A Fas-Associated Death Domain Protein/Caspase-8-Signaling Axis Promotes S-Phase Entry and Maintains S6 Kinase Activity in T Cells Responding to IL-2 This information is current as of September 30, 2021. Adrian F. Arechiga, Bryan D. Bell, Sabrina Leverrier, Brian M. Weist, Melissa Porter, Zhengqi Wu, Yuka Kanno, Stephanie J. Ramos, S. Tiong Ong, Richard Siegel and Craig M. Walsh J Immunol 2007; 179:5291-5300; ; Downloaded from doi: 10.4049/jimmunol.179.8.5291 http://www.jimmunol.org/content/179/8/5291 http://www.jimmunol.org/ References This article cites 68 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/179/8/5291.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Fas-Associated Death Domain Protein/Caspase-8-Signaling Axis Promotes S-Phase Entry and Maintains S6 Kinase Activity in T Cells Responding to IL-21 Adrian F. Arechiga,2* Bryan D. Bell,* Sabrina Leverrier,* Brian M. Weist,* Melissa Porter,† Zhengqi Wu,† Yuka Kanno,‡ Stephanie J. Ramos,* S. Tiong Ong,§ Richard Siegel,† and Craig M. Walsh3* Fas-associated death domain protein (FADD) constitutes an essential component of TNFR-induced apoptotic signaling. Paradox- ically, FADD has also been shown to be crucial for lymphocyte development and activation. In this study, we report that FADD is necessary for long-term maintenance of S6 kinase (S6K) activity. S6 phosphorylation at serines 240 and 244 was only observed after long-term stimulation of wild-type cells, roughly corresponding to the time before S-phase entry, and was poorly induced in Downloaded from T cells expressing a dominantly interfering form of FADD (FADDdd), viral FLIP, or possessing a deficiency in caspase-8. Defects in S6K1 phosphorylation were also observed. However, defective S6K1 phosphorylation was not a consequence of a wholesale defect in mammalian target of rapamycin function, because 4E-BP1 phosphorylation following T cell activation was unaffected by FADDdd expression. Although cyclin D3 up-regulation and retinoblastoma hypophosphorylation occurred normally in FADDdd T cells, cyclin E expression and cyclin-dependent kinase 2 activation were markedly impaired in FADDdd T cells. These results demonstrate that a FADD/caspase-8-signaling axis promotes T cell cycle progression and sustained S6K activity. The Journal of http://www.jimmunol.org/ Immunology, 2007, 179: 5291–5300. as-associated death domain protein (FADD)4 is an adaptor way, FADD serves as an adaptor between the ligated death recep- protein for death receptors of the TNF family (including tor and the caspases that directly convey apoptotic signals. Indeed, F Fas, TNFR1, and TRAIL) (1, 2). A death domain motif FADD oligomerization plays a key role in the assembly of a (DD), present within the C terminus of FADD, binds to the cyto- caspase-8-activating platform necessary for death receptor-depen- plasmic tail of TNFR family members, or to the DD-containing dent apoptosis (9–11). In the past, the cellular function of this adaptor TNFR-associated death domain protein (3–5). At the N adaptor has been described as exclusively apoptosis promoting. by guest on September 30, 2021 terminus, FADD contains a death effector domain which homo- However, parallel work has demonstrated that the function of typically interacts with analogous motifs present within the prodo- FADD, like other molecules recruited to ligated death receptors, is main of caspase-8, and in humans, its close relative caspase-10 (6). far more complex (12, 13). When monomers of caspase-8 are brought into close proximity, Although one would predict that mice with a deficiency in they become activated by autoproteolytic processing (7, 8). In this FADD would lack death receptor-mediated apoptosis, thus leading to an accumulation of nonapoptotic cells, transgenic mice express- *Department of Molecular Biology and Biochemistry, and Center for Immunology, ing a dominant-negative form of FADD (denoted FADDdd) do not University of California, Irvine, CA 92697; †Immunoregulation Unit, Autoimmunity manifest lymphoproliferative diseases (12). Paradoxically, previ- Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health, Bethesda, MD 20892; ‡Molecular Immunol- ous results have shown that FADD mutant T cells fail to clonally ogy and Inflammation Branch, NIAMS, National Institutes of Health, Bethesda, MD expand following mitogenic stimulation (14–21). Patients possess- 20892; and §Division of Hematology/Oncology, Department of Medicine, College of ing a mutant form of caspase-8 have an