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. 2020 Aug 9;12(4):261–278. doi: 10.1007/s13238-020-00751-5

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Lys241 and Lys692 deacetylation mediated by HDAC3 are critical for TBK1 kinase activity. (A) Immunoblot analysis of endogenous total TBK1 and acetylated TBK1 (Ac-TBK1) in HEK293T cells, assessed before (10% input) or after (IP) immunoprecipitation with IgG (control) or anti-acetyl-lysine. (B) Immunoblot analysis of acetylated TBK1 (Ac-TBK1) in HEK293T cells (1.5 × 10⁶) transiently co-transfected for 48 h with Flag-tagged TBK1 (3 μg) and empty vectors or Myc-tagged HDAC3 (1 μg) expression plasmids before co-immunoprecipitation (with anti-Flag or IgG as a control) and immunoblot analysis (with antibody to all acetylated lysine residues, anti-Flag, anti-Myc, anti-GAPDH). (C) Effect of HDAC3 specific inhibitor (RGFP966, 100 nmol/L) on TBK1 acetylation. Immunoblot analysis of acetylated TBK1 (Ac-TBK1) in HEK293T cells (1.5 × 10⁶), transiently transfected for 48 h with Flag-tagged TBK1 (3 μg) and treated for 24 h with RGFP966 before immunoprecipitation (with anti-Flag or IgG as a control) and immunoblot analysis (with antibody to all acetylated lysine residues, anti-Flag, anti-ACTIN). (D) Identification of lysine residues in TBK1 in HEK293T HDAC3-deficient cells, by immunoprecipitation combined with mass spectrometry, and Luciferase activity of an IFN-β reporter in HEK293T cells transfected with control vector (VEC) or wild-type TBK1 (WT) or mutants TBK1 (30R, 30Q, 154R, 154Q, 236R, 236Q, 241R, 241Q, 251R, 251Q, 607R, 607Q, 646R, 646Q, 691R, 691Q, 692R, 692Q). (E) Immunoblot analysis of phosphorylated and total TBK1, IRF3 in HEK293T cells transfected with control vector (VEC) or wild-type TBK1 (WT) or K241R K241Q, K692R, K692Q. (F) TBK1 kinase activity assay. HEK293T TBK1-knockout cells (4 × 10⁵) transiently transfected for 48 h with Flag-tagged empty vector (VEC), wild-type TBK1 (WT) or TBK1 mutants (K241R, K241Q, K692R or K692Q) before immunoprecipitation (with anti-Flag), then according the TBK1 kinase activity kit manufacture. (G) Interaction between HDAC3 and wild type (WT) or TBK1 mutants. Immunoblot analysis of HEK293T cells (1.5 × 10⁶) transiently co-transfected for 48 h with Myc-tagged HDAC3 and Flag-tagged empty vector, wild type (WT) or TBK1 mutants (K241R, K241Q, K692R, K692Q) (3 μg each) before co-immunoprecipitation (with anti-Flag) and immunoblot analysis were performed with the indicated antibodies. (H and I) Immunoblot analysis of specific sites of acetylated TBK1 (Ac-K241, Ac-K692) in HDAC3 knockdown HEK293T cells (1.5 × 10⁶) (H) or HDAC3 overexpressed HEK293T cells (1.5 × 10⁶) (I) transiently transfected for 48 h with Flag-tagged TBK1 (3 μg each) and empty vectors or Myc-tagged HDAC3 (1 μg in I) expression plasmids before immunoprecipitation (with anti-Flag or IgG as a control) and immunoblot analysis (with antibody to TBK1 acetylated at Lys241 (K241Ac) or K692Ac, anti-Flag, anti-HDAC3 or anti-Myc). Data are representative of three independent experiments. Graphs show mean ± SD; n = 3. **P < 0.01; ***P < 0.001 (Student’s t-test)