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.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (PAN-GO) inference that NAP1 is active in the cytoplasm, consistent with experimental localization and its cytoplasmic adaptor role for TBK1/IKBKE. Reason: Correct core compartment; NAP1 acts as a cytoplasmic adaptor that binds and activates TBK1, consistent with the experimental cytoplasmic localization (PMID:14560022). Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14560022}. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic transfer of cytoplasmic localization (UniProt subcellular location / mouse ortholog), redundant with but consistent with the experimental and IBA cytoplasm annotations. Reason: Correct core cytoplasmic localization; redundant electronic assignment that agrees with the EXP (PMID:14560022) and IBA evidence. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14560022}. |
| GO:0005515 protein binding | IPI PMID:14743216 A physical and functional map of the human TNF-alpha/NF-kapp... | KEEP AS NON CORE | 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. 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. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402; |
| GO:0005515 protein binding | IPI PMID:21903422 Mapping a dynamic innate immunity protein interaction networ... | KEEP AS NON CORE | Summary: IntAct interaction with TBK1 (Q9UHD2) from a dynamic innate-immunity (type I IFN) interaction network. Bare protein binding is uninformative. Reason: Records the real NAP1-TBK1 interaction in the type I IFN network but bare protein binding is uninformative. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402; |
| GO:0005515 protein binding | IPI PMID:21931555 Vaccinia virus protein C6 is a virulence factor that binds T... | KEEP AS NON CORE | 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. 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. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Q9H6S1; P17362: OPG029; Xeno; NbExp=2; IntAct=EBI-359973, EBI-9519257; |
| GO:0005515 protein binding | IPI PMID:22014111 Flavivirus NS3 and NS5 proteins interaction network: a high-... | KEEP AS NON CORE | Summary: IntAct interaction with a flavivirus protein (Q9E7P0) from a flavivirus NS3/NS5 yeast two-hybrid screen. Bare protein binding is uninformative. Reason: High-throughput host-virus Y2H interaction; bare protein binding is uninformative and the interaction is not central to the core function. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Q9H6S1; Q9E7P0; Xeno; NbExp=2; IntAct=EBI-359973, EBI-11361108; |
| GO:0005515 protein binding | IPI PMID:29251827 Quantitative Proteomics Identified TTC4 as a TBK1 Interactor... | KEEP AS NON CORE | Summary: IntAct interaction with TBK1 (Q9UHD2) from the TBK1/STING/MDA5 interactome (TTC4 study). Bare protein binding is uninformative. Reason: Records the NAP1-TBK1 interaction in the antiviral innate-immune interactome but bare protein binding is uninformative. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402; |
| GO:0005515 protein binding | IPI PMID:32707033 Kinase Interaction Network Expands Functional and Disease Ro... | KEEP AS NON CORE | Summary: IntAct interaction with TBK1 (Q9UHD2) from a human kinase interaction network. Bare protein binding is uninformative. Reason: Records the NAP1-TBK1 interaction but bare protein binding is uninformative. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Q9H6S1; Q9UHD2: TBK1; NbExp=7; IntAct=EBI-359973, EBI-356402; |
| GO:0043124 negative regulation of canonical NF-kappaB signal transduction | IEA GO_REF:0000107 | KEEP AS NON CORE | 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. 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. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Promotes TBK1-induced as well as TNF or PMA-induced activation of NF-kappa-B |
| GO:0005737 cytoplasm | EXP PMID:14560022 Identification of NAP1, a regulatory subunit of IkappaB kina... | ACCEPT | Summary: Experimental evidence that NAP1 localizes to the cytoplasm (foundational identification paper). Core localization where NAP1 binds and activates TBK1/IKBKE. Reason: Experimentally supported core cytoplasmic localization, the compartment in which NAP1 acts as a TBK1/IKBKE adaptor. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14560022}. |
| GO:0051607 defense response to virus | NAS PMID:17142768 NAK-associated protein 1 participates in both the TLR3 and t... | ACCEPT | 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. 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. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Adapter protein which binds TBK1 and IKBKE playing a role in antiviral innate immunity PMID:17142768 NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction file:human/AZI2/AZI2-deep-research-falcon.md 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. |
| GO:0060337 type I interferon-mediated signaling pathway | NAS PMID:17142768 NAK-associated protein 1 participates in both the TLR3 and t... | KEEP AS NON CORE | 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. 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. Supporting Evidence: PMID:17142768 NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction |
| GO:1902554 serine/threonine protein kinase complex | NAS PMID:17142768 NAK-associated protein 1 participates in both the TLR3 and t... | ACCEPT | 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. 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. Supporting Evidence: file:human/AZI2/AZI2-uniprot.txt Activates serine/threonine-protein kinase TBK1 and facilitates its oligomerization file:human/AZI2/AZI2-deep-research-falcon.md It binds and activates TANK-binding kinase 1 (TBK1), inducing conformational changes that enable TBK1 autophosphorylation at Ser172—essential for kinase activation |
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Download this section (compressed HTML)Q: 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?
Q: 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?
Q: 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?
Experiment: 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).
Experiment: 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.
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