title: Cell type-specific subcellular localization of phospho-TBK1 in response to cytoplasmic viral DNA.
abstract: Cytoplasmic viral RNA and DNA are recognized by RIG-I-like receptors and DNA sensors that include DAI, IFI16, DDX41, and cGAS. The RNA and DNA sensors evoke innate immune responses through the IPS-1 and STING adaptors. IPS-1 and STING activate TBK1 kinase. TBK1 is phosphorylated in its activation loop, leading to IRF3/7 activation and Type I interferon (IFN) production. IPS-1 and STING localize to the mitochondria and endoplasmic reticulum, respectively, whereas it is unclear where phosphorylated TBK1 is localized in response to cytoplasmic viral DNA. Here, we investigated phospho-TBK1 (p-TBK1) subcellular localization using a p-TBK1-specific antibody. Stimulation with vertebrate DNA by transfection increased p-TBK1 levels. Interestingly, stimulation-induced p-TBK1 exhibited mitochondrial localization in HeLa and HepG2 cells and colocalized with mitochondrial IPS-1 and MFN-1. Hepatitis B virus DNA stimulation or herpes simplex virus type-1 infection also induced p-TBK1 mitochondrial localization in HeLa cells, indicating that cytoplasmic viral DNA induces p-TBK1 mitochondrial localization in HeLa cells. In contrast, p-TBK1 did not show mitochondrial localization in RAW264.7, L929, or T-23 cells, and most of p-TBK1 colocalized with STING in response to cytoplasmic DNA in those mammalian cells, indicating cell type-specific localization of p-TBK1 in response to cytoplasmic viral DNA. A previous knockout study showed that mouse IPS-1 was dispensable for Type I IFN production in response to cytoplasmic DNA. However, we found that knockdown of IPS-1 markedly reduced p-TBK1 levels in HeLa cells. Taken together, our data elucidated the cell type-specific subcellular localization of p-TBK1 and a cell type-specific role of IPS-1 in TBK1 activation in response to cytoplasmic viral DNA.
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Oshiumi
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Moeko
authors.initials: M
authors.lastname: Miyashita
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Hussein Hassan
authors.initials: HH
authors.lastname: Aly
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Misako
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Seya
mesh_terms: D000818:Animals; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D056945:Hep G2 Cells; D006509:Hepatitis B / Q000201:enzymology* / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D014418:Tupaiidae
chemical_list: D004279:DNA, Viral; D010750:Phosphoproteins; C498588:Tbk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human