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TBKBP1 and TBK1 form a growth factor signalling axis mediating immunosuppression and tumourigenesis

Nature Cell Biology volume 21, pages 1604–1614 (2019)Cite this article

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Abstract

TANK-binding kinase 1 (TBK1) responds to microbial stimuli and mediates the induction of type I interferon (IFN). Here, we show that TBK1 is also a central mediator of growth factor signalling; this function of TBK1 relies on a specific adaptor—TBK-binding protein 1 (TBKBP1). TBKBP1 recruits TBK1 to protein kinase C-theta (PKCθ) through a scaffold protein, CARD10. This enables PKCθ to phosphorylate TBK1 at Ser 716, a crucial step for TBK1 activation by growth factors but not by innate immune stimuli. Although the TBK1–TBKBP1 signalling axis is not required for the induction of type I IFN, it mediates mTORC1 activation and oncogenesis. Conditional deletion of either TBK1 or TBKBP1 in lung epithelial cells inhibits tumourigenesis in a mouse model of lung cancer. In addition to promoting tumour growth, the TBK1–TBKBP1 axis facilitates tumour-mediated immunosuppression through a mechanism that involves induction of the checkpoint molecule PD-L1 and stimulation of glycolysis. These findings suggest a PKCθ–TBKBP1–TBK1 growth factor signalling axis that mediates both tumour growth and immunosuppression.

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Fig. 1: Lung-epithelial-cell-specific deletion of TBK1 inhibits Kras-induced lung tumourigenesis.
Fig. 2: Growth factors activate TBK1 in a TBKBP1-dependent manner.
Fig. 3: TBKBP1 and TBK1 form a growth factor signalling axis that mediates mTORC1 activation and lung tumourigenesis.
Fig. 4: PKCθ mediates growth factor stimulated TBK1 activation through phosphorylating TBK1 at Ser 716.
Fig. 5: EGF-stimulated TBK1 activation involves assembly of a signalling complex that is composed of PKCθ, CARD10, TBKBP1 and TBK1.
Fig. 6: A small-molecule inhibitor of TBK1 inhibits lung tumourigenesis and promotes antitumour T-cell responses.
Fig. 7: TBK1 and TBKBP1 regulate tumour-mediated immunosuppression by facilitating EGF-induced PD-L1 expression.

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Data availability

Source data are available online for Figs. 1e–g,i,k, 3i,j, 4k,l, 6c–e,g and 7a,c,e–g,i–k, and Extended Data Figs. 1b–e and 7a–c,g, and unprocessed blots are provided for Figs. 1–7 and Extended Data Figs. 1–6. The mass spectrometry data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org/cgi/GetDataset) through the MASSIVE repository (MSV000084505) under the accession code PXD016025. All other data that support the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank F. Zhu, C. Wang and X. Lin for plasmid DNAs; S. Akira for Tank knockout mice and F. J. DeMayo for the Ccspcre mice; the personnel from the flow cytometry, DNA analysis and animal facilities at The MD Anderson Cancer Center and the Mass Spectrometry Proteomics Core at Baylor College of Medicine for technical assistance. This study was supported by a National Institutes of Health grant (AI057555) and partially supported by a seed fund from the Center for Inflammation and Cancer at the MD Anderson Cancer Center. T.G. was a visiting student supported by a scholarship from the China Scholarship Council (grant number 201906380080). The MD Anderson core facilities are supported by a NIH/NCI Cancer Center Support Grant (CCSG; P30CA016672), and the Baylor College of Medicine Mass Spectrometry Proteomics Core is supported by a CPRIT Core Facility Award (RP170005) and a P30 Cancer Center Support Grant (NCI-CA125123).

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Authors and Affiliations

  1. Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Lele Zhu, Yanchuan Li, Xiaoping Xie, Xiaofei Zhou, Meidi Gu, Zuliang Jie, Chun-Jung Ko, Tianxiao Gao, Blanca E. Hernandez, Xuhong Cheng & Shao-Cong Sun

  2. The University of Texas Graduate School of Biomedical Sciences, Houston, TX, USA

    Shao-Cong Sun

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Contributions

L.Z. designed and performed the research, prepared the figures and wrote part of the manuscript. Y.L., X.X., X.Z., M.G., Z.J., C.-J.K., T.G., B.E.H. and X.C. contributed to the experiments. S.-C.S. supervised the work and wrote the manuscript.