analogous defect in clonal Medicine, University of California, Irvine, CA 92697 expansion (22). However, the defect in human caspase-8-deficient Received for publication March 7, 2007. Accepted for publication August 7, 2007. T cells appears to be broader that that observed in FADDdd mice. The costs of publication of this article were defrayed in part by the payment of page Whereas in the human T cells, caspase-8 activity was necessary for charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. IL-2 expression and NF-␬B activation following TCR cross-link- 1 This work was supported by National Institutes of Health Grant AI050606 (to ing (22, 23), FADDdd T cells expressed normal levels of IL-2 and C.M.W.) and Training Grants T32GM007311, T32CA09054, and T32AI060573 (to induced the NF-␬B-signaling cascade to a similar extent as wild- A.F.A., B.D.B., and S.J.R., respectively). type cells (24). Despite overtly normal TCR-proximal signaling, 2 Current address: Benaroya Research Institute at Virginia Mason, Seattle, WA FADDdd T cells are highly defective in clonal expansion. This 98101. reduced clonal expansion is a consequence of diminished cell cy- 3 Address correspondence and reprint requests to Dr. Craig M. Walsh, Department of Molecular Biology and Biochemistry, and Center for Immunology, University of cle entry and survival, particularly at low doses of TCR cross- California, Irvine, CA 92697-3900. E-mail address: [email protected] linking Abs with exogenously supplied IL-2 (21). These findings 4 Abbreviations used in this paper: FADD, Fas-associated death domain protein; DD, suggest that proliferative responses to IL-2, a cytokine known to death domain protein; FADDdd, dominant-negative form of FADD; v-FLIP, viral regulate both T cell proliferation as well as apoptosis (25), are FLIP; S6K, S6 kinase; CDK, cyclin-dependent kinase; Rap, rapamycin; mTOR, mam- malian target of Rap; Rb, retinoblastoma; Rosc, roscovitine; ChIP, chromatin immu- defective in FADDdd T cells. Mice with IL-2R deficiencies have noprecipitation; 7AAD, 7-aminoactinomycin D; SGK, serum and glucocorticoid-reg- proliferative as well as apoptotic deficiencies, reminiscent of defects ulated kinase. observed in mice with a deficiency in Fas (25, 26). These findings Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 have lead to the hypothesis that there is an important connection www.jimmunol.org 5292 DEFECTIVE S6 KINASE ACTIVITY IN FADDdd T CELLS between IL-2 signaling and death receptor signaling for the regulation all experiments, anti-CD3 was plate-bound by first binding anti-hamster of T cell homeostasis (26, 27). IgG in carbonate buffer, followed by 1 h incubation with the appropriate ␮ As previously reported, FADDdd-expressing T cells had dra- amount of 2C11. zVAD-FMK was used at 150 M (Alexis Biochemi- cals). For inhibition experiments, cells were pretreated before activation matic defects in clonal expansion to anti-CD3 and exogenously for 20 min at 37°C with either LY294002 (LY; 10 ␮M) or rapamycin supplied IL-2, despite normal STAT5 phosphorylation following (Rap; 10 ␮M); these inhibitors were left in the culture for the indicated IL-2R stimulation (21). Similar defects as well as normal STAT5 times. LY and Rap were obtained from Cell Signaling Technology. In phosphorylation have also been seen in transgenic mice expressing some experiments, Rap was added after 18 h stimulation to block mam- malian target of Rap (mTOR) signaling after initial TCR stimulation, the viral protein MC159-viral FLIP (v-FLIP), which blocks acti- but before the initial entry into S phase (18 h). vation of caspase-8 in the Fas-signaling complex (28). A second pathway activated by the IL-2R involves the recruitment of the Src Western blot analysis homology 2-containing adaptor SHC, and the consequent induc- tion of PI3K (25). As assessed by anti-phospho-Akt immunoblot- Following activation, splenocytes were harvested at times indicated. CD4 ting, PI3K activity in response to IL-2 was overtly normal in and CD8 T cells were isolated using anti-CD4 and/or anti-CD8-coated Dynabeads (Dynal Biotech), followed by cell lysis (150 mM NaCl, 50 mM FADDdd T cells. However, using a proteomics approach with an Ab NaF, 10 mM ␤-glycerophosphate, 20 mM HEPES (pH 7.4), 1% Triton generated to detect the phosphorylated substrates of Akt and other X-100, 1 mM PMSF, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 1 mM members of the serum and glucocorticoid-regulated kinase (SGK) NaVO4).