Cell type-specific subcellular localization of phospho-TBK1 in response to cytoplasmic viral DNA

PLoS One. 2013 Dec 9;8(12):e83639. doi: 10.1371/journal.pone.0083639. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA, Viral / genetics
  • DNA, Viral / metabolism*
  • HeLa Cells
  • Hep G2 Cells
  • Hepatitis B / enzymology*
  • Hepatitis B / genetics
  • Hepatitis B virus / genetics
  • Hepatitis B virus / metabolism*
  • Humans
  • Mice
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein Transport
  • Tupaiidae

Substances

  • DNA, Viral
  • Phosphoproteins
  • Tbk1 protein, mouse
  • Protein Serine-Threonine Kinases
  • TBK1 protein, human

Grant support

This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan, and the Ministry of Health Labor and Welfare of Japan, the Kato Memorial Bioscience Foundation, Yasuda Cancer Foundation, and the Ono Foundation. Financial supports by a MEXT Gran-in-Project “the Carcinogenic Spiral”, the Natinal Cancer Center Research and Development Fund (23-A-44), and the Japan Initiative for Global Research Network on Infectious Disease (J-GRID) are gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.