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Extended data

Extended Data Fig. 1 TBKBP1 is not required for TBK1 activation or type I IFN induction by TLR ligands and viruses.

a, Immunoblot analysis of the indicated phosphorylated (P-) and total proteins in whole-cell lysates of A549 cells stably infected with a control shRNA or a Tbkbp1-specific shRNA, stimulated with the indicated inducers. b-e, qRT-PCR analysis of Ifna and Ifnb mRNA expression in A549 cells stably infected with a control shRNA (shCtrl) or shRNAs for Tbk1 (b,c) or Tbkbp1 (d,e), stimulated with the indicated inducers. Data are representative of three independent experiments. n = 3 (b,d,e) or 5 (c) per group. Two-sided unpaired Student’s t-test (b-e). Source data for graphs are provided in Statistical Source Data Fig. 1 and unprocessed blots are shown in Unprocessed Blots Fig. 1.

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Extended Data Fig. 2 TBK1, but not IKKε, mediates growth factor-stimulated mTORC1 activation.

a, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated lung cells from Tbk1WT-KrasLA2 and Tbk1cKO-KrasLA2 mice. b-e, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of control or Tbk1-knockdown H157 (b), H460 (c), or A549 (d,e) cells, stimulated with the indicated inducers. f, g, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of WT or Tbk1-deficient primary MEF cells stimulated by EGF (f) or essential amino acids (EAAs, g). h, Immunoblot analysis of the indicated proteins in the cytoplasmic extracts (Cyt Ext) or nuclear extracts (Nucl Ext) of control or Tbk1-knockdown A549 cells, stimulated with EGF. i, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of EGF-stimulated A549 cells stably infected with a control shRNA (shCtrl) or two different IKKe-specific shRNAs. Data are representative of three independent experiments. Unprocessed blots are shown in Unprocessed Blots Fig. 2.

Source data

Extended Data Fig. 3 TBK1 mediates mTORC1 activation by EGF and has cell type-specific functions in mTORC1 activation by TLR ligands.

a, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of A549 cells stably infected with a control shRNA (Ctrl) or two different Tbk1-specific shRNAs (D5 and D9), stimulated with Pam3Csk4. b-h, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of primary lung cells from WT and Tbk1cKO mice (b),Tbk1WT-KrasLA2 (WT-Kras) and Tbk1cKO-KrasLA2 (cKO-Kras) mice (c,d), bone marrow-derived macrophages (BMDM) from WT or Tbk1 myeloid cell-conditional KO (MKO) mice (e,f), or WT and Tbk1 KO primary MEFs (g,h), stimulated with the indicated TLR ligands. Data are representative of three independent experiments. Unprocessed blots are shown in Unprocessed Blots Fig. 3.

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Extended Data Fig. 4 TBK1 is associated with mTORC1 via Raptor.

a, Schematic of Myc-BioID (Myc-tagged BirA R118G) and Myc-BioID-TBK1 constructs cloned into the pCLXSN(GFP) retroviral vector. b, Scatter plot showing several known TBK1-binding proteins and a novel TBK1-binding protein, Raptor (also called RPTOR) identified by BioID screening. c,d, Immunoblot analysis of Raptor and Rictor pulled down by TBK1 IP (c, upper) or mTOR and TBK1 pulled down by Rictor IP (d, upper), with protein expression being monitored by direct immunoblot assays (lower), using whole-cell lysates of HEK293 cells transfected with the indicated expression vectors. e, Co-IP analysis of endogenous TBK1-Raptor and TBK1-Rictor interactions in EGF-stimulated A549 cells (upper). IgG was used as a negative control for IP. f,g, Co-IP analysis of TBK1-mTORC1 interactions (upper) and direct immunoblot analyses of the indicated proteins (lower) in control or Tbk1-knockdown A549 cells stimulated with EGF. The Lysates were subjected to IP with antibody against TBK1 (f) or Raptor (g). Data are representative of three independent experiments. Unprocessed blots are shown in Unprocessed Blots Fig. 4.

