DOK3 Is Required for IFN-β Production by Enabling TRAF3/TBK1 Complex Formation and IRF3 Activation This information is current as Susana Soo-Yeon Kim, Koon-Guan Lee, Ching-Siang Chin, of September 25, 2021. Say-Kong Ng, Natasha Ann Pereira, Shengli Xu and Kong-Peng Lam J Immunol 2014; 193:840-848; Prepublished online 13 June 2014; doi: 10.4049/jimmunol.1301601 Downloaded from http://www.jimmunol.org/content/193/2/840 Supplementary http://www.jimmunol.org/content/suppl/2014/06/13/jimmunol.130160 Material 1.DCSupplemental http://www.jimmunol.org/ References This article cites 33 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/193/2/840.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! 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The Journal of Immunology DOK3 Is Required for IFN-b Production by Enabling TRAF3/TBK1 Complex Formation and IRF3 Activation Susana Soo-Yeon Kim,*,† Koon-Guan Lee,† Ching-Siang Chin,† Say-Kong Ng,† Natasha Ann Pereira,† Shengli Xu,† and Kong-Peng Lam*,†,‡ The downstream of kinase (DOK) family of adaptors is generally involved in the negative regulation of signaling pathways. DOK1, 2, and 3 were shown to attenuate TLR4 signaling by inhibiting Ras-ERK activation. In this study, we elucidated a novel role for DOK3 in IFN-b production. Macrophages lacking DOK3 were impaired in IFN-b synthesis upon influenza virus infection or polyinosinic-polyribocytidylic acid stimulation. In the absence of DOK3, the transcription factor IFN regulatory factor 3 was not phosphorylated and could not translocate to the nucleus to activate ifn-b gene expression. Interestingly, polyinosinic- polyribocytidylic acid–induced formation of the upstream TNFR-associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 2 2 complex was compromised in dok3 / macrophages. DOK3 was shown to bind TBK1 and was required for its activation. Downloaded from Furthermore, we demonstrated that overexpression of DOK3 and TBK1 could significantly enhance ifn-b promoter activity. DOK3 was also shown to bind TRAF3, and the binding of TRAF3 and TBK1 to DOK3 required the tyrosine-rich C-terminal domain of DOK3. We further revealed that DOK3 was phosphorylated by Bruton’s tyrosine kinase. Hence, DOK3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-b production. The Journal of Immunology, 2014, 193: 840–848. http://www.jimmunol.org/ he downstream of kinase (DOK) family of adaptors (6). DOK3 was also shown to have an inhibitory role as it restricts comprises seven structurally related proteins with each Ca2+ (7) and JNK (8) activation in B cell receptor signaling. T possessing an NH2-terminal pleckstrin homology (PH), Unlike B cells, which express DOK1 and 3, and T cells, which a central phosphotyrosine-binding (PTB), and a C-terminal express DOK1 and 2, myeloid cells express all three DOK adap- tyrosine-rich domain (1). DOK1–3 are preferentially expressed tors (1). The role of DOK proteins in myeloid cells is beginning in hematopoietic cells whereas DOK4–7 are found in neural and to be unraveled, and there is increasing evidence to indicate that other cell types (2). As these proteins have no catalytic activity, they are involved in TLR signaling and innate immunity. TLRs are they function mainly as adaptors to facilitate protein–protein pattern recognition receptors that bind pathogen-associated mo- by guest on September 25, 2021 interactions and possibly also as scaffolds to nucleate protein lecular patterns found on microbes (9). Examples of pathogen- complexes in signal transduction pathways. Recently, DOK1–3 associated molecular patterns include the bacterial cell wall com- were identified as tumor suppressors for lung cancer in human and ponent LPS that is recognized by TLR4 and viral dsRNA that is mice (3) and shown to play a role in preventing the development detected by TLR3 (10). DOK1 and 2 have been shown to participate of aggressive histiocytic sarcoma (4). in TLR4 signaling by negatively regulating ERK activation and Accumulating data suggest that DOK1–3 function to limit protein TNF-a production in LPS-stimulated macrophages (11). Recently, tyrosine kinase–mediated signaling in immune cells (5). In partic- DOK3 was also demonstrated to negatively regulate LPS-induced ular, DOK1 and 2 have been characterized as negative regulators of ERK activation and the production of inflammatory cytokines in Ras-ERK signaling downstream of Ag receptors in B and T cells macrophages (12). Thus, it seems that DOK1–3 act similarly to inhibit TLR4 signaling in macrophages. *Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Whereas DOK1 and 2 were shown not to