Tumor Suppressor p14ARF Enhances IFN- −γ Activated Immune Response by Inhibiting PIAS1 via SUMOylation This information is current as Jennifer Alagu, Yoko Itahana, Faizal Sim, Sheng-Hao Chao, of September 27, 2021. Xuezhi Bi and Koji Itahana J Immunol published online 30 May 2018 http://www.jimmunol.org/content/early/2018/05/29/jimmun ol.1800327 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/05/29/jimmunol.180032 Material 7.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 May 30, 2018, doi:10.4049/jimmunol.1800327 The Journal of Immunology Tumor Suppressor p14ARF Enhances IFN-g–Activated Immune Response by Inhibiting PIAS1 via SUMOylation Jennifer Alagu,* Yoko Itahana,* Faizal Sim,† Sheng-Hao Chao,‡,x Xuezhi Bi,‡ and Koji Itahana* The ability of cells to induce the appropriate transcriptional response to inflammatory stimuli is crucial for the timely induction of host defense mechanisms. Although a role for tumor suppressor p14ARF (ARF) in the innate immune response was previously demonstrated, the underlying mechanism is still unclear. ARF is a potent upregulator of protein SUMOylation; however, no as- sociation of this function with the immune system has been made. In this study, we show the unique role of ARF in IFN-g–induced immune response using human cell lines. Through a systematic search of proteins SUMOylated by ARF, we identified PIAS1, an inhibitor of IFN-activated transcription factor STAT1, as a novel ARF-binding partner and SUMOylation target. In response to IFN-g treatment, ARF promoted PIAS1 SUMOylation to inhibit the ability of PIAS1 to attenuate IFN-g response. Wild-type, but Downloaded from not ARF mutants unable to enhance PIAS1 SUMOylation, prevented the PIAS1-mediated inhibition of IFN-g response. Con- versely, the SUMO-deconjugase SENP1 deSUMOylated PIAS1 to reactivate PIAS1 that was inhibited by ARF. These findings suggest that PIAS1 function is negatively modulated by SUMO modification and that SUMOylation by ARF is required to inhibit PIAS1 activity and restore IFN-g–induced transcription. In the presence of ARF, in which case PIAS1 is inhibited, depletion of PIAS1 did not have an additive effect on IFN-g response, suggesting that ARF-mediated enhancement of IFN-g response is mainly due to PIAS1 inhibition. Our findings reveal a novel function of ARF to inhibit PIAS1 by enhancing SUMOylation to promote the http://www.jimmunol.org/ robust induction of IFN-g response. The Journal of Immunology, 2018, 201: 000–000. nflammation is a protective mechanism regulated by the inflammatory signaling is a critical factor in determining whether host’s immune system against potential or further harm. inflammation is resolved or prolonged. A chronic inflamma- I Detection of injury, infection, or transforming agents triggers tory microenvironment promotes tumorigenesis as well as other the release of chemokines from affected and surrounding cells for chronic illnesses (1). the acute mounting of immune cells. Activated immune cells se- The IFNs mediate antitumor and antiviral responses. Type I IFNs crete proinflammatory cytokines such as IFNs and TNF-a and (IFN-a and IFN-b) are produced by all nucleated cells and bind by guest on September 27, 2021 chemokines to promote cytotoxicity toward infected cells and IFN-a/b receptor subunits to induce either the homodimerization microorganisms until clearance of threat is achieved. The ability of STAT1 or heterodimerization of STAT1 and STAT2. Type II of host cells to appropriately sense, respond to, and relay IFN (IFN-g) is secreted by immune cells and binds to the IFN-g receptor to induce STAT1 homodimerization. The activated *Cancer and Stem Cell Biology Program, Duke–NUS Medical School, Singapore dimers transcriptionally initiate an inflammatory response from 169857, Singapore; †Temasek Polytechnic School of Applied Science, Singapore the promoters of IFN-stimulated genes (ISGs) that contain IFN- ‡ 529757, Singapore; Proteomics Groups, Bioprocessing Technology Institute, stimulated response elements (ISRE) and/or IFN-g–activated site Agency for Science, Technology and Research, Singapore 138668, Singapore; and xDepartment of Microbiology, National University of Singapore, Singapore 117597, (GAS) elements (1, 2). Singapore The tumor suppressor protein p14ARF (ARF) is expressed ORCIDs: 0000-0003-3560-4560 (Y.I.); 0000-0001-9475-8709 (S.