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Extended Data Fig. 5 TANK and NAP1 are dispensable for EGF-induced mTORC1 activation.

a-d, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of A549 cells stably infected with a control shRNA (shCtrl) or shRNAs specific for Tank (a), Nap1 (b), or Tbkbp1 (c,d), stimulated with the indicated inducers. e,f, Genotyping PCR (e) and immunoblot (f) analyses of Tbkbp1+/+Ccsp-Cre (WT), Tbkbp1+/flCcsp-Cre (Het), and Tbkbp1fl/flCcsp-Cre (cKO) mice, showing the flox and WT Tbk1 alleles and Ccsp-Cre (e) and the TBKBP1 protein expression (f). Data are representative of three independent experiments. Unprocessed blots are shown in Unprocessed Blots Fig. 5.

Source data

Extended Data Fig. 6 Role of TRAFs and PKC in TBK1 phosphorylation and activation.

a,b, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of EGF-stimulated A549 cells stably infected with a control shRNA (shCtrl) or two different shRNAs targeting Traf6 (a) or Traf3 (b). c,d, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of A549 cells stimulated with EGF in the presence of the PKC inhibitor GF109203X or solvent control DMSO. e, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of control or PKCe-knockdown A549 cells, stimulated with EGF for the indicated time periods. f,g, Immunoblot analysis of T538-phosphorylated (p-) and total PKCq in the lysates of A549 cells stimulated with insulin (f) or FBS (g). h,i, immunoblot analysis of TBK1 and S172- or S716-phosphorylated (p-) TBK1 following HA IP from A549 cells stably transduced with HA-TBK1 and stimulated with either EGF (h) or Insulin (i). j, Immunoblot analysis of S172-phosphorylated (p-) and total TBK1 in whole-cell lysates of HEK293T cells transfected with the indicated expression vectors. k, CoIP assays to detect TBK1 dimerization based interaction of HA-TBK1 with Flag-TBK1 (upper) and immunoblot analysis of protein expression level in lysates (lower). l, Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in LPS-stimulated control shRNA-transduced A549 cells or Tbk1 shRNA (D5)-transduced A549 cells that were reconstituted with and an empty vector or shRNA-resistant expression vectors encoding HA-tagged TBK1 wildtype (WT) or S716A. Data are representative of three independent experiments. Unprocessed blots are shown in Unprocessed Blots Fig. 6.

Source data

Extended Data Fig. 7 TBK1 deletion in lung epithelial cells promotes T cell activation.

a, Immunohistochemical staining of T cells using the indicated antibodies in lung sections of Tbk1WT-KrasLA2 (WT-Kras) and Tbk1cKO-KrasLA2 (cKO-Kras) mice. Scale bar, 100 mm. Data are presented as a representative image (upper) or summary graphs (lower, n = 7 mice per group). b,c, Flow cytometric analysis of the frequency of CD4 and CD8 T cells in lung cells (b) or IFNg+ effector T cells within lung CD4 or CD8 T cell populations (c). n = 5 mice per group. d-g, Experimental design (d), efficiency of T cell depletion in the lung and spleen (e), a representative image of lung tumours (f, scale bar, 100 mm), and summary graphs of lung weight, tumour numbers, and tumour size (g) of Tbk1WT-KrasLA2 and Tbk1cKO-KrasLA2 mice treated with anti-CD3 or an IgG isotype control (150 mg/mouse). n = 11 mice per group. Data are representative of three independent experiments, and bar graphs are presented as mean±s.d. values. Two-sided unpaired Student’s t-test (a,b,c,g). Source data for graphs are provided in Statistical Source Data Fig. 7.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: summary of TBK1-binding proteins identified by BioID. Supplementary Table 2: primers used in quantitative RT–PCR and mouse genotyping PCR. Supplementary Table 3: list of antibodies used.

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Zhu, L., Li, Y., Xie, X. et al. TBKBP1 and TBK1 form a growth factor signalling axis mediating immunosuppression and tumourigenesis. Nat Cell Biol 21, 1604–1614 (2019). https://doi.org/10.1038/s41556-019-0429-8

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