id: Q9H6S1 gene_symbol: AZI2 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: >- AZI2 (5-azacytidine-induced protein 2), better known as NAP1 (NAK-associated protein 1; also TBKBP2, TILP), is a cytoplasmic adapter protein that binds and positively regulates the IkappaB kinase (IKK)-related serine/threonine kinases TBK1 (NAK) and IKBKE (IKKepsilon). The protein has an N-terminal homodimerization region and two coiled-coil segments, and a C-terminal TBK1/IKBKE-binding domain (the Pfam TBD module, residues ~216-257) shared with the related adapters TANK and TBKBP1/SINTBAD. By binding TBK1, NAP1 promotes TBK1 activation and oligomerization and thereby couples upstream innate-immune signals to TBK1 kinase output. NAP1 functions as a shared adaptor in type I interferon induction downstream of both the endosomal Toll-like receptor 3 (via the TRIF/TICAM1 adaptor) and the cytoplasmic RIG-I/MDA5 RNA-sensing pathway, driving IRF3 activation and IFN-beta production, and it also potentiates NF-kappaB activation, including TBK1-dependent phosphorylation of the p65/RELA subunit. NAP1 is a constituent of the TBK1-IKKepsilon-NAP1 complex. Structurally, its N-terminal region binds the SKICH domains of the selective-autophagy cargo receptors NDP52/CALCOCO2 and TAX1BP1, allowing NAP1 (with its paralog SINTBAD) to bridge these receptors to TBK1 and recruit/ activate TBK1 at autophagic cargo. NAP1 is itself a phosphoprotein and is subject to TRIM38-mediated K48-linked polyubiquitination and degradation. alternative_products: - name: 1 (Long) id: Q9H6S1-1 - name: 2 (Short) id: Q9H6S1-3 sequence_note: VSP_023817, VSP_023818 - name: '3' id: Q9H6S1-4 sequence_note: VSP_047087, VSP_047090 - name: '4' id: Q9H6S1-5 sequence_note: VSP_047088, VSP_047089 existing_annotations: - term: id: GO:0005737 label: cytoplasm evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in review: summary: Phylogenetic (PAN-GO) inference that NAP1 is active in the cytoplasm, consistent with experimental localization and its cytoplasmic adaptor role for TBK1/IKBKE. action: ACCEPT reason: Correct core compartment; NAP1 acts as a cytoplasmic adaptor that binds and activates TBK1, consistent with the experimental cytoplasmic localization (PMID:14560022). supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14560022}.' - term: id: GO:0005737 label: cytoplasm evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: located_in review: summary: Electronic transfer of cytoplasmic localization (UniProt subcellular location / mouse ortholog), redundant with but consistent with the experimental and IBA cytoplasm annotations. action: ACCEPT reason: Correct core cytoplasmic localization; redundant electronic assignment that agrees with the EXP (PMID:14560022) and IBA evidence. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14560022}.' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:14743216 qualifier: enables review: summary: IntAct interaction with TBK1 (Q9UHD2) from a TNF-alpha/NF-kappaB pathway interaction map. Records the functionally central TBK1 interaction but bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: Captures the key NAP1-TBK1 interaction underlying its adaptor function, but bare protein binding (GO:0005515) is uninformative per curation guidelines; the functional MF/CC is better captured by the kinase-complex and IFN annotations. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402;' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:21903422 qualifier: enables review: summary: IntAct interaction with TBK1 (Q9UHD2) from a dynamic innate-immunity (type I IFN) interaction network. Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: Records the real NAP1-TBK1 interaction in the type I IFN network but bare protein binding is uninformative. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402;' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:21931555 qualifier: enables review: summary: IntAct interactions from the vaccinia virus C6 study (NAP1 binds vaccinia C6/OPG029 and TBK1). C6 is a viral antagonist that targets TBK1 adaptors to block IRF3/IRF7. Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: Records a real host-virus interaction (vaccinia C6 targeting NAP1 as a TBK1 adaptor) and the TBK1 interaction, but bare protein binding is uninformative. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'Q9H6S1; P17362: OPG029; Xeno; NbExp=2; IntAct=EBI-359973, EBI-9519257;' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:22014111 qualifier: enables review: summary: IntAct interaction with a flavivirus protein (Q9E7P0) from a flavivirus NS3/NS5 yeast two-hybrid screen. Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: High-throughput host-virus Y2H interaction; bare protein binding is uninformative and the interaction is not central to the core function. