Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

ARTICLE Received 25 Jun 2015 | Accepted 30 Oct 2015 | Published 10 Dec 2015 DOI: 10.1038/ncomms10072 OPEN Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis Smitha Pillai1, Jonathan Nguyen1, Joseph Johnson1, Eric Haura2, Domenico Coppola3 & Srikumar Chellappan1 TANK Binding Kinase 1 (TBK1) is a non-canonical IkB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis. 1 Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida 33612, USA. 2 Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida 33612, USA. 3 Department of Anatomic Pathology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida 33612, USA. Correspondence and requests for materials should be addressed to S.C. (email: Srikumar.Chellapan@moffitt.org). NATURE COMMUNICATIONS | 6:10072 | DOI: 10.1038/ncomms10072 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10072 T BK1 is an IKK (IkB Kinase)-related kinase that is activated by phosphorylation of Serine-172 by TLR and RIG1 signalling, and this circuit triggers phosphorylation of IRF3 and IRF7, activation of NFkB and the expression of proinflammatory genes and interferons1–6. In addition to the crucial role TBK1 plays in regulating innate immunity, recent studies suggest that TBK1 participates in pathways leading to survival and cellular transformation7. RalB-mediated activation of TBK1 promotes TBK1 assembly with the exocyst complex through its interaction with Sec5 leading to inflammatory responses and prosurvival signalling by directly phosphorylating multiple sites on Akt8. TBK1 is essential for the survival of non-small cell lung cancers driven by oncogenic KRAS9–11; this synthetic lethal interaction of TBK1 with mutant K-Ras was governed by its ability to activate NFkB anti-apoptotic signalling through c-Rel and BCL-XL. TBK1 also contributes to prostate cancer dormancy and drug resistance by inhibiting mTOR12, and to tamoxifen resistance of breast cancer cells by enhancing transcriptional activity of ERa7. TBK1 has been reported to phosphorylate the mitotic kinase PLK1 (ref. 13), but roles for TBK1 in mitosis have not been investigated. Here we demonstrate direct roles for TBK1 in regulating mitosis, where it binds to and phosphorylates CEP170, a forkhead domain and centrosome- and spindle microtubuleassociated protein14, as well as NuMA, which associates with the pericentrosomal domains of the spindle apparatus and is necessary for cytokinesis15. Here we demonstrate that TBK1 regulates microtubule dynamics and also mitotic progression by modulating CEP170 and NuMA functions. Results pS172 TBK1 localizes to centrosomes and mitotic spindles. Immunofluorescence experiments using a phospho-TBK1 (pS-172) specific antibody on A549, H1650, Calu-6 and PC9 non-small cell lung cancer (NSCLC) cell lines as well as the immortalized human tracheobronchial epithelial cell line AALE established that phospho-TBK1 localized to centrosomal regions during prophase and prometaphase, where it co-localized with alpha tubulin (Fig. 1a, Supplementary Fig. 1a–d). Similar findings were manifest in U937 myeloid leukaemia cells and Daudi Burkitt lymphoma cells (Supplementary Fig. 1e), and a second phospho-TBK1 antibody showed similar localization of pTBK1 (Supplementary Fig. 2). Further, phospho-TBK1 associated with spindle microtubules during metaphase and with the midbody during telophase and cytokinesis (Fig. 1a). Finally, depletion of TBK1-related IKKe kinase using siRNAs (Fig. 1b) or inhibition of mitotic kinase PLK1 using the inhibitor BI2536 (Fig. 1c) did not alter the centrosomal localization of phospho-TBK1. To further confirm the centrosomal localization of phosphoTBK1, centrosomes were isolated from A549 and H460 NSCLC cells by discontinuous sucrose gradient fractionation16,17 and subjected to western blot analysis. Phospho-TBK1 and total TBK1 were principally found in centrosomal fraction (Fraction 4, Fig. 1d,e), which also contained g-tubulin, phospho-PLK1, PLK1 and CEP170. Phospho-TBK1 and total TBK1 were also present in additional fractions; correlating with the observation that phospho-TBK1 also is associated with spindle apparatus during mitosis. Interestingly, pTBK1 localization to centrosomes did not depend on microtubule integrity, as pTBK1 localized to centrosomes when microtubules were hyperstabilized or depolymerized (Supplementary Fig. 3). TBK1 is necessary for progression through mitosis. Given the centrosomal localization of phospho-TBK1, we assessed if TBK1 contributes to mitosis. Depletion of TBK1 by two different 2 TBK1-selective siRNAs or by a lentiviral small-hairpin RNA (shRNA; Supplementary Fig. 4) significantly reduced the number of mitotic cells (Fig. 2a,b). Centrosomal structures start to get organized towards the end of the S-phase18. To assess if this was associated with localization of active phospho-TBK1 to centrosomes, A549 NSCLC cells were arrested at the G1/S transition by double-thymidine block. Release from arrest showed that TBK1 is activated at late S-phase, 4 h after release from the double-thymidine block (Fig. 2c). Finally, maximal levels of phospho-TBK1 phosphorylation coincided with increased levels of phosphorylation of histone H3 at serine 10 (pH3S10), an indicator for progression into mitosis (Fig. 2c). To test whether TBK1 inhibition prevents cell cycle progression, A549 and H1650 NSCLC cells were arrested in the G1/S phase transition by double-thymidine block and released for 9 h in the presence or absence of BX795. Untreated cells, but not BX795-treated cells, entered mitosis as seen by elevated levels of pH3S10 in western blots (Fig. 2d). Similarly selective knockdown of TBK1 using siRNAs prevented entry of cells into mitosis following double-thymidine block and release, as seen by low levels of pH3S10 (Fig. 2e). A recent study suggested that PLK1, a well-established mitotic kinase, might be a TBK1 substrate13. Hence, we tested whether TBK1 regulates mitosis via PLK (...truncated)