibition","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutant analysis with imaging readout, pharmacological and genetic dissection, single lab","pmids":["34099552"],"is_preprint":false},{"year":2024,"finding":"NAP1 and SINTBAD, two TBK1 adaptors, restrict OPTN-driven mitophagy initiation by competing with OPTN for TBK1 binding; they promote NDP52-driven mitophagy progression by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200; OPTN is the primary recruiter of TBK1 during mitophagy initiation.","method":"Co-immunoprecipitation, competitive binding assays, knockout cells, mitophagy flux assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple KO lines, competition binding assay, mechanistic model validated","pmids":["38918639"],"is_preprint":false},{"year":2024,"finding":"A lysosome-localized pool of TBK1 is recruited upon elevated amino acid levels and phosphorylates Rab7 at Ser72 to relieve Rab7-dependent inhibition of mTORC1; an ALS-FTD-associated TBK1 mutant (E696K) constitutively accumulates at lysosomes, causing elevated Rab7 phosphorylation and increased mTORC1 activation.","method":"Subcellular fractionation, co-immunoprecipitation, phosphorylation assays, mTORC1 activity assays, disease mutant analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — lysosomal localization experimentally defined, substrate phosphorylation and mTORC1 consequence validated, disease mutant mechanism identified","pmids":["39103493"],"is_preprint":false},{"year":2024,"finding":"FIP200 controls the TBK1 activation threshold at SQSTM1/p62-positive condensates; TBK1 is recruited to p62 aggregates via TAX1BP1 and phosphorylates p62 at Ser403; removal of FIP200 or disruption of FIP200-TAX1BP1 interaction strongly increases TBK1 activation at these condensates.","method":"Co-immunoprecipitation, phosphorylation assays, FIP200 KO cells, aggregate imaging","journal":"Scientific reports (2021)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, KO with defined activation phenotype, p62 phosphosite assay, single lab","pmids":["34226595"],"is_preprint":false},{"year":2023,"finding":"PTK2B directly phosphorylates TBK1 at Tyr591 to increase TBK1 oligomerization and activation; PTK2B also promotes STING oligomerization in a kinase-independent manner; Ptk2b-deficient mice are more susceptible to viral infection.","method":"Co-immunoprecipitation, in vitro kinase assay, mutagenesis of Tyr591, Ptk2b KO mice, viral infection","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with phosphosite identification, in vivo KO validation, single lab","pmids":["37989995"],"is_preprint":false},{"year":2024,"finding":"SIRT5 desuccinylates TBK1 at Lys137, leading to TBK1 dephosphorylation and suppression of downstream inflammatory signaling; reduced SIRT5 in aged primate skeletal muscle increases TBK1 succinylation and promotes inflammation.","method":"In vitro succinylation/desuccinylation assays, site mutagenesis (K137), SIRT5 KO/overexpression in myotubes, primate muscle tissue analysis","journal":"Nature metabolism","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with site identification, cellular KO validation, single lab","pmids":["40087407"],"is_preprint":false},{"year":2024,"finding":"SIRT5 desuccinylates TBK1 at K38, K154, and K692 in macrophages; succinylation at these sites inhibits TBK1 binding to IRF3 and TRAF2, suppressing inflammatory signaling; macrophage adoptive transfer of succinylation-resistant TBK1-2KR (K154/692R) exacerbates sepsis-induced lung injury.","method":"Succinylation site mapping, mutagenesis, Co-IP, macrophage adoptive transfer, sepsis model","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis, Co-IP disruption, in vivo adoptive transfer, single lab","pmids":["39673708"],"is_preprint":false},{"year":2021,"finding":"Myeloid cell TBK1 suppresses NF-κB and MAP kinase signaling pathways in macrophages to attenuate induction of proinflammatory cytokines, particularly IL-1β; myeloid-specific TBK1 KO causes spontaneous adipose inflammation and exacerbates colitis, and IL-1R1 ablation eliminates these inflammatory symptoms.","method":"Myeloid-conditional TBK1 KO mice, NF-κB/MAPK signaling assays, cytokine measurements, IL-1R1 double KO","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined phenotype and epistasis via IL-1R1 KO rescue, single lab","pmids":["35074921"],"is_preprint":false},{"year":2024,"finding":"CDK4/6 phosphorylates TBK1 at Ser527 to inactivate STING signaling in prostate cancer; CDK4/6-mediated phosphorylation of RB1 at Ser249/Thr252 also induces RB1-TBK1 interaction, reducing TBK1 Ser172 phosphorylation and suppressing the STING pathway.","method":"Phosphorylation assays, Co-immunoprecipitation, CDK4/6 inhibitor treatment, prostate cancer cell models","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphosite identification, Co-IP, CDK4/6 inhibitor treatment, both RB1-dependent and independent pathways tested, single lab","pmids":["38861362"],"is_preprint":false},{"year":2023,"finding":"Tiliroside directly binds TBK1 (confirmed by molecular docking and biolayer interferometry) and inhibits its enzymatic activity, reducing p62 Ser349 phosphorylation and Keap1-Nrf2 axis activity to induce ferroptosis in hepatocellular carcinoma cells.","method":"Biolayer interferometry binding assay, enzymatic activity assay, Co-IP, xenograft tumor models","journal":"Phytomedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding confirmed by BLI, enzymatic inhibition assay, in vivo validation, single lab","pmids":["36657316"],"is_preprint":false},{"year":2023,"finding":"NLRC4 facilitates the interaction between TBK1 and the E3 ubiquitin ligase CBL to enhance K63-linked polyubiquitination of TBK1, promoting cGAS-STING pathway activation and TBK1 phosphorylation.","method":"Co-immunoprecipitation, ubiquitination assays (K63-specific), NLRC4 KO cells and Nlrc4-/- mice, viral infection","journal":"Journal of medical virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, K63-specific ubiquitination, genetic KO mice with viral phenotype, single lab","pmids":["37537877"],"is_preprint":false},{"year":2021,"finding":"Loss of TBK1 kinase function protects against diet-induced metabolic dysfunction through loss of an inhibitory interaction between TBK1 and the insulin receptor.","method":"Global Tbk1 kinase-dead mutant mice, metabolic profiling, insulin receptor interaction assay","journal":"Molecular metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mouse model with defined metabolic phenotype and mechanistic interaction assay, single lab","pmids":["29935921"],"is_preprint":false},{"year":2022,"finding":"The MAM-specific E3 ubiquitin ligase AMFR ubiquitinates nascent proteins at the MAM to activate TBK1; TBK1 activation at the MAM results in ribosomal protein degradation under proteostatic stress; MAM or TBK1 deficiency impairs this response and increases cellular vulnerability.","method":"MAM fractionation, TBK1 KO, AMFR knockdown, ribosomal degradation assay, in vitro and in vivo models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation with functional consequence, KO phenotype, E3 ligase identification, single lab","pmids":["37967220"],"is_preprint":false},{"year":2020,"finding":"Vimentin interacts with TBK1 and IKKε to disrupt TBK1-IRF3 and IKKε-IRF3 interactions, inhibiting IRF3 phosphorylation and nuclear translocation and suppressing type I IFN production.","method":"Co-immunoprecipitation, vimentin KO mice, overexpression, IRF3 localization assay","journal":"Cell reports (2022)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP showing disruption of TBK1-IRF3 interaction, KO mice with in vivo viral phenotype, single lab","pmids":["36223739"],"is_preprint":false}],"current_model":"TBK1 is a serine/threonine kinase that functions as a central node in innate immunity, autophagy, and cell survival: it is activated by scaffolds including STING (via its C-terminal tail), MAVS/TRAFs, and optineurin at mitochondria, whereupon TBK1 phosphorylates IRF3/IRF7 to induce type I interferons, directly phosphorylates RIPK1 to suppress cell death, phosphorylates autophagy adaptors (optineurin S177, p62 S403, Stx17 S202) and organelle regulators (RAB7A S72, DRP1) to control selective autophagy and mitochondrial dynamics, and phosphorylates multiple metabolic targets (AMPK, NIK, mTOR S2159, Raptor S877) to modulate energy homeostasis and NF-κB signaling; its activity is regulated by post-translational modifications including K48/K63/K27-linked ubiquitination, arginine methylation (PRMT1 at R54/R134/R228), tyrosine phosphorylation (PTK2B at Y591), and lysine succinylation (SIRT5 at K137/K154/K692), with haploinsufficiency causing ALS/FTD and gain-of-function duplications causing normal-tension glaucoma."