regulate TLR3 signaling Singapore, Singapore 119228; †Bioprocessing Technology Institute, Agency for Sci- ence, Technology and Research, Singapore 138668; and ‡Department of Microbiol- (11), the role of DOK3 in TLR3 signal transduction remains to be ogy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore determined. TLR3 recognition of viral dsRNA or its synthetic an- 119228 alog, polyinosinic-polyribocytidylic acid [poly(I:C)] activates the Received for publication June 19, 2013. Accepted for publication May 13, 2014. TRIF-dependent signaling pathway, resulting in IFN-b production. This work was supported by the Agency for Science Technology and Research/ This signaling cascade leads to the formation of the TNFR- Biomedical Research Council. associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 com- S.S.-Y.K., K.-G.L., C.-S.C., S.-K.N., N.A.P., and S.X. performed research; K.-P.L. conceptualized the project; and S.S.-Y.K., K.-G.L., and K.-P.L. analyzed data and plex and activation of TBK1 that subsequently leads to the phos- wrote the paper. phorylation and activation of the transcription factor IFN regulatory Address correspondence and reprint requests to Dr. Kong-Peng Lam, Bioprocessing Tech- factor (IRF) 3 (13). Activated IRF3 dimerizes and translocates from nology Institute, Agency for Science, Technology and Research, No. 06-01, Centros, 20 the cytoplasm to the nucleus to drive ifn-b mRNA synthesis (14). Biopolis Way, Singapore 138668. E-mail address: [email protected] Interestingly, dok3 mRNA was reported to be upregulated in The online version of this article contains supplemental material. virus-infected cells (15). This is in contrast to the demonstrated Abbreviations used in this article: BTK, Bruton’s tyrosine kinase; DOK, down- degradation of DOK1–3 proteins in LPS-stimulated macrophages stream of kinase; HA, hemagglutinin; HDAC1, histone deacetylase 1; IRF, IFN regula- tory factor; NS, nonstructural; PH, pleckstrin homology; poly(I:C), polyinosinic- (12), suggesting that DOK3 could have a different role in TLR3 polyribocytidylic acid; PTB, phosphotyrosine-binding; TBK, TANK-binding kinase; activation in macrophages. In this study, we examined the involve- TRAF, TNFR-associated factor; WT, wild-type. ment of DOK3 in TLR3 signaling and showed that the adaptor was Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 phosphorylated upon poly(I:C) stimulation and played a critical www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301601 The Journal of Immunology 841 role in TLR3-triggered IFN-b production. More importantly, we virus, 59-AAGGGCTTTCACCGAAGAGG-39 and 59-CCCATTCTCAT- demonstrate that DOK3 is needed for the induction of IRF3 by TACTGCTTC-39. facilitating the formation of the upstream TRAF3/TBK1 complex Transfection and reporter assays and the activation of TBK1 and IRF3. HEK293T cells (1 3 105) were seeded in 48-well dishes (Costar) and transfected the following day using Lipofectamine 2000 (Invitrogen). Empty control plasmid was added to ensure that each transfection received Materials and Methods the same amount of total DNA. To normalize for transfection efficiency, Mice, cells, and plasmids 100 ng pRL-TK (Renilla luciferase) reporter plasmid was added to each 2 2 transfection. Approximately 18 h after transfection, luciferase assays were Wild-type C57BL/6 and dok3 / mice were bred in our facilities 2 2 performed using a dual-specific luciferase assay kit (Promega). The IFN-b whereasBruton’styrosinekinase(btk) / mice were obtained from The 2 2 2 2 promoter luciferase reporter plasmid was obtained from Dr. Di Wang Jackson Laboratory (Bar Harbor, ME). tlr3 / and trif / mice were (Zhejiang University, Zhejiang, China). provided by Osamu Takeuchi and Shizuo Akira (Osaka University, Osaka, Japan). Experiments with mice were performed according to guide ELISA lines from the National Advisory Committee on Laboratory Animal Research. Macrophages were differentiated from bone marrow as pre- Untreated or naked poly(I:C)-stimulated or influenza virus–infected WT 2 2 viously described (16). HEK293T cells were transfected with recombi- and dok3 / macrophages were cultured in six-well plates at 5 3 106 nant vectors encoding murine TLR3, DOK3, TRAF, TBK1, TRIF, BTK,or cells/ml. Supernatants were harvested and assayed for cytokine produc- various truncated and site-directed mutants (Stratagene) with hemag- tion. The concentrations of IL-12p40, TNF-a (BD Pharmingen, San Diego, glutinin (HA), FLAG, or GFP tag using Lipofectamine 2000 (Invitrogen, CA), IFN-b (PBL InterferonSource), and RANTES (R&D Systems) were Grand Island, NY).