-H.C.); 0000-0001- ubiquitously at very low levels and upregulated in response to 6702-0805 (X.B.); 0000-0002-7241-2894 (K.I.). oncogenic stress (3). The canonical function of ARF is to inhibit Received for publication March 5, 2018. Accepted for publication May 7, 2018. the E3 ligase activity of MDM2 toward p53, resulting in stabili- This work was supported by a Duke–NUS Medical School core grant, Singapore zation of p53 to induce cell cycle arrest, senescence, or apoptosis Ministry of Health’s National Medical Research Council Grant NMRC/OFIRG/ 15nov049/2016, National Research Foundation Competitive Research Program upon oncogenic stress. As an obstacle for transformation, ARF Grant NRF2012NRF-CRP001-056, and Singapore Ministry of Education Academic is lost in ∼40% of human cancers (4). Recently, several reports Research Fund Tier 2 Grants MOE2013-T2-2-123 and MOE2017-T2-1-081 (to K.I.). demonstrated that ARF has a noncanonical function to sense J.A., F.S., and X.B. performed experiments. J.A., Y.I., S.-H.C., and K.I. designed the inflammatory stimuli (5) and promote the robust induction of experiments and J.A. analyzed the results. J.A., Y.I., and K.I. interpreted the results and wrote the manuscript. proinflammatory genes independently of p53 (6). The underlying Address correspondence and reprint requests to Prof. Koji Itahana, Cancer and Stem mechanisms, however, are still unclear. Cell Biology Program, Duke–NUS Medical School, Room 07-18, Level 7, 8 College The promotion of small ubiquitin-like modifier (SUMO) con- Road, Singapore 169857, Singapore. E-mail address: [email protected] jugation on various proteins is another p53-independent function of The online version of this article contains supplemental material. ARF (7). SUMOylation is a posttranslational process by which Abbreviations used in this article: ARF, p14ARF; co-IP, coimmunoprecipitation; proteins are modified via the sequential actions of E1 (SAE1/2), GAS, IFN-g–activated site; ISRE, IFN-stimulated response element; LC-MS, liquid chromatography–mass spectrometry; MS, mass spectrometry; NF-kB-RE, NF-kB- E2 (Ubc9), and E3 enzymes in a manner analogous to ubiquitination. response element; PIAS, protein inhibitor of activated STAT; SENP, sentrin- SUMOylation serves to regulate protein activity, stability, and specific protease; siRNA, small interfering RNA; STUbL, SUMO-targeted ubiquitin localization. The conjugation of SUMO2 and SUMO3 is pre- ligase, SUMO, small ubiquitin-like modifier. dominantly induced as a protective response to cellular stresses Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 such as infection and inflammatory stimuli (8, 9). Although ARF www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800327 2 ARF PROMOTES IFN-g RESPONSE BY INHIBITING PIAS1 is not a SUMO E3 ligase, ARF has been shown to promote the CTGCGGATCCAG-39 and 59-CTGGATCCGCAGTCAACCTCTCTT- SUMOylation of its interaction partners such as p53 (10, 11), TAATTAAAGCTC-39; K368R, 59-CATTCAGATGAATGAGAGAAA- MDM2 (12), NPM1 (13), and Werner’s helicase (14). We have ACCAACCTGGGTTTG-39 and 59-CAAACCCAGGTTGGTTTTCTC- TCATTCATCTGAATG-39; K408, 59-GATGAAATACAATTTAGGGA- previously reported that ARF enhances NPM1 SUMOylation to GGATGGCACTTGGG-39 and 59-CCCAAGTGCCATCCTCCCTAAA- repress centrosome amplification (13). However, none have been TTGTATTTCATC-39; and K456, 59-GTCCTCAAATAAAAACAAGA- characterized for immunological roles. GAGTAGAAGTGATTGACCTAAC-3 9 and 59-GTTAGGTCAATCAC- Protein inhibitor of activated STAT (PIAS) family proteins were TTCTACTCTCTTGTTTTTATTTGAGGAC-39. ARF constructs were previously described elsewhere (13). Adenoviral ARF was generated originally identified as inhibitors of STATactivity that also possess by ViraQuest. All transfections were carried out via calcium phosphate an E3 SUMO ligase property. Four members constitute the PIAS precipitation. family and share more than 40% sequence similarity and conserved functional domains (15, 16). During IFN signaling, PIAS1 is phos- Cell culture and reagents phorylated to suppress STAT1 activity (17, 18). PIAS1 inhibits H1299 lung carcinoma, U2OS osteosarcoma, HEK 293T kidney cells, and STAT1 activity by several mechanisms such as through binding the WI-38 fibroblast cell lines (ATCC) were cultured in DMEM supplemented