},"narrative":{"mechanistic_narrative":"TBK1 is a serine/threonine kinase that functions as a central signaling hub coupling innate immune sensing, selective autophagy, cell-death suppression, and metabolic control [PMID:12692549, PMID:30146158, PMID:29425491]. In antiviral signaling, TBK1 is recruited to upstream scaffolds—STING via its C-terminal tail, and MAVS via TRAFs that engage the TBK1 SDD domain—whereupon it phosphorylates IRF3 to drive type I interferon induction, while STING-dependent NF-κB output is comparatively kinase-independent and redundant with IKKε [PMID:12692549, PMID:22394562, PMID:32268090, PMID:29125880, PMID:34901991]. Cryo-EM of the cGAMP-STING–TBK1 complex established that substrate IRF3/STING phosphorylation requires trans-oligomerization, since the STING phosphosite cannot reach the active site of the TBK1 subunit that binds it [PMID:30842653]. TBK1 enforces cell survival by directly phosphorylating RIPK1 within the TNFR1 complex—to which it is recruited through LUBAC-generated linear ubiquitin and the NEMO/TANK/NAP1 adaptors—to restrain RIPK1-driven apoptosis and necroptosis; this activity underlies the embryonic lethality of Tbk1 loss and human TBK1-deficiency autoinflammation [PMID:30146158, PMID:30420664, PMID:34363755]. In selective autophagy, TBK1 is activated on cargo by adaptor platforms (optineurin, NDP52, TAX1BP1, with NAP1/SINTBAD tuning recruitment) and phosphorylates autophagy regulators including optineurin Ser177, p62/SQSTM1, Syntaxin 17 Ser202, and RAB7A Ser72 to control xenophagy, mitophagy, and autophagosome initiation [PMID:30627666, PMID:30853401, PMID:30827897, PMID:21868362, PMID:38918639]. TBK1 additionally phosphorylates metabolic and mTOR-axis targets—AMPK, NIK, mTOR Ser2159, and Raptor Ser877—to govern energy homeostasis and growth signaling [PMID:29425491, PMID:31530866, PMID:34245780]. Its activity is set by an extensive PTM code: activating arginine methylation by PRMT1 and tyrosine phosphorylation by PTK2B promote oligomerization and Ser172 autophosphorylation, whereas SIRT5-reversible succinylation and stabilizing/destabilizing ubiquitination further modulate the pool of active kinase [PMID:29138248, PMID:34257412, PMID:34551290, PMID:37989995, PMID:39673708]. Genetically, TBK1 haploinsufficiency and kinase/dimerization-disrupting mutations cause familial ALS and frontotemporal dementia, in part by impairing optineurin binding and mitophagy [PMID:25803835, PMID:30033073, PMID:34099552].","teleology":[{"year":2000,"claim":"Established TBK1 as an IKK-activating kinase, placing it upstream of NF-κB before its interferon role was known.","evidence":"In vitro kinase assay and dominant-negative overexpression with IκB degradation readout","pmids":["10783893"],"confidence":"High","gaps":["Did not define physiological stimuli or scaffolds linking TBK1 to IKK in vivo","Direct vs indirect IKKβ phosphorylation under endogenous conditions not resolved"]},{"year":2003,"claim":"Identified the canonical antiviral function: TBK1 (with IKKε) phosphorylates IRF3 downstream of dsRNA sensing to induce type I interferon.","evidence":"Dominant-negative/siRNA knockdown with IRF3 phosphorylation and interferon reporter readouts","pmids":["12692549"],"confidence":"High","gaps":["Upstream scaffold recruiting TBK1 to TLR3 not defined","Division of labor between TBK1 and IKKε left open"]},{"year":2009,"claim":"Revealed a non-immune survival role: TBK1 is selectively required by KRAS-mutant cancer cells through NF-κB anti-apoptotic signaling.","evidence":"Systematic RNAi screen with shRNA apoptosis follow-up across cell lines","pmids":["19847196"],"confidence":"High","gaps":["Direct TBK1 substrates driving the survival signal not identified","Mechanism of selective dependence on mutant KRAS unresolved"]},{"year":2012,"claim":"Defined STING as the scaffold that physically couples TBK1 to its substrate IRF3, explaining substrate selection in cytosolic DNA sensing.","evidence":"In vitro reconstitution, mutagenesis, and co-immunoprecipitation separating TBK1 activation from IRF3 binding","pmids":["22394562"],"confidence":"High","gaps":["Structural basis of activation not yet resolved","Whether STING also gates NF-κB output unaddressed"]},{"year":2015,"claim":"Connected TBK1 to human neurodegeneration, showing loss-of-function mutations cause ALS/FTD via haploinsufficiency and disrupted optineurin binding.","evidence":"Exome sequencing of ALS/FTD families with in vitro binding assays","pmids":["25803835"],"confidence":"High","gaps":["Which TBK1 substrate(s) drive neurodegeneration not established","Cell-type-specific basis of motor neuron vulnerability unknown"]},{"year":2018,"claim":"Established the essential survival mechanism: TBK1 directly suppresses RIPK1 kinase to prevent apoptosis/inflammation, explaining Tbk1-null embryonic lethality.","evidence":"Genetic epistasis (Tbk1-/- × RIPK1 kinase-dead), TNFR1 complex Co-IP, and in vivo TNF shock models","pmids":["30146158","30420664"],"confidence":"High","gaps":["RIPK1 phosphosite(s) targeted by TBK1 not fully mapped here","Relative contribution of IKKε in this complex left partial"]},{"year":2018,"claim":"Expanded TBK1 into metabolic and autophagy substrate space, identifying AMPK/NIK inhibition and direct RAB7A Ser72 phosphorylation for mitophagy.","evidence":"Conditional adipocyte KO mice, phosphoproteomics, in vitro kinase assays, and mitophagy imaging with phosphomutants","pmids":["29425491","30627666"],"confidence":"High","gaps":["How TBK1 is targeted to specific metabolic vs autophagic substrates not unified","In vivo significance of RAB7A Ser72 for organismal mitophagy unresolved"]},{"year":2019,"claim":"Solved the activation mechanism structurally: STING tail insertion and the requirement for trans-oligomerization for substrate phosphorylation.","evidence":"Cryo-EM of cGAMP-STING–TBK1 complex with mutational validation","pmids":["30842653"],"confidence":"High","gaps":["Structural basis for non-STING scaffold activation not addressed","How oligomerization is achieved at other organelles unclear"]},{"year":2019,"claim":"Defined TBK1's role in autophagy initiation through autophagy receptors and substrate phosphorylation (Stx17 Ser202, p62) that recruit the ULK1 machinery.","evidence":"Chemically inducible dimerization, CRISPR KO, in vitro kinase assays, and live-cell imaging","pmids":["30853401","30827897","28871090"],"confidence":"High","gaps":["Hierarchy among multiple autophagy substrates not resolved","Whether all receptors use the same TBK1 activation route unclear"]},{"year":2019,"claim":"Mapped scaffold- and PTM-controlled activation modes distinguishing growth-factor from immune signaling, and linked TBK1 to mTOR and cancer phenotypes.","evidence":"Reciprocal Co-IP, phosphosite identification (TBK1 Ser716, Raptor Ser877), conditional KO tumor models, and VHL/PPM1B regulation","pmids":["31792381","31810986","31530866","30938713"],"confidence":"High","gaps":["How distinct upstream adaptors select downstream substrate programs not integrated","Some axes (Axl-RalB, Raptor) rest on single-lab evidence"]},{"year":2020,"claim":"Separated IRF3 (kinase-dependent) from NF-κB (kinase-tolerant, IKKε-redundant) STING outputs and added DRP1/mitochondrial dynamics control.","evidence":"Single/double KO, pharmacological inhibition, in vivo validation, and DRP1 knock-in mimics with mitochondrial morphology assays","pmids":["32268090","33171123"],"confidence":"High","gaps":["Kinase-independent NF-κB scaffolding function of TBK1 not mechanistically defined","How DRP1 phosphorylation integrates with antiviral MAVS aggregation in vivo unresolved"]},{"year":2021,"claim":"Resolved scaffold-encoded pathway choice in vivo and established that human TBK1 loss drives TNF/RIPK1-dependent autoinflammation treatable by anti-TNF.","evidence":"STING knock-in alleles with disease rescue in DNase II-/- mice; patient fibroblast necroptosis assays and anti-TNF treatment","pmids":["34901991","34363755"],"confidence":"High","gaps":["Why human TBK1 loss tolerates interferon hypomorphism but not cell-death control not fully explained","Tissue-specific thresholds for autoinflammation unknown"]},{"year":2021,"claim":"Defined the optineurin-centered platform and PTM code (PRMT1 methylation) that drive TBK1 oligomerization and feedback autoactivation during mitophagy and interferon responses.","evidence":"Live-cell imaging, monobody inhibition, in