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'Q9H6S1; Q9E7P0; Xeno; NbExp=2; IntAct=EBI-359973, EBI-11361108;' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:29251827 qualifier: enables review: summary: IntAct interaction with TBK1 (Q9UHD2) from the TBK1/STING/MDA5 interactome (TTC4 study). Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: Records the NAP1-TBK1 interaction in the antiviral innate-immune interactome but bare protein binding is uninformative. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402;' - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32707033 qualifier: enables review: summary: IntAct interaction with TBK1 (Q9UHD2) from a human kinase interaction network. Bare protein binding is uninformative. action: KEEP_AS_NON_CORE reason: Records the NAP1-TBK1 interaction but bare protein binding is uninformative. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402;' - term: id: GO:0043124 label: negative regulation of canonical NF-kappaB signal transduction evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: involved_in review: summary: Ensembl Compara transfer from the mouse ortholog (Q9QYP6) of a negative regulation of canonical NF-kappaB role. The human-characterized role of NAP1 is predominantly to potentiate NF-kappaB (PMID:14560022), so this orthology-based term is at best context-specific and conflicts with the dominant positive-regulatory role. action: KEEP_AS_NON_CORE reason: Orthology-transferred (mouse) annotation; not contradicted by clearly identified human evidence so retained, but the dominant, experimentally characterized human role is positive regulation/potentiation of NF-kappaB, so this negative-regulation term is non-core and possibly context-specific. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: Promotes TBK1-induced as well as TNF or PMA-induced activation of NF-kappa-B - term: id: GO:0005737 label: cytoplasm evidence_type: EXP original_reference_id: PMID:14560022 qualifier: located_in review: summary: Experimental evidence that NAP1 localizes to the cytoplasm (foundational identification paper). Core localization where NAP1 binds and activates TBK1/IKBKE. action: ACCEPT reason: Experimentally supported core cytoplasmic localization, the compartment in which NAP1 acts as a TBK1/IKBKE adaptor. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: 'SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14560022}.' - term: id: GO:0051607 label: defense response to virus evidence_type: NAS original_reference_id: PMID:17142768 qualifier: involved_in review: summary: ComplexPortal author-statement (TBK1-IKKepsilon-NAP1 complex, CPX-6038) that NAP1 participates in antiviral defense. NAP1 acts as a shared adaptor downstream of TLR3 and the cytoplasmic RIG-I/MDA5 pathway in type I IFN induction. action: ACCEPT reason: Core biological process; NAP1 is an established adaptor in antiviral innate immunity feeding IRF3 activation and IFN-beta induction via both TLR3/TRIF and the cytoplasmic RNA-sensing pathways. Recent literature compiled in the falcon deep-research report reinforces NAP1 as a positive regulator of the TBK1-IRF3/IRF7 type I IFN axis downstream of the major nucleic-acid sensing pathways, although the specific sensor branches (RLR-MAVS, cGAS-STING, TLR3-TRIF) are general TBK1-pathway context rather than uniquely AZI2-specific findings. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: Adapter protein which binds TBK1 and IKBKE playing a role in antiviral innate immunity - reference_id: PMID:17142768 supporting_text: NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction - reference_id: file:human/AZI2/AZI2-deep-research-falcon.md supporting_text: NAP1 is critical for linking TBK1 to IRF3/IRF7 and the type I interferon response, acting downstream of RLR–MAVS, cGAS–STING, and TLR3–TRIF pathways. - term: id: GO:0060337 label: type I interferon-mediated signaling pathway evidence_type: NAS original_reference_id: PMID:17142768 qualifier: involved_in review: summary: ComplexPortal author-statement that NAP1 (in the TBK1-IKKepsilon-NAP1 complex) participates in type I interferon signaling/induction. NAP1 couples TLR3 and cytoplasmic RNA sensing to IRF3/type I IFN. action: KEEP_AS_NON_CORE reason: NAP1's documented role is in type I IFN induction (driving IRF3 activation and IFN-beta production upstream), rather than in the downstream IFN-receptor (JAK/STAT) signaling that GO:0060337 (type I interferon-mediated signaling pathway) most precisely denotes; retained as a correct but imprecise pathway-context annotation. The defense-response-to-virus and IFN-beta-production-related roles are the core capture. supported_by: - reference_id: PMID:17142768 supporting_text: NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction - term: id: GO:1902554 label: serine/threonine protein kinase complex evidence_type: NAS original_reference_id: PMID:17142768 qualifier: part_of review: summary: ComplexPortal author-statement that NAP1 is part of a serine/threonine protein kinase complex (the TBK1-IKKepsilon-NAP1 complex, CPX-6038). Core cellular component capturing NAP1's defining role as a subunit of the TBK1/IKBKE kinase complex. action: ACCEPT reason: Core cellular component; NAP1 is a constituent/regulatory subunit of the TBK1-IKKepsilon kinase complex, which is the structural basis of its adaptor and kinase-activating function. The falcon deep-research report corroborates the mechanistic basis, with NAP1 binding promoting TBK1 activation via Ser172 autophosphorylation. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: Activates serine/threonine-protein kinase TBK1 and facilitates its oligomerization - reference_id: file:human/AZI2/AZI2-deep-research-falcon.md supporting_text: It binds and activates TANK-binding kinase 1 (TBK1), inducing conformational changes that enable TBK1 autophosphorylation at Ser172—essential for kinase activation core_functions: - description: Acts as an adapter and positive regulator of the IKK-related serine/threonine kinases TBK1 and IKBKE, binding them via its C-terminal TBK1-binding domain to promote TBK1 activation and oligomerization as a subunit of the TBK1-IKKepsilon-NAP1 kinase complex. supported_by: - reference_id: file:human/AZI2/AZI2-uniprot.txt supporting_text: Adapter protein which binds TBK1 and IKBKE playing a role in antiviral innate immunity. Activates serine/threonine-protein kinase TBK1 and facilitates its oligomerization. - reference_id: PMID:14560022 supporting_text: NAP1 activates NAK and facilitates its oligomerization locations: - id: GO:0005737 label: cytoplasm - description: Functions as a shared adaptor in innate antiviral immunity, coupling TLR3 (via TRIF/TICAM1) and the cytoplasmic RIG-I/MDA5 RNA-sensing pathway to TBK1-driven IRF3 activation and type I interferon (IFN-beta) induction, and potentiating NF-kappaB activation including TBK1-dependent phosphorylation of p65/RELA. supported_by: - reference_id: PMID:17142768 supporting_text: NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction - reference_id: PMID:14560022 supporting_text: the NAK-NAP1 complex itself effectively phosphorylated serine 536 of the p65/RelA subunit of NF-kappaB directly_involved_in: - id: GO:0051607 label: defense response to virus references: - id: file:human/AZI2/AZI2-deep-research-falcon.md title: Falcon deep research report for AZI2 findings: - statement: AZI2/NAP1 is a scaffold/adaptor (not an enzyme) that binds TBK1 and promotes its activation via adaptor-driven assembly/oligomerization and TBK1 Ser172 autophosphorylation, linking TBK1 to multiple signaling platforms and cargo-receptor complexes (innate immunity, selective autophagy/mitophagy, TNF signaling, mitosis). reference_section_type: OTHER reference_review: relevance: HIGH correctness: UNVERIFIED review_notes: >- LLM-synthesized deep-research report; treat as a lead, not as primary evidence. AZI2-SPECIFIC claims it surfaces (anchored to retrievable primary papers not in the current GOA) include: NAP1/AZI2 is required for TBK1 activation at centrosomes during mitosis/cytokinesis, with TBK1 phosphorylating NAP1 at Ser318 to trigger its degradation (Paul et al. 2023, J Cell Biol, doi:10.1083/jcb.202303082); AZI2 mediates TBK1 activation at unresolved selective-autophagy cargo-receptor complexes upon RB1CC1/FIP200 loss, driving IRF3 chemokine signaling and CD8 T-cell infiltration in breast cancer (Yeo et al. 2024, Autophagy, doi:10.1080/15548627.2023.2259775; Okamoto et al. 2020, Cancer Res); AZI2/NAP1 with SINTBAD controls mitophagy initiation/progression by tuning TBK1 recruitment to OPTN/NDP52 (Adriaenssens et al. 2024); AZI2 and TANK cooperatively recruit TBK1 to the TNF receptor to restrain RIPK1-driven cell death (Ujevic et al. 2024, Nat Commun, doi:10.1038/s41467-024-54399-4). GENERAL TBK1-PATHWAY / ADAPTOR-FAMILY inference (TBK1 as the central kinase downstream of RLR-MAVS, cGAS-STING, TLR3-TRIF; competition among NAP1/TANK/SINTBAD for TBK1) is shared pathway/family context, not necessarily AZI2-specific. The Glon et al. 2025 condensate model is a non-peer-reviewed bioRxiv preprint and is treated as speculative. None of these primary papers are cited in the current GOA, so no existing-annotation action was changed on the basis of this report alone; it is recorded to flag candidate functions (mitosis/cytokinesis, selective autophagy/mitophagy, TNF-receptor cell-death checkpoint) for future curation. - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000107 title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:14560022 title: Identification of NAP1, a regulatory subunit of IkappaB kinase-related kinases that potentiates NF-kappaB signaling. findings: - statement: NAP1 interacts with the IKK-related kinases NAK/TBK1 and IKBKE/IKKepsilon, activates TBK1 and facilitates its oligomerization, and the NAK-NAP1 complex phosphorylates Ser536 