Fig 2: Tbkbp1 facilitates AMPKa-mediated Ulk1 activation by antagonizing mTORC1. a, b IB analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of IL-15-stimulated WT and Tbkbp1-KO thymic NKT cells (a) or NKT hybridoma cells transduced with a non-silencing control shRNA or two different Tbkbp1-specific shRNAs (b). c IB analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of IL-15-stimulated NKT hybridoma cells. d, e IB analysis of phosphorylated Ulk1 on Ser757 or Ser555 using WT or Tbkbp1-KO NKT cells incubated for 60 min with the mTOR inhibitor Torin or solvent control DMSO (d) or IL-15-stimulated NKT hybridoma cells transduced with a nonsilencing control shRNA or two different Tbkbp1-specific shRNAs (e). f IB analysis of phosphorylated Ulk1 on Ser757 or Ser 555 in IL-15-stimulated NKT hybridoma cells transduced with an empty vector or HA-Tbkbp1 expression vector. g Co-IP analyses of AMPKα-Ulk1 interactions (upper panels) and direct IB analyses of the indicated proteins (lower panels) in NKT hybridoma cells transduced with a control or Tbkbp1-specific shRNA (#1) incubated with IL-15 as indicated. IgG was used as a negative control for IP. Data are representative of three or more independent experiments

Fig 3: Tbkbp1 mediates IL-15-stimulated autophagy in NKT cells. a–d IB analysis of LC3 modification and p62 degradation (a, c) and Ulk1 phosphorylation (b, d) in whole-cell lysates of IL-15-stimulated WT or Tbkbp1-KO thymic NKT cells (a, b) or NKT hybridoma cells transduced with a non-silencing control shRNA (shCtrl) or two different Tbkbp1-specific shRNAs that were stimulated with IL-15 (d) or IL-15 together with DMSO or bafilomycin A (c). e Confocal microscopic analysis of LC3 puncta, DAPI (nuclear staining), and merged picture of untreated (NT) or IL-15-stimulated control or Tbkbp1-knockdown NKT hybridoma cells. Data are presented as representative images (left) and summary graph of quantified LC3 puncta (right). Scale bar, 5 μm. f Flow cytometric analysis of intracellular Bcl2 expression level in WT or Tbkbp1-KO thymic NKT cells cultured for 2 days with medium control, IL-15, or IL-15 plus the autophagy inhibitor 3MA. g Summary graph of flow cytometric analysis of AnnexinV+ apoptotic cells in enriched thymic NKT cells that were either untreated (NT) or cultured for 1 day with the indicated agents. h, i Co-IP analysis of Tbkbp1-FIP200 (h) or Tbkbp1-Ulk1(i) interaction (upper) and direct IB assays (lower) using lysates of HEK293 cells transfected with the indicated expression vectors. j IB analysis of the indicated proteins in IP samples of control IgG, anti-FIP200, or anti-Ulk1 or direct lysates of NKT hybridoma cells that were stimulated as indicated. k Schematic of Tbkbp1 WT and mutants showing the coiled-coil domains (CCs), TBK-binding domain (TBD), proline-rich domain, and zinc fingers (ZF). l, m Co-IP analysis of Ulk1 binding with the indicated Tbkbp1 mutants (upper) and direct IB assays (lower) using HEK293 cells transfected with Myc-ULk1 along with Tbkbp1 truncation mutants (l) and point mutants (m). n IB analysis of the indicated phosphorylated (P-) or total proteins in whole-cell lysates of Tbkbp1-knockdown NKT hybridoma cells reconstituted with a vector control (Ctrl) or the indicated Tbkbp1 point mutants, stimulated as indicated. Data are representative of three or more independent experiments. **P < 0.01; ***P < 0.001. Two-way ANOVA (e), Student’s t-test (g)