vitro methylation with site mutagenesis, oligomerization assays, and myeloid Prmt1 KO mice","pmids":["38287189","34099552","34551290"],"confidence":"High","gaps":["Quantitative interplay between activating and inhibitory PTMs not modeled","How ALS mutations perturb the OPTN feedback loop in neurons untested"]},{"year":2024,"claim":"Refined adaptor logic and organelle-localized pools controlling TBK1 substrate choice, including a lysosomal pool acting on Rab7–mTORC1 with disease-mutant consequences.","evidence":"Competitive binding/Co-IP for NAP1/SINTBAD vs OPTN, KO mitophagy assays, subcellular fractionation, and ALS-FTD mutant (E696K) analysis","pmids":["38918639","39103493"],"confidence":"High","gaps":["General rules linking TBK1 subcellular localization to substrate selection not unified","How disease mutants shift the localization equilibrium in patient neurons unresolved"]},{"year":null,"claim":"How the dense, partly antagonistic PTM and scaffold network is integrated to select among TBK1's many substrate programs in a given cell state remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of how methylation, succinylation, tyrosine phosphorylation, and ubiquitination jointly set TBK1 activity","Substrate prioritization across immune, autophagic, survival, and metabolic outputs not reconciled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,3,7,8,11,18,19,20]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,7,11,18,19,30]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,8,31]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[7,18,37,38]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[40]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[14]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[50]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,3,17,22,25]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[7,10,11,20,39]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,6,26]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,13,19]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[8,49]}],"complexes":["IKK complex","TNFR1 signaling complex"],"partners":["STING1","OPTN","IRF3","RIPK1","NDP52","TBKBP1","MAVS","TAX1BP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UHD2","full_name":"Serine/threonine-protein kinase TBK1","aliases":["NF-kappa-B-activating kinase","T2K","TANK-binding kinase 1"],"length_aa":729,"mass_kda":83.6,"function":"Serine/threonine kinase that plays an essential role in regulating inflammatory responses to foreign agents (PubMed:10581243, PubMed:11839743, PubMed:12692549, PubMed:12702806, PubMed:14703513, PubMed:15367631, PubMed:15485837, PubMed:18583960, PubMed:21138416, PubMed:23453971, PubMed:23453972, PubMed:23746807, PubMed:25636800, PubMed:26611359, PubMed:32404352, PubMed:34363755, PubMed:32298923). Following activation of toll-like receptors by viral or bacterial components, associates with TRAF3 and TANK and phosphorylates interferon regulatory factors (IRFs) IRF3 and IRF7 as well as DDX3X (PubMed:12692549, PubMed:12702806, PubMed:14703513, PubMed:15367631, PubMed:18583960, PubMed:25636800). This activity allows subsequent homodimerization and nuclear translocation of the IRFs leading to transcriptional activation of pro-inflammatory and antiviral genes including IFNA and IFNB (PubMed:12702806, PubMed:15367631, PubMed:25636800, PubMed:32972995). In order to establish such an antiviral state, TBK1 form several different complexes whose composition depends on the type of cell and cellular stimuli (PubMed:23453971, PubMed:23453972, PubMed:23746807). Plays a key role in IRF3 activation: acts by first phosphorylating innate adapter proteins MAVS, STING1 and TICAM1 on their pLxIS motif, leading to recruitment of IRF3, thereby licensing IRF3 for phosphorylation by TBK1 (PubMed:25636800, PubMed:30842653, PubMed:37926288). Phosphorylated IRF3 dissociates from the adapter proteins, dimerizes, and then enters the nucleus to induce expression of interferons (PubMed:25636800). Thus, several scaffolding molecules including FADD, TRADD, MAVS, AZI2, TANK or TBKBP1/SINTBAD can be recruited to the TBK1-containing-complexes (PubMed:21931631). Under particular conditions, functions as a NF-kappa-B effector by phosphorylating NF-kappa-B inhibitor alpha/NFKBIA, IKBKB or RELA to translocate NF-Kappa-B to the nucleus (PubMed:10783893, PubMed:15489227). Restricts bacterial proliferation by phosphorylating the autophagy receptor OPTN/Optineurin on 'Ser-177', thus enhancing LC3 binding affinity and antibacterial autophagy (PubMed:21617041). Phosphorylates SMCR8 component of the C9orf72-SMCR8 complex, promoting autophagosome maturation (PubMed:27103069). Phosphorylates ATG8 proteins MAP1LC3C and GABARAPL2, thereby preventing their delipidation and premature removal from nascent autophagosomes (PubMed:31709703). Seems to play a role in energy balance regulation by sustaining a state of chronic, low-grade inflammation in obesity, which leads to a negative impact on insulin sensitivity (By similarity). Attenuates retroviral budding by phosphorylating the endosomal sorting complex required for transport-I (ESCRT-I) subunit VPS37C (PubMed:21270402). Phosphorylates Borna disease virus (BDV) P protein (PubMed:16155125). Plays an essential role in the TLR3- and IFN-dependent control of herpes virus HSV-1 and HSV-2 infections in the central nervous system (PubMed:22851595). Acts both as a positive and negative regulator of the mTORC1 complex, depending on the context: activates mTORC1 in response to growth factors by catalyzing phosphorylation of MTOR, while it limits the mTORC1 complex by promoting phosphorylation of RPTOR (PubMed:29150432, PubMed:31530866). Acts as a positive regulator of the mTORC2 complex by mediating phosphorylation of MTOR, leading to increased phosphorylation and activation of AKT1 (By similarity). Phosphorylates and activates AKT1 (PubMed:21464307). Involved in the regulation of TNF-induced RIPK1-mediated cell death, probably acting via CYLD phosphorylation that in turn controls RIPK1 ubiquitination status (PubMed:34363755). Also participates in the differentiation of T follicular regulatory cells together with the receptor ICOS (PubMed:27135603)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9UHD2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TBK1","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"MIF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TBK1","total_profiled":1310},"omim":[{"mim_id":"621413","title":"PHOSPHOLIPASE A2 INHIBITOR AND LY6/PLAUR DOMAIN-CONTAINING PROTEIN; PINLYP","url":"https://www.omim.org/entry/621413"},{"mim_id":"620995","title":"SET DOMAIN-CONTAINING PROTEIN 4; SETD4","url":"https://www.omim.org/entry/620995"},{"mim_id":"620880","title":"AUTOINFLAMMATION WITH ARTHRITIS AND VASCULITIS; AIARV","url":"https://www.omim.org/entry/620880"},{"mim_id":"620676","title":"COILED-COIL DOMAIN-CONTAINING PROTEIN 61; CCDC61","url":"https://www.omim.org/entry/620676"},{"mim_id":"619890","title":"GOLGI-ASSOCIATED RAB2 INTERACTOR 5A; GARIN5A","url":"https://www.omim.org/entry/619890"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TBK1"},"hgnc":{"alias_symbol":["NAK"],"prev_symbol":[]},"alphafold":{"accession":"Q9UHD2","domains":[{"cath_id":"3.30.200.20","chopping":"2-89","consensus_level":"medium","plddt":90.2116,"start":2,"end":89},{"cath_id":"3.10.20.90","chopping":"304-384","consensus_level":"medium","plddt":98.2072,"start":304,"end":384},{"cath_id":"1.20.1270.420","chopping":"409-661","consensus_level":"medium","plddt":92.2971,"start":409,"end":661}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHD2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHD2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UHD2-F1-predicted_aligned_error_v6.png","plddt_mean":89.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TBK1","jax_strain_url":"https://www.jax.org/strain/search?query=TBK1"},"sequence":{"accession":"Q9UHD2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UHD2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UHD2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UHD2"}},"corpus_meta":[{"pmid":"19847166","id":"PMC_19847166","title":"Systematic 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IκB degradation and NF-κB activation; endogenous NAK is activated by phorbol esters and growth factors, and catalytically inactive NAK specifically inhibits NF-κB activation by PKCε.