A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation This information is current as of October 1, 2021. Di Peng, Zining Wang, Anfei Huang, Yong Zhao and F. Xiao-Feng Qin J Immunol published online 12 December 2016 http://www.jimmunol.org/content/early/2016/12/09/jimmun ol.1601009 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/12/09/jimmunol.160100 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published December 12, 2016, doi:10.4049/jimmunol.1601009 The Journal of Immunology A Novel Function of F-Box Protein FBXO17 in Negative Regulation of Type I IFN Signaling by Recruiting PP2A for IFN Regulatory Factor 3 Deactivation Di Peng,*,† Zining Wang,*,† Anfei Huang,‡ Yong Zhao,*,† and F. Xiao-Feng Qin‡ The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the sta- bility of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimer- ization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the Downloaded from protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A. The Journal of Immunology, 2017, 198: 000–000. uring studies of cell cycle regulators, the F-box motif was studies have reported that F-box proteins regulate specific sub- first found in cyclin F and was shown to interact with strates in diverse biological processes, including cell growth and http://www.jimmunol.org/ D S-phase kinase-associated protein 1 (SKP1) (1). The division, cell development and differentiation, and cell survival family of F-box proteins, which is characterized by this novel and death (6). Consequently, dysfunction of these proteins is motif, was identified through screening proteins that bind SKP1 closely linked with cancers and other diseases (6–9). However, the (2, 3). Traditionally, F-box proteins function as a component of role for F-box proteins in innate immunity remain poorly studied, the SKP1-Cullin1-F-box (SCF) E3 ligase complex to initiate and thus far there is no report about involvement of F-box proteins ubiquitin-mediated targeted substrate destruction (4, 5). Previous in antiviral events through type I IFN (IFN-I) signaling. In innate immunity, the IFN-Is induced by the activation of IFN-I signaling cascade play a central role in monitoring and defending by guest on October 1, 2021 *State Key Laboratory of Biocontrol, Sun Yat-Sen University, Guangzhou 510275, against viral infection as well as guiding the subsequent adaptive † China; Key Laboratory of Gene Engineering of the Ministry of Education, Sun Yat- immune responses (10–13). During the processes of viral infection Sen University, Guangzhou 510275, China; ‡Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union and replication, host pattern recognition receptors sense the Medical College, Beijing 100005, China; and Suzhou Institute of Systems Medicine, nucleic acid and other components from viruses, termed pathogen- Suzhou, Jiangsu 215123, China associated molecular patterns, to mount a series of signaling Received for publication June 13, 2016. Accepted for publication November 11, cascade resulting in the production of IFN-Is and other cytokines 2016. (13–15). These secreted IFNs further induce and amplify the This work was supported by the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (Grant 2016-I2M-1-005), the Ministry of Health (Grant downstream transcriptional activation of a large number of IFN- 201302018), the National Major Scientific and Technological Special Project for stimulated genes through the JAK-STAT pathway, which cumu- Significant New Drugs Development (Grant 2015ZX09102023), the National Natural late to an effective restriction and elimination of pathogens by the Science Foundation of Key Projects (Grant 81590765), and the Guangdong Innova- tive Research Team Program (Grant 201001Y0104687244). host (16, 17). In addition, induction of IFN-I signaling pathway is k Address correspondence and reprint requests to Prof. F. Xiao-Feng Qin or Prof. often accompanied by NF- B activation during microbial infection, Yong Zhao, Center of Systems Medicine, Institute of Basic Medical Sciences, which act in concert to induce a number of other proinflammatory Chinese Academy of Medical Sciences and Peking Union Medical College, molecules required for successful host defense (18). Weak IFN-I No. 5, Dong Dan San Tiao Street, Beijing 100005, China (F.X.-F.Q.) or State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of the signaling results in insufficient IFN-I output needed for antiviral Ministry of Education, Sun Yat-Sen University, No. 135, Xingang Xi Road, immunity (19). In contrast, chronic activation of cellular antiviral Guangzhou 510275, China (Y.Z.). E-mail addresses: [email protected] (F.X.-F.Q.) responses might lead to the prolonged and excess production of or [email protected] (Y.Z.) IFN-Is, which is deleterious and can provoke autoimmune disorders The online version of this article contains supplemental material. (19–22). Therefore, it is critical to build and integrate multiple Abbreviations used in this article: Co-IP, coimmunoprecipitation; FBA, F-box asso- ciated; HA, HRP-anti-hemagglutinin; IAD, IFN regulatory factor association do- positive and negative circuitries for regulating IFN-I signaling to main; IFN-I, type I IFN; IKKi, IkB kinase ε; IRF3, IFN regulatory factor 3; ISRE, ensure the homeostasis of IFN-I production by the host. IFN-sensitive response element; MAVS, mitochondrial antiviral-signaling protein; IFN regulatory factor 3 (IRF3) is a member of the IFN regulatory MOI, multiplicity of infection; poly(dA:dT), poly(deoxyadenylic-deoxythymidylic) acid; poly(I:C), polyinosinic-polycytidylic acid; PP2A, protein phosphatase 2; SCF, factors family, which plays a central role in IFN-I induction and SKP1-Cullin1-F-box; SeV, Sendai virus; sgRNA, short guide RNA; siRNA, small antiviral response (13, 23, 24). Upon activation of RNA and DNA interfering RNA; TBK1, TANK-binding kinase 1; VSV, vesicular stomatitis virus. sensors, the key adaptor molecules TIR domain-containing This article is distributed under The American Association of Immunologists, Inc., adapter inducing IFN-b (TRIF), mitochondrial antiviral signal- Reuse Terms and Conditions for Author Choice articles. ing protein (MAVS), and stimulator of IFN genes (STING) in Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 the downstream pathway recruit and activate the TANK-binding www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601009 2 FBXO17 NEGATIVELY REGULATES TYPE I IFN SIGNALING kinase 1 (TBK1) or IkB kinase ε (IKKi) to phosphorylate IRF3 Luciferase reporter kit (Promega) in a Synergy II luminometer (Biotech) (25–27). The phosphorylated IRF3 undergoes dimerization and according to the manufacturer’s protocol. then translocates from the cell cytoplasm to the nucleus to initiate RNA preparation and real-time PCR the transcription of IFN-Is and other target genes (28–30). Hence, Total RNA was extracted from HEK293T, THP-1, and A549 cells using controlling IRF3 activation is critical for the early regulation of TRIzol (Invitrogen) following the manufacturer’s instructions. cDNAs IFN-I signaling. Indeed, a body of previous studies have demon- were transcribed from the extracted total RNA using the PrimeScript RT strated that different molecules and mechanisms have been Reagent Kit with gDNA Eraser (Takara). Real-time PCR was performed employed for regulating the activation of IRF3. For example,