Caspase-3 Is a Component of Fas Death-Inducing Signaling Complex in Lipid Rafts and Its Activity Is Required for Complete Caspase-8 Activation during This information is current as Fas-Mediated Cell Death of October 2, 2021. Salah M. Aouad, Luchino Y. Cohen, Ehsan Sharif-Askari, Elias K. Haddad, Antoine Alam and Rafick-Pierre Sekaly J Immunol 2004; 172:2316-2323; ; doi: 10.4049/jimmunol.172.4.2316 Downloaded from http://www.jimmunol.org/content/172/4/2316 References This article cites 47 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/172/4/2316.full#ref-list-1 http://www.jimmunol.org/ 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 October 2, 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Caspase-3 Is a Component of Fas Death-Inducing Signaling Complex in Lipid Rafts and Its Activity Is Required for Complete Caspase-8 Activation during Fas-Mediated Cell Death1 Salah M. Aouad,* Luchino Y. Cohen,* Ehsan Sharif-Askari,* Elias K. Haddad,*† Antoine Alam,* and Rafick-Pierre Sekaly2*† Since its discovery, caspase-8 has been placed at the apex of the proteolytic cascade triggered by death receptor (DR) cross-linking. Because of its capacity to interact with the cytoplasmic portion of DR, it has been suggested that caspase-8 acts independently of other caspases in the initiation of Fas and other DR signaling. In this study, we demonstrate that in Jurkat cells, caspase-3 cleavage is an early step during Fas-induced apoptosis. We show that caspase-3 processing into its p20 occurs rapidly after Fas cross- linking, in the absence of mitochondrial depolarization and caspase-9 activation. Moreover, caspase-3 is present in lipid rafts of untreated Jurkat cells and peripheral T lymphocytes. Caspase-3, caspase-8, and Fas-associated death domain are further recruited Downloaded from to lipid rafts of Jurkat cells following anti-Fas treatment. Fas immunoprecipitation reveals that caspase-3 is a component of the death-inducing signaling complex, suggesting that this cysteine protease is in close proximity to caspase-8. Furthermore, trans- duction of Jurkat cells with a caspase-3 dominant-negative form inhibits caspase-8 processing and results in inhibition of apo- ptosis, suggesting that caspase-3 activity is required for caspase-8 activation. Overall, these findings support a model whereby caspase-3 is a component of the death-inducing signaling complex located in lipid rafts, and as such, is involved in the amplification of caspase-8 activity by the mitochondrion. The Journal of Immunology, 2004, 172: 2316–2323. http://www.jimmunol.org/ poptosis or programmed cell death plays a fundamental Fas ligation leads to caspase activation via the Fas-associated role in the development and homeostasis of the immune death domain (FADD) protein, an adaptor molecule that recruits A system. Alteration of the apoptotic machinery in periph- caspase-8 to Fas because FADD interacts with Fas through its eral T lymphocytes leads to abnormal T cell homeostasis and the death domain and with caspase-8 through a death effector domain development of autoimmune diseases in human and mice (1). (DED). This results in the formation of a death-inducing signaling Death receptors (DR)3 are TNFR family members such as TNFRI, complex (DISC) (14–17). Recently, it has been reported that Fas Fas (CD95/APO-1), DR3, DR4, and DR5 that harbor a death do- engagement on murine thymocytes induces the clustering of this main on their intracellular portion (2, 3) and are potent inducers of receptor as well as the recruitment of FADD and caspase-8 in the by guest on October 2, 2021 apoptosis. Ligation of these receptors with their respective ligands lipid-rich plasma membrane compartments called lipid rafts (18). or agonistic Abs leads to apoptosis through the activation of pro- These plasma membrane structures are highly ordered microdo- teases of the caspase family (4, 5). These cysteine proteases trigger mains containing sphingolipids, cholesterol, transmembrane pro- the apoptotic response by cleaving many substrates after aspartic teins, and lipid-anchored proteins (19). They have long been pro- acid residues. To date, 14 members have been identified within the posed as the host of signal initiation of several immune receptors, caspase family (6–8). Cells from mice deficient in some caspases including the TCR, the B cell receptor, and Fc⑀RI (20–23). NF-␬B or expressing viral caspase inhibitors, such as the cytokine re- activation by TNFRI and Fas-mediated apoptosis in thymocytes sponse modifier A or