of p65/RELA; NAP1 potentiates NF-kappaB-dependent gene expression and localizes to the cytoplasm. reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text available. Foundational identification of NAP1 as a TBK1/IKBKE adaptor and activator; source of the core adaptor function and cytoplasmic localization (EXP). - id: PMID:14743216 title: A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Pathway-scale interaction map; source of an IntAct NAP1-TBK1 protein binding annotation. - id: PMID:17142768 title: NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction. findings: - statement: NAP1 acts as a shared adaptor downstream of TLR3 (via TRIF/TICAM1) and the cytoplasmic RIG-I/MDA5 dsRNA-sensing pathway to drive IRF3 activation and type I interferon induction. reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: Abstract-only in cache (full_text_available false). Source of the ComplexPortal NAS annotations (defense response to virus, type I IFN signaling, ser/thr kinase complex) for the TBK1-IKKepsilon-NAP1 complex. - id: PMID:21903422 title: Mapping a dynamic innate immunity protein interaction network regulating type I interferon production. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Innate-immunity interaction network; source of an IntAct NAP1-TBK1 protein binding annotation. - id: PMID:21931555 title: Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7. findings: - statement: Vaccinia virus C6 binds the TBK1 adaptor proteins (NAP1, TANK, SINTBAD) and inhibits IRF3/IRF7 activation, identifying NAP1 as a TBK1 adaptor targeted by a viral immune antagonist. reference_section_type: ABSTRACT reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text available. Source of host-virus IntAct protein binding annotations (vaccinia C6, TBK1); corroborates NAP1 as a bona fide TBK1 adaptor in antiviral signaling. - id: PMID:22014111 title: 'Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.' findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: High-throughput host-virus Y2H screen; source of a bare protein binding annotation with a flavivirus protein. - id: PMID:29251827 title: Quantitative Proteomics Identified TTC4 as a TBK1 Interactor and a Positive Regulator of SeV-Induced Innate Immunity. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: TBK1/STING/MDA5 interactome study; source of an IntAct NAP1-TBK1 protein binding annotation. - id: PMID:30459273 title: Mechanistic insights into the interactions of NAP1 with the SKICH domains of NDP52 and TAX1BP1. findings: - statement: Crystal structures define how the N-terminal region of NAP1 binds the SKICH domains of the selective-autophagy cargo receptors NDP52/CALCOCO2 and TAX1BP1, allowing NAP1 to bridge these receptors to TBK1. reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text available. Structural basis for NAP1 acting as a TBK1-recruiting adaptor for autophagy cargo receptors NDP52 and TAX1BP1. - id: PMID:32707033 title: Kinase Interaction Network Expands Functional and Disease Roles of Human Kinases. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: Kinase interaction network; source of an IntAct NAP1-TBK1 protein binding annotation. suggested_questions: - question: Does human NAP1 have a genuine negative-regulatory role in canonical NF-kappaB signaling (as inferred by orthology from mouse), or is the IEA GO:0043124 annotation context-specific given that the foundational human study shows NAP1 potentiates NF-kappaB? - question: How is NAP1's adaptor activity partitioned between TBK1 activation in antiviral type I IFN induction and TBK1 recruitment to NDP52/TAX1BP1 in selective autophagy, and do its four isoforms (which differ in the TBK1-binding region) have distinct functions? - question: Recent primary literature (compiled in the falcon deep-research report but not yet reflected in GOA) implicates AZI2/NAP1 in TBK1 activation at centrosomes during mitosis/cytokinesis (Paul et al. 2023), in selective autophagy/mitophagy via OPTN/NDP52 and FIP200/RB1CC1 (Adriaenssens et al. 2024; Yeo et al. 2024), and in a TBK1-dependent TNF-receptor cell-death checkpoint with TANK (Ujevic et al. 2024). Should these become curated GO annotations (e.g. mitotic cell cycle, macroautophagy/mitophagy, negative regulation of programmed cell death), and which are AZI2-specific versus shared TBK1-adaptor functions? suggested_experiments: - description: Reconstitute TBK1 activation in vitro with purified TBK1 and wild-type versus TBK1-binding-domain (residues 216-257) mutant NAP1 to quantify how NAP1 promotes TBK1 trans-autophosphorylation and oligomerization, and test substrate phosphorylation (IRF3, p65/RELA). - description: Use NAP1 (AZI2) knockout cells reconstituted with isoform-specific or domain-mutant constructs to dissect NAP1's contribution to TLR3- versus RIG-I/MDA5-driven IFN-beta induction and to NDP52/TAX1BP1-dependent selective autophagy/xenophagy.