\",\n      \"method\": \"In vitro kinase assay, dominant-negative overexpression, IκB degradation assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay demonstrating direct phosphorylation, dominant-negative functional validation, replicated across multiple stimuli\",\n      \"pmids\": [\"10783893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TBK1 and IKKε are essential components of the IRF3 signaling pathway downstream of TLR3 and viral dsRNA; both kinases phosphorylate IRF3 to drive type I interferon induction.\",\n      \"method\": \"Dominant-negative and siRNA knockdown, reporter assays, phosphorylation assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function and dominant-negative approaches with IRF3 phosphorylation readout, widely replicated\",\n      \"pmids\": [\"12692549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TBK1 is selectively essential in KRAS-mutant cancer cells; TBK1 activates NF-κB anti-apoptotic signals involving c-Rel and BCL-XL that are required for survival in these cells.\",\n      \"method\": \"Systematic RNAi screen, shRNA knockdown with apoptosis readout, NF-κB pathway analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic RNAi screen with mechanistic follow-up across multiple cell lines, widely replicated\",\n      \"pmids\": [\"19847196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"STING functions as a scaffold protein that binds both TBK1 and IRF3; the C-terminal region of STING is necessary and sufficient to activate TBK1 and promote IRF3 phosphorylation; STING mutations that selectively disrupt IRF3 binding abrogate IRF3 phosphorylation without impairing TBK1 activation.\",\n      \"method\": \"In vitro reconstitution system, mutagenesis, co-immunoprecipitation\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis validating interaction mode and functional consequences\",\n      \"pmids\": [\"22394562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Loss-of-function mutations in TBK1 cause familial ALS and frontotemporal dementia via haploinsufficiency; C-terminal coiled-coil domain (CCD2) mutations disrupt TBK1 interaction with the adaptor protein optineurin.\",\n      \"method\": \"Exome sequencing of ALS/FTD patients, in vitro expression and binding assays, linkage analysis\",\n      \"journal\": \"Nature neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic and in vitro functional validation across multiple families and mutation types\",\n      \"pmids\": [\"25803835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TBK1 is an endogenous inhibitor of RIPK1 kinase; embryonic lethality of Tbk1-/- mice is rescued by inactivating RIPK1 kinase activity, establishing TBK1 as a suppressor of RIPK1-driven apoptosis and inflammation.\",\n      \"method\": \"Genetic epistasis (Tbk1-/- × RIPK1 kinase-dead mice), embryonic lethality rescue assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis with complete rescue, multiple phenotypic readouts\",\n      \"pmids\": [\"30146158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TBK1 and IKKε are recruited to the TNFR1 signaling complex via LUBAC-dependent linear ubiquitin; they phosphorylate RIPK1 in the TNFR1-SC to prevent TNF-induced cell death. NEMO mediates recruitment via adaptors TANK and NAP1.\",\n      \"method\": \"Co-immunoprecipitation of signaling complex, genetic knockout, in vivo TNF lethal shock model\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple KO cell lines, in vivo rescue experiment\",\n      \"pmids\": [\"30420664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TBK1 directly phosphorylates RAB7A at Ser72 upon mitochondrial depolarization to promote mitophagy; this modification reduces RAB GDI association and increases FLCN-FNIP1 recruitment, and non-phosphorylatable RAB7A impairs mitophagy and ATG9A vesicle recruitment to damaged mitochondria.\",\n      \"method\": \"Quantitative phosphoproteomics, in vitro kinase assay, interaction proteomics with phosphomutants, mitophagy imaging\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, phosphoproteomics, and functional mutant analysis in single study\",\n      \"pmids\": [\"30627666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TBK1 directly inhibits AMPK to repress cellular respiration and increase energy storage in adipocytes; conversely, AMPK can increase TBK1 activity via its downstream target ULK1; TBK1 also suppresses inflammation by phosphorylating and inducing degradation of the IKK kinase NIK.\",\n      \"method\": \"Adipocyte-specific TBK1 knockout mice, in vitro kinase assays, phosphorylation and degradation assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with metabolic phenotype plus in vitro kinase assays for multiple substrates\",\n      \"pmids\": [\"29425491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structure of TBK1 in complex with cGAMP-bound STING reveals that the STING C-terminal tail inserts as a β-strand into a groove between the TBK1 kinase domain and SDD of the second subunit; phosphorylation site Ser366 cannot reach the active site of bound TBK1, indicating that STING phosphorylation requires oligomerization of both proteins.\",\n      \"method\": \"Cryo-electron microscopy structure determination, mutational analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with mutational validation, mechanistic model experimentally confirmed\",\n      \"pmids\": [\"30842653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NDP52 initiates selective autophagy by localizing and activating the ULK1 complex in a TBK1-dependent manner; ectopic placement of NDP52 on organelles is sufficient to induce selective autophagy; tethering ULK1 directly to cargo bypasses both autophagy receptors and TBK1 requirement.\",\n      \"method\": \"Chemically inducible dimerization (CID) assays, CRISPR KO lines, live-cell imaging\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic CID approach with CRISPR KO controls and rescue experiments\",\n      \"pmids\": [\"30853401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TBK1 phosphorylates Syntaxin 17 at Ser202; phospho-Stx17 translocates from the Golgi to ATG13+FIP200+ pre-autophagosomal structures to control autophagy initiation; TBK1 or Stx17 KO blocks ATG13/FIP200 puncta formation and reduces ULK1 complex assembly.\",\n      \"method\": \"Phosphorylation mapping, in vitro kinase assay, CRISPR KO, live-cell imaging, co-immunoprecipitation\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay identifying substrate site, KO with defined phenotypic rescue, localization experiments\",\n      \"pmids\": [\"30827897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TBK1 is activated by VHL loss or hypoxia in cancer cells; VHL-mediated hydroxylation of TBK1 at Pro48 promotes PPM1B phosphatase binding and TBK1 dephosphorylation; TBK1 phosphorylates p62/SQSTM1 at Ser366, which is essential for p62 stability and cancer cell proliferation.\",\n      \"method\": \"Genetic ablation, pharmacological inhibition, PROTAC degradation, phosphorylation assays, xenograft model\",\n      \"journal\": \"Cancer discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic and pharmacological tools, mechanistic pathway mapping, in vivo validation\",\n      \"pmids\": [\"31810986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TBKBP1 recruits TBK1 to PKCθ via scaffold protein CARD10; PKCθ phosphorylates TBK1 at Ser716, a step required for TBK1 activation by growth factors but not innate immune stimuli; the TBK1-TBKBP1 axis mediates mTORC1 activation, oncogenesis, and PD-L1 induction.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assays, conditional knockout mice, lung cancer model\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, phosphosite identification, conditional KO with tumor phenotype, multiple orthogonal approaches\",\n      \"pmids\": [\"31792381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PINK1/Parkin-mediated TBK1 activation at damaged mitochondria during mitophagy sequesters TBK1 from its physiological role at centrosomes, causing a block in mitosis.\",\n      \"method\": \"Genetic interaction screen, live-cell imaging, mitosis assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic interaction with mitosis readout and TBK1 localization data, single lab\",\n      \"pmids\": [\"31577952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Axl receptor tyrosine kinase induces TBK1 activity through a Ras-RalB-dependent mechanism to drive epithelial-mesenchymal transition in KRAS-mutant pancreatic cancer.