Fig 4: Interaction of TBK1 with the scaffold protein TANK is crucial for IKKϵ protein degradation. (A) Immunoblot (IB) analysis of WT cells, TBK1-deficient cells (clone #2), and TBK1-deficient cells transduced with either Flag-tagged TBK1 WT or TBK1 L704 mutant described to be unable to interact with the scaffold proteins TANK, NAP1, and SINTBAD. One representative blot of n = 4 independent lysate preparations is shown. (B, C) Quantification of IBs from (A). TBK1 (B) and IKKϵ (C) protein expression levels normalized to the expression of the housekeeping protein β-Actin are shown as the fold change of WT cells (mean ± SD of n = 4 lysate preparations). Individual values are visualized as dots. (C) One-way ANOVA followed by Dunnett’s multiple comparisons test was used to compare IKKϵ expression of TBK1-transduced with that of untransduced TBK1-deficient cells (ns – not significant, ** p ≤ 0.01). (D) Type I IFN reporter gene expression in THP1 monocytes after stimulation for 24 h with the RIG-I/MDA5 ligand polyI:C (100 ng/ml), the cGAS ligands pDNA (100 ng/ml) or G3-YSD (500 ng/ml), IFNα (1000 U/ml), or vehicle only (Lipo). Shown is the mean ± SD of n = 5 independent experiments. Individual values are visualized as dots. (E) Whole-cell lysates of TBK1-deficient cells ectopically expressing Flag-tagged TBK1 WT, TBK1 L693A, TBK1 L694E, or TBK1 L704A were prepared and the TBK1 variants were immunoprecipitated (IP) with anti-Flag beads. Interacting proteins were detected by IB with the indicated antibodies. As input control, 2% of the lysate employed for immunoprecipitation was used. One representative blot of n = 3 independent experiments is shown. (F). Immunoblot (IB) analysis of WT cells, TBK1-deficient cells (clone #2), and TBK1-deficient cells transduced with either Flag-tagged TBK1 WT, TBK1 L693A mutant that specifically lacks the ability to interact with TANK, or TBK1 K694E mutant unable to interact with the scaffold proteins TANK and SINTBAD. One representative blot of n = 4 independent lysate preparations is shown. (G, H) Quantification of IBs from (F) TBK1 (G) and IKKϵ (H) protein expression levels normalized to the expression of the housekeeping protein β-Actin are shown as the fold change of WT cells (mean ± SD of n = 4 lysate preparations). Individual values are visualized as dots. (H) One-way ANOVA followed by Dunnett’s multiple comparisons test was used to compare IKKϵ expression of TBK1-transduced with that of untransduced TBK1-deficient cells (ns – not significant, * p ≤ 0.05).

Fig 5: Tbkbp1 recruits PP6 to Ulk1 to oppose mTORC1-mediated Ulk1 phosphorylation. a Co-IP analysis of mTOR-Ulk1 interactions (upper panels) and direct IB analysis of the indicated proteins (lower panels) in NKT hybridoma cells transduced with a control or Tbkbp1-specific shRNA, either not treated (NT) or incubated with IL-15 or the mTORC1 inhibitor rapamycin (Rap). IgG was used as a negative control for IP. b Co-IP analysis of Tbkbp1-ANKRD28 interactions and direct IB assays using lysates of HEK293 cells transfected with the indicated expression vectors. c Co-IP analysis of ULK1 association with Tbkbp1 and ANKRD28 using lysates of HEK293 cells transfected with the indicated expression vectors. d Co-IP analysis of Ulk1 interaction with FIP200, Tbkbp1, and ANKRD28 using lysates of IL-15-stimulated NKT hybridoma cells transduced with a control or Tbkbp1-specific shRNA. IgG was used as a negative control for IP. e, f IB analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of IL-15-stimulated NKT hybridoma cells transduced with a nonsilencing control shRNA, two different ANKRD28-specific shRNAs (e) or two different PP6c-specific shRNAs (f). Data are representative of three independent experiments