p35, are resistant to apoptosis mediated by are dependent on relocalization of these DR to lipid rafts (24). DR, confirming the requirements of these cysteine proteases in Two pathways observed in different cell types have been pro- DR-mediated apoptosis (9–13). posed for subsequent steps of Fas signaling. In type I cells, the highly efficient recruitment of caspase-8 to the DISC allows this *Laboratoire d’Immunologie, De´partement de Microbiologie et Immunologie, Uni- caspase to act directly on downstream caspases such as caspase-3, versite´de Montre´al, Montreal, Quebec, Canada; and †Department of Microbiology which is responsible for the cleavage of death substrates and ex- and Immunology, McGill University, Montreal, Quebec, Canada ecution of apoptosis (25). In type II cells, recruitment of caspase-8 Received for publication July 14, 2003. Accepted for publication November 26, 2003. to the DISC is weak, but sufficient to trigger mitochondrial events The costs of publication of this article were defrayed in part by the payment of page through processing of the proapoptotic Bcl-2 family member, Bid charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (25, 26). These events culminate in the release of cytochrome c in the cytosol, the formation of the apoptosome, a multimeric cata- 1 This work was supported by the Medical Research Council of Canada, Grant MOP38105 (to R.-P.S.). R.-P.S. holds a Canada Research Chair in Human Immu- lytic complex consisting of cytochrome c, Apaf-1, and caspase-9 nology, and is a senior scientist of the Canadian Institutes for Health Research. that serves to amplify the weak initiating signal resulting from the 2 Address correspondence and reprint requests to Dr. Rafick-Pierre Sekaly, Universite´ altered DISC (25) and the release of second mitochondria-derived de Montre´al, Pavillon principal, De´partement de Microbiologie et Immunologie, C.P. 6128 Succursale Centre-ville, Montreal, Quebec, H3C 3J7, Canada. E-mail address: activator of caspases (Smac)/direct inhibitor of apoptosis-binding rafi[email protected] protein with a low isoelectric point, which antagonizes XIAP-me- 3 Abbreviations used in this paper: DR, death receptor; AnV, annexin V; DED, death diated caspase-3 inhibition (27). Regardless of the cell type, effector domain; DFF, DNA fragmentation factor; DISC, death-inducing signaling caspase-8 is still considered to act independently of other caspases complex; DOX, doxycyclin; FADD, Fas-associated death domain; FLIPL, FLIP-long isoform; PARP, poly(ADP-ribose) polymerase; XIAP, X-linked inhibitor of apoptosis in the initiation phase of Fas-induced apoptosis. The poor recruit- protein; Smac, second mitochondria-derived activator of caspases. ment of caspase-8 to the DISC in type II cells prompted us to dissect Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 2317 events that occur before Fas-mediated mitochondrial alteration and to (BD Biosciences, Mississauga, Ontario, Canada). Assessment of the mi- seek the contribution of another major caspase, caspase-3, to Fas sig- tochondrial transmembrane potential was performed by incubation of cell naling. Our results reveal that this caspase is a component of the DISC suspensions for 30 min at 37°C in PBS containing 12 nM of dihexylox- acarbocyanine iodide (DIOC6) (Molecular Probes, Eugene, OR). The stain- along with caspase-8, that caspase-3 colocalizes in lipid rafts with ing was monitored by FACS using the FL-1 detector following extensive caspase-8, and that caspase-3 activity is required for complete washing in PBS buffer. At least 104 cells were acquired for each sample. caspase-8 activation following Fas cross-linking. Western blotting Materials and Methods Cell lysates (20–50 ␮g protein/lane), immune complexes (for experiments Cells and reagents on DISC collection), or lipid raft fractions were resolved by SDS-PAGE on The Jurkat cell line (clone E6-1) was purchased from the American Type 12–14% gels and transferred to nitrocellulose HyBond-C membranes (Am- Culture Collection (Manassas, VA). The CH11 and M3 anti-Fas Abs were ersham Pharmacia Biotech, Baie d’Urfe´, Quebec, Canada). Membranes obtained from Beckman Coulter (Fullerton, CA) and Immunex (Seattle, were blocked with 5% skimmed milk in PBS/0.05% Tween 20 (PBST) for WA), respectively. Anti-FADD, anti-caspase-8, and anti-caspase-9 Abs 2 h at room temperature, then incubated with the appropriate Ab in the were obtained from BD Transduction Laboratories (Mississauga, Ontario, same solution overnight at 4°C.