\",\n      \"method\": \"Genetic knockdown, pharmacological inhibition, EMT marker analysis, invasion/metastasis assays\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with defined pathway placement and functional EMT readout, single lab\",\n      \"pmids\": [\"30938713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TBK1 promotes Raptor Ser877 phosphorylation in vitro and in cells; this modification limits mTORC1 activity, as a non-phosphorylatable Raptor S877A mutant increases mTORC1 signaling.\",\n      \"method\": \"In vitro kinase assay coupled with mass spectrometry, phosphosite mutagenesis, mTORC1 activity assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with phosphosite identification and functional mutagenesis, single lab\",\n      \"pmids\": [\"31530866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TBK1 is redundant with IKKε for STING-induced NF-κB responses in myeloid cells; IRF3 activation is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition.\",\n      \"method\": \"Genetic KO (single and double), pharmacological inhibition, reporter assays, in vivo validation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic and pharmacological approaches, human and mouse cells, and in vivo validation distinguishing IRF3 vs NF-κB dependency\",\n      \"pmids\": [\"32268090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TBK1 directly phosphorylates DRP1/DNM1L upon RLR-MAVS signaling activation; this phosphorylation prevents DRP1 oligomerization and mitochondrial fragmentation, and is required for assembly of large MAVS aggregates and efficient antiviral immunity.\",\n      \"method\": \"In vitro kinase assay, knockin (KI) strategy mimicking TBK1-DRP1 signaling, co-immunoprecipitation, mitochondrial morphology assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, KI dominant-negative phenotype, KI rescue, multiple orthogonal methods in one study\",\n      \"pmids\": [\"33171123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TBK1 directly interacts with mTORC2 and phosphorylates mTOR at Ser2159 within mTORC2; this increases mTORC2 intrinsic catalytic activity and Akt phosphorylation; TBK1-mTOR S2159 phosphorylation also promotes mTORC1 signaling.\",\n      \"method\": \"Co-immunoprecipitation of mTORC2, in vitro kinase assay, Mtor S2159A knock-in MEFs and mice\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted kinase assay, co-IP, and knock-in allele validating the TBK1-mTOR axis\",\n      \"pmids\": [\"34245780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TBK1 directly phosphorylates the autophagy adaptor optineurin (OPTN) at Ser177 after Salmonella invasion, enhancing OPTN interaction with Atg8 family proteins and recruiting the autophagic machinery to intracellular bacteria for xenophagy.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, bacterial clearance assays\",\n      \"journal\": \"Science signaling (review citing primary data); primary mechanism originally described 2011\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with phosphosite identification, supported by multiple labs\",\n      \"pmids\": [\"21868362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM23 undergoes K27-linked auto-ubiquitination on its ARF domain, enabling GTP hydrolysis activity and facilitating TBK1 dimerization; active TBK1 then phosphorylates p62 to promote selective autophagy.\",\n      \"method\": \"RNAi screen, mutagenesis of ubiquitination sites, TBK1 dimerization assay, p62 phosphorylation assay\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic dissection with mutagenesis of ubiquitination site and functional readouts, multiple viral pathogens tested\",\n      \"pmids\": [\"28871090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MAVS recruits TBK1/IKKε via TRAFs; TRAFs interact directly with the SDD domain of TBK1/IKKε via their coiled-coil domain; TRAF E3 ligase activity synthesizes ubiquitin chains to activate NEMO; NEMO-activated IKKα/β then further phosphorylate and activate TBK1/IKKε.\",\n      \"method\": \"TRAF quadruple knockout cells, co-immunoprecipitation, ubiquitination assays, domain mapping\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quadruple KO cells with complete loss of viral response, Co-IP domain mapping, multiple orthogonal methods\",\n      \"pmids\": [\"29125880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"USP1-UAF1 deubiquitinase complex binds TBK1, removes K48-linked polyubiquitination, and prevents TBK1 proteasomal degradation, thereby stabilizing TBK1 and enhancing antiviral IFN responses.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, USP1-UAF1 inhibitor ML323, in vitro and in vivo viral infection\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, pharmacological inhibitor, single lab\",\n      \"pmids\": [\"29138248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TBK1 controls AKT ubiquitination and degradation to regulate T-cell activation and migration; T-cell-specific TBK1 ablation deregulates AKT-mTORC1 signaling, enhancing T-cell activation and impairing effector T-cell egress from draining lymph nodes.\",\n      \"method\": \"T-cell-conditional TBK1 knockout mice, AKT ubiquitination assay, mTORC1 activity assays, EAE model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined cellular phenotype and mechanistic follow-up, single lab\",\n      \"pmids\": [\"25606824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBK1 is recruited to STING via the STING C-terminal tail; STING S365A mutation (abolishing IRF3 binding) allows TBK1 recruitment and NF-κB activation but not IRF3 activation; disruption of TBK1 binding to STING (L373A or C-terminal truncation) prevents both pathways and rescues autoinflammatory disease in DNase II-/- mice.\",\n      \"method\": \"Genetic knockin mice (STING S365A, L373A, C-terminal truncation), disease rescue experiments in DNase II-/- mice, cytokine neutralization\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic knockin alleles with in vivo disease rescue, orthogonal experiments dissecting IRF3 vs NF-κB pathway\",\n      \"pmids\": [\"34901991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBK1 loss in humans causes autoinflammation driven by TNF-induced RIPK1-dependent cell death (necroptosis); patient-derived fibroblasts with homozygous TBK1 loss show elevated necroptosis in vitro; IFN-I induction via RIG-I/MDA5 is hypomorphic but sufficient in the absence of TBK1.\",\n      \"method\": \"Patient-derived fibroblasts, necroptosis assays, ex vivo cytokine profiling, anti-TNF treatment\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human genetic loss-of-function with functional cellular assays and in vivo treatment response\",\n      \"pmids\": [\"34363755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBK1 phosphorylates full-length and N-terminal huntingtin at Ser13/Ser16 in vitro and at Ser13 in cells and in vivo; TBK1-mediated phosphorylation inhibits mutant HTT aggregation and increases autophagic clearance, reducing cytotoxicity.\",\n      \"method\": \"In vitro kinase assay, cell-based phosphorylation assay, aggregation assays, autophagy flux assay, C. elegans model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, in vivo validation, multiple model systems\",\n      \"pmids\": [\"32757223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBK1 interacts with tau in human AD brain tissue and phosphorylates tau in vitro at multiple sites; TBK1 activation in Drosophila tauopathy model triggers tau hyperphosphorylation and enhanced neurodegeneration, while TBK1 ortholog knockdown suppresses tau toxicity.\",\n      \"method\": \"Immunoaffinity enrichment + mass spectrometry from human brain, in vitro kinase assay + MS, Drosophila genetic epistasis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with MS mapping, Drosophila genetic validation, human brain Co-IP, single lab\",\n      \"pmids\": [\"33965374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NEDD4 E3 ligase catalyzes K27-linked polyubiquitination of TBK1 at K344, targeting TBK1 for NDP52-mediated selective autophagic degradation; this limits TBK1 protein levels at late stages of viral infection to prevent excessive immune response.\",\n      \"method\": \"Ubiquitination assays (K27 linkage), co-immunoprecipitation, autophagy flux assays, viral infection\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific ubiquitin linkage and lysine site identified, autophagic degradation pathway confirmed, single lab\",\n      \"pmids\": [\"34257412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PRMT1 directly interacts with TBK1 and catalyzes asymmetric arginine methylation at R54, R134, and R228; this modification enhances TBK1 oligomerization and subsequent Ser172 autophosphorylation and downstream type I IFN production.\",\n      \"method\": \"Co-immunoprecipitation, in vitro methylation assay, site-directed mutagenesis, oligomerization assay, myeloid-specific Prmt1 KO mice\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with site mutagenesis, functional KO validation in vivo, mechanistic link to oligomerization confirmed\",\n      \"pmids\": [\"34551290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TBK1 interacts with NLRP3 and acts as a phospho-switch to limit NLRP3 inflammasome activation; TBK1 and IKKε together constitute an OFF switch for NLRP3 via phosphorylation at a site distinct from Ser3; simultaneous deletion of both kinases is required to fully release NLRP3 activation.\",\n      \"method\": \"Single and double KO macrophages, pharmacological inhibition, NLRP3 mutagenesis, co-immunoprecipitation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological approaches, NLRP3 mutagenesis, mechanistic interaction confirmed by Co-IP, single lab\",\n      \"pmids\": [\"34518217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TBK1 phosphorylates METTL3 at Ser67 upon viral infection; phospho-METTL3 interacts with the translational complex to enhance protein translation and m6A modification, stabilizing IRF3 mRNA to facilitate antiviral responses.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation mapping, METTL3 KO cells and Mettl3 conditional KO mice, viral infection models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation site identified, functional KO validation with viral phenotype, mechanistic link to translational complex by Co-IP, single lab\",\n      \"pmids\": [\"35172162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TBC1D9, a Ca2+-binding protein, mediates TBK1 activation during xenophagy and mitophagy; upon bacterial infection, Ca2+ increase promotes TBC1D9 binding to ubiquitin-coated bacteria via its UBR and Ca2+-binding motif, and TBC1D9 KO suppresses TBK1 activation and ULK1 complex recruitment.\",\n      \"method\": \"Co-immunoprecipitation, TBC1D9 knockout cells, Ca2+ chelation experiments, live-cell imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with defined phenotype, Co-IP, Ca2+ perturbation experiments, single lab\",\n      \"pmids\": [\"32034138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TBK1 controls an antiapoptotic response downstream of TNF by triggering a specific RelA/p65 phosphorylation event, resulting in PAI-2 expression; PAI-2 limits caspase-3 activation by stabilizing transglutaminase 2, which cross-links and inactivates procaspase-3.\",\n      \"method\": \"Genetic studies in Tg2-/- mice, RelA phosphorylation mapping, caspase-3 activation assay, TNF liver injury models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO model with defined pathway, mechanistic assays linking TBK1→RelA phosphorylation→PAI-2→TG2→procaspase-3 axis\",\n      \"pmids\": [\"22203995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288, and Ser293, converting it into an active E3 ubiquitin ligase; endogenous Pellino 1 E3 ligase activity is activated in LPS/poly(I:C)-stimulated macrophages in a TBK1/IKKε-dependent manner.\",\n      \"method\": \"In vitro kinase assay, two-hybrid screen, E3 ligase activity assay, IKKε/TBK1 pharmacological inhibition in macrophages\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with phosphosite mapping, functional E3 ligase activity measured, single lab\",\n      \"pmids\": [\"21204785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALS-associated TBK1 missense mutations (G217R, R357X) abolish TBK1 ability to phosphorylate IRF3 and optineurin, impair TBK1 homodimerization (G217R), and reduce optineurin binding; proportion of active (phosphorylated) TBK1 is reduced in lymphoblastoid cell lines from mutation carriers.\",\n      \"method\": \"In vitro kinase assay with IRF3 and OPTN substrates, dimerization assay, patient-derived cell lines\",\n      \"journal\": \"Neurobiology of aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assays with substrates, patient-derived cells, single lab\",\n      \"pmids\": [\"30033073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Optineurin provides a unique platform for TBK1 activation at mitophagy contact sites between damaged mitochondria and autophagosome formation sites; both OPTN-ubiquitin and OPTN-PAS interaction axes facilitate OPTN-TBK1 complex assembly, initiating a positive feedback loop of TBK1 hetero-autophosphorylation.\",\n      \"method\": \"Live-cell imaging, engineered monobodies blocking OPTN, Co-IP, mitophagy flux assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — monobody functional inhibition, live imaging, Co-IP, and defined mechanistic feedback loop, multiple orthogonal approaches\",\n      \"pmids\": [\"38287189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALS-linked TBK1 mutations that abolish both dimerization and kinase activity are most disruptive to mitophagy; TBK1 recruitment and OPTN phosphorylation at S177 are each necessary for engulfment of damaged mitochondria; ULK1 activity also contributes to OPTN S177 phosphorylation.\",\n      \"method\": \"Quantitative imaging of mitophagy in cells expressing ALS mutants, kinase assays, ULK1 inhibition\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutant analysis with imaging readout, pharmacological and genetic dissection, single lab\",\n      \"pmids\": [\"34099552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NAP1 and SINTBAD, two TBK1 adaptors, restrict OPTN-driven mitophagy initiation by competing with OPTN for TBK1 binding; they promote NDP52-driven mitophagy progression by recruiting TBK1 to NDP52 and stabilizing its interaction with FIP200; OPTN is the primary recruiter of TBK1 during mitophagy initiation.\",\n      \"method\": \"Co-immunoprecipitation, competitive binding assays, knockout cells, mitophagy flux assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple KO lines, competition binding assay, mechanistic model validated\",\n      \"pmids\": [\"38918639\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A lysosome-localized pool of TBK1 is recruited upon elevated amino acid levels and phosphorylates Rab7 at Ser72 to relieve Rab7-dependent inhibition of mTORC1; an ALS-FTD-associated TBK1 mutant (E696K) constitutively accumulates at lysosomes, causing elevated Rab7 phosphorylation and increased mTORC1 activation.\",\n      \"method\": \"Subcellular fractionation, co-immunoprecipitation, phosphorylation assays, mTORC1 activity assays, disease mutant analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lysosomal localization experimentally defined, substrate phosphorylation and mTORC1 consequence validated, disease mutant mechanism identified\",\n      \"pmids\": [\"39103493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FIP200 controls the TBK1 activation threshold at SQSTM1/p62-positive condensates; TBK1 is recruited to p62 aggregates via TAX1BP1 and phosphorylates p62 at Ser403; removal of FIP200 or disruption of FIP200-TAX1BP1 interaction strongly increases TBK1 activation at these condensates.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assays, FIP200 KO cells, aggregate imaging\",\n      \"journal\": \"Scientific reports (2021)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, KO with defined activation phenotype, p62 phosphosite assay, single lab\",\n      \"pmids\": [\"34226595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PTK2B directly phosphorylates TBK1 at Tyr591 to increase TBK1 oligomerization and activation; PTK2B also promotes STING oligomerization in a kinase-independent manner; Ptk2b-deficient mice are more susceptible to viral infection.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, mutagenesis of Tyr591, Ptk2b KO mice, viral infection\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with phosphosite identification, in vivo KO validation, single lab\",\n      \"pmids\": [\"37989995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SIRT5 desuccinylates TBK1 at Lys137, leading to TBK1 dephosphorylation and suppression of downstream inflammatory signaling; reduced SIRT5 in aged primate skeletal muscle increases TBK1 succinylation and promotes inflammation.\",\n      \"method\": \"In vitro succinylation/desuccinylation assays, site mutagenesis (K137), SIRT5 KO/overexpression in myotubes, primate muscle tissue analysis\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with site identification, cellular KO validation, single lab\",\n      \"pmids\": [\"40087407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SIRT5 desuccinylates TBK1 at K38, K154, and K692 in macrophages; succinylation at these sites inhibits TBK1 binding to IRF3 and TRAF2, suppressing inflammatory signaling; macrophage adoptive transfer of succinylation-resistant TBK1-2KR (K154/692R) exacerbates sepsis-induced lung injury.\",\n      \"method\": \"Succinylation site mapping, mutagenesis, Co-IP, macrophage adoptive transfer, sepsis model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis, Co-IP disruption, in vivo adoptive transfer, single lab\",\n      \"pmids\": [\"39673708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Myeloid cell TBK1 suppresses NF-κB and MAP kinase signaling pathways in macrophages to attenuate induction of proinflammatory cytokines, particularly IL-1β; myeloid-specific TBK1 KO causes spontaneous adipose inflammation and exacerbates colitis, and IL-1R1 ablation eliminates these inflammatory symptoms.\",\n      \"method\": \"Myeloid-conditional TBK1 KO mice, NF-κB/MAPK signaling assays, cytokine measurements, IL-1R1 double KO\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined phenotype and epistasis via IL-1R1 KO rescue, single lab\",\n      \"pmids\": [\"35074921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CDK4/6 phosphorylates TBK1 at Ser527 to inactivate STING signaling in prostate cancer; CDK4/6-mediated phosphorylation of RB1 at Ser249/Thr252 also induces RB1-TBK1 interaction, reducing TBK1 Ser172 phosphorylation and suppressing the STING pathway.\",\n      \"method\": \"Phosphorylation assays, Co-immunoprecipitation, CDK4/6 inhibitor treatment, prostate cancer cell models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphosite identification, Co-IP, CDK4/6 inhibitor treatment, both RB1-dependent and independent pathways tested, single lab\",\n      \"pmids\": [\"38861362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Tiliroside directly binds TBK1 (confirmed by molecular docking and biolayer interferometry) and inhibits its enzymatic activity, reducing p62 Ser349 phosphorylation and Keap1-Nrf2 axis activity to induce ferroptosis in hepatocellular carcinoma cells.\",\n      \"method\": \"Biolayer interferometry binding assay, enzymatic activity assay, Co-IP, xenograft tumor models\",\n      \"journal\": \"Phytomedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding confirmed by BLI, enzymatic inhibition assay, in vivo validation, single lab\",\n      \"pmids\": [\"36657316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NLRC4 facilitates the interaction between TBK1 and the E3 ubiquitin ligase CBL to enhance K63-linked polyubiquitination of TBK1, promoting cGAS-STING pathway activation and TBK1 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays (K63-specific), NLRC4 KO cells and Nlrc4-/- mice, viral infection\",\n      \"journal\": \"Journal of medical virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, K63-specific ubiquitination, genetic KO mice with viral phenotype, single lab\",\n      \"pmids\": [\"37537877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of TBK1 kinase function protects against diet-induced metabolic dysfunction through loss of an inhibitory interaction between TBK1 and the insulin receptor.\",\n      \"method\": \"Global Tbk1 kinase-dead mutant mice, metabolic profiling, insulin receptor interaction assay\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mouse model with defined metabolic phenotype and mechanistic interaction assay, single lab\",\n      \"pmids\": [\"29935921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The MAM-specific E3 ubiquitin ligase AMFR ubiquitinates nascent proteins at the MAM to activate TBK1; TBK1 activation at the MAM results in ribosomal protein degradation under proteostatic stress; MAM or TBK1 deficiency impairs this response and increases cellular vulnerability.\",\n      \"method\": \"MAM fractionation, TBK1 KO, AMFR knockdown, ribosomal degradation assay, in vitro and in vivo models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation with functional consequence, KO phenotype, E3 ligase identification, single lab\",\n      \"pmids\": [\"37967220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Vimentin interacts with TBK1 and IKKε to disrupt TBK1-IRF3 and IKKε-IRF3 interactions, inhibiting IRF3 phosphorylation and nuclear translocation and suppressing type I IFN production.\",\n      \"method\": \"Co-immunoprecipitation, vimentin KO mice, overexpression, IRF3 localization assay\",\n      \"journal\": \"Cell reports (2022)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP showing disruption of TBK1-IRF3 interaction, KO mice with in vivo viral phenotype, single lab\",\n      \"pmids\": [\"36223739\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TBK1 is a serine/threonine kinase that functions as a central node in innate immunity, autophagy, and cell survival: it is activated by scaffolds including STING (via its C-terminal tail), MAVS/TRAFs, and optineurin at mitochondria, whereupon TBK1 phosphorylates IRF3/IRF7 to induce type I interferons, directly phosphorylates RIPK1 to suppress cell death, phosphorylates autophagy adaptors (optineurin S177, p62 S403, Stx17 S202) and organelle regulators (RAB7A S72, DRP1) to control selective autophagy and mitochondrial dynamics, and phosphorylates multiple metabolic targets (AMPK, NIK, mTOR S2159, Raptor S877) to modulate energy homeostasis and NF-κB signaling; its activity is regulated by post-translational modifications including K48/K63/K27-linked ubiquitination, arginine methylation (PRMT1 at R54/R134/R228), tyrosine phosphorylation (PTK2B at Y591), and lysine succinylation (SIRT5 at K137/K154/K692), with haploinsufficiency causing ALS/FTD and gain-of-function duplications causing normal-tension glaucoma.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TBK1 is a serine/threonine kinase that functions as a central signaling hub coupling innate immune sensing, selective autophagy, cell-death suppression, and metabolic control [#1, #5, #8]. In antiviral signaling, TBK1 is recruited to upstream scaffolds—STING via its C-terminal tail, and MAVS via TRAFs that engage the TBK1 SDD domain—whereupon it phosphorylates IRF3 to drive type I interferon induction, while STING-dependent NF-\\u03baB output is comparatively kinase-independent and redundant with IKK\\u03b5 [#1, #3, #17, #22, #25]. Cryo-EM of the cGAMP-STING\\u2013TBK1 complex established that substrate IRF3/STING phosphorylation requires trans-oligomerization, since the STING phosphosite cannot reach the active site of the TBK1 subunit that binds it [#9]. TBK1 enforces cell survival by directly phosphorylating RIPK1 within the TNFR1 complex—to which it is recruited through LUBAC-generated linear ubiquitin and the NEMO/TANK/NAP1 adaptors—to restrain RIPK1-driven apoptosis and necroptosis; this activity underlies the embryonic lethality of Tbk1 loss and human TBK1-deficiency autoinflammation [#5, #6, #26]. In selective autophagy, TBK1 is activated on cargo by adaptor platforms (optineurin, NDP52, TAX1BP1, with NAP1/SINTBAD tuning recruitment) and phosphorylates autophagy regulators including optineurin Ser177, p62/SQSTM1, Syntaxin 17 Ser202, and RAB7A Ser72 to control xenophagy, mitophagy, and autophagosome initiation [#7, #10, #11, #20, #39]. TBK1 additionally phosphorylates metabolic and mTOR-axis targets—AMPK, NIK, mTOR Ser2159, and Raptor Ser877—to govern energy homeostasis and growth signaling [#8, #16, #19]. Its activity is set by an extensive PTM code: activating arginine methylation by PRMT1 and tyrosine phosphorylation by PTK2B promote oligomerization and Ser172 autophosphorylation, whereas SIRT5-reversible succinylation and stabilizing/destabilizing ubiquitination further modulate the pool of active kinase [#23, #29, #30, #42, #44]. Genetically, TBK1 haploinsufficiency and kinase/dimerization-disrupting mutations cause familial ALS and frontotemporal dementia, in part by impairing optineurin binding and mitophagy [#4, #36, #38].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established TBK1 as an IKK-activating kinase, placing it upstream of NF-\\u03baB before its interferon role was known.\",\n      \"evidence\": \"In vitro kinase assay and dominant-negative overexpression with I\\u03baB degradation readout\",\n      \"pmids\": [\"10783893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define physiological stimuli or scaffolds linking TBK1 to IKK in vivo\", \"Direct vs indirect IKK\\u03b2 phosphorylation under endogenous conditions not resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified the canonical antiviral function: TBK1 (with IKK\\u03b5) phosphorylates IRF3 downstream of dsRNA sensing to induce type I interferon.\",\n      \"evidence\": \"Dominant-negative/siRNA knockdown with IRF3 phosphorylation and interferon reporter readouts\",\n      \"pmids\": [\"12692549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream scaffold recruiting TBK1 to TLR3 not defined\", \"Division of labor between TBK1 and IKK\\u03b5 left open\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed a non-immune survival role: TBK1 is selectively required by KRAS-mutant cancer cells through NF-\\u03baB anti-apoptotic signaling.\",\n      \"evidence\": \"Systematic RNAi screen with shRNA apoptosis follow-up across cell lines\",\n      \"pmids\": [\"19847196\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct TBK1 substrates driving the survival signal not identified\", \"Mechanism of selective dependence on mutant KRAS unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined STING as the scaffold that physically couples TBK1 to its substrate IRF3, explaining substrate selection in cytosolic DNA sensing.\",\n      \"evidence\": \"In vitro reconstitution, mutagenesis, and co-immunoprecipitation separating TBK1 activation from IRF3 binding\",\n      \"pmids\": [\"22394562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of activation not yet resolved\", \"Whether STING also gates NF-\\u03baB output unaddressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected TBK1 to human neurodegeneration, showing loss-of-function mutations cause ALS/FTD via haploinsufficiency and disrupted optineurin binding.\",\n      \"evidence\": \"Exome sequencing of ALS/FTD families with in vitro binding assays\",\n      \"pmids\": [\"25803835\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which TBK1 substrate(s) drive neurodegeneration not established\", \"Cell-type-specific basis of motor neuron vulnerability unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established the essential survival mechanism: TBK1 directly suppresses RIPK1 kinase to prevent apoptosis/inflammation, explaining Tbk1-null embryonic lethality.\",\n      \"evidence\": \"Genetic epistasis (Tbk1-/- \\u00d7 RIPK1 kinase-dead), TNFR1 complex Co-IP, and in vivo TNF shock models\",\n      \"pmids\": [\"30146158\", \"30420664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RIPK1 phosphosite(s) targeted by TBK1 not fully mapped here\", \"Relative contribution of IKK\\u03b5 in this complex left partial\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Expanded TBK1 into metabolic and autophagy substrate space, identifying AMPK/NIK inhibition and direct RAB7A Ser72 phosphorylation for mitophagy.\",\n      \"evidence\": \"Conditional adipocyte KO mice, phosphoproteomics, in vitro kinase assays, and mitophagy imaging with phosphomutants\",\n      \"pmids\": [\"29425491\", \"30627666\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TBK1 is targeted to specific metabolic vs autophagic substrates not unified\", \"In vivo significance of RAB7A Ser72 for organismal mitophagy unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Solved the activation mechanism structurally: STING tail insertion and the requirement for trans-oligomerization for substrate phosphorylation.\",\n      \"evidence\": \"Cryo-EM of cGAMP-STING\\u2013TBK1 complex with mutational validation\",\n      \"pmids\": [\"30842653\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for non-STING scaffold activation not addressed\", \"How oligomerization is achieved at other organelles unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined TBK1's role in autophagy initiation through autophagy receptors and substrate phosphorylation (Stx17 Ser202, p62) that recruit the ULK1 machinery.\",\n      \"evidence\": \"Chemically inducible dimerization, CRISPR KO, in vitro kinase assays, and live-cell imaging\",\n      \"pmids\": [\"30853401\", \"30827897\", \"28871090\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy among multiple autophagy substrates not resolved\", \"Whether all receptors use the same TBK1 activation route unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mapped scaffold- and PTM-controlled activation modes distinguishing growth-factor from immune signaling, and linked TBK1 to mTOR and cancer phenotypes.\",\n      \"evidence\": \"Reciprocal Co-IP, phosphosite identification (TBK1 Ser716, Raptor Ser877), conditional KO tumor models, and VHL/PPM1B regulation\",\n      \"pmids\": [\"31792381\", \"31810986\", \"31530866\", \"30938713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How distinct upstream adaptors select downstream substrate programs not integrated\", \"Some axes (Axl-RalB, Raptor) rest on single-lab evidence\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Separated IRF3 (kinase-dependent) from NF-\\u03baB (kinase-tolerant, IKK\\u03b5-redundant) STING outputs and added DRP1/mitochondrial dynamics control.\",\n      \"evidence\": \"Single/double KO, pharmacological inhibition, in vivo validation, and DRP1 knock-in mimics with mitochondrial morphology assays\",\n      \"pmids\": [\"32268090\", \"33171123\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase-independent NF-\\u03baB scaffolding function of TBK1 not mechanistically defined\", \"How DRP1 phosphorylation integrates with antiviral MAVS aggregation in vivo unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved scaffold-encoded pathway choice in vivo and established that human TBK1 loss drives TNF/RIPK1-dependent autoinflammation treatable by anti-TNF.\",\n      \"evidence\": \"STING knock-in alleles with disease rescue in DNase II-/- mice; patient fibroblast necroptosis assays and anti-TNF treatment\",\n      \"pmids\": [\"34901991\", \"34363755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why human TBK1 loss tolerates interferon hypomorphism but not cell-death control not fully explained\", \"Tissue-specific thresholds for autoinflammation unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the optineurin-centered platform and PTM code (PRMT1 methylation) that drive TBK1 oligomerization and feedback autoactivation during mitophagy and interferon responses.\",\n      \"evidence\": \"Live-cell imaging, monobody inhibition, in vitro methylation with site mutagenesis, oligomerization assays, and myeloid Prmt1 KO mice\",\n      \"pmids\": [\"38287189\", \"34099552\", \"34551290\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative interplay between activating and inhibitory PTMs not modeled\", \"How ALS mutations perturb the OPTN feedback loop in neurons untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Refined adaptor logic and organelle-localized pools controlling TBK1 substrate choice, including a lysosomal pool acting on Rab7\\u2013mTORC1 with disease-mutant consequences.\",\n      \"evidence\": \"Competitive binding/Co-IP for NAP1/SINTBAD vs OPTN, KO mitophagy assays, subcellular fractionation, and ALS-FTD mutant (E696K) analysis\",\n      \"pmids\": [\"38918639\", \"39103493\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"General rules linking TBK1 subcellular localization to substrate selection not unified\", \"How disease mutants shift the localization equilibrium in patient neurons unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the dense, partly antagonistic PTM and scaffold network is integrated to select among TBK1's many substrate programs in a given cell state remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of how methylation, succinylation, tyrosine phosphorylation, and ubiquitination jointly set TBK1 activity\", \"Substrate prioritization across immune, autophagic, survival, and metabolic outputs not reconciled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 3, 7, 8, 11, 18, 19, 20]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 7, 11, 18, 19, 30]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 8, 31]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [7, 18, 37, 38]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [40]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [50]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 3, 17, 22, 25]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7, 10, 11, 20, 39]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 6, 26]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 13, 19]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [8, 49]}\n    ],\n    \"complexes\": [\"IKK complex\", \"TNFR1 signaling complex\"],\n    \"partners\": [\"STING1\", \"OPTN\", \"IRF3\", \"RIPK1\", \"NDP52\", \"TBKBP1\", \"MAVS\", \"TAX1BP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}