Fas-Mediated Cell Death Complete Caspase-8 Activation During Rafts

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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

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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, transduction of Jurkat cells with a caspase-3 dominant-negative form inhibits caspase-8 processing and results in inhibition of apoptosis, 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) (14Ð17). 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 (6Ð8). Cells from mice deficient in some caspases including the TCR, the B cell receptor, and Fc⑀RI (20Ð23). 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 (9Ð13). 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

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 (20Ð50 ␮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 12Ð14% 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. After three washes in PBST, blots were Canada) and BD PharMingen (San Diego, CA). The anti-caspase-3 (28), incubated for1hatroom temperature with HRP-conjugated goat anti- anti-caspase-8, anti-LCK, anti-FLIP, and anti-DNA fragmentation factor rabbit IgG or goat anti-mouse IgG. Blots were then washed three times (DFF) (29) Abs were generated in rabbits in our laboratory, using GST with PBST, revealed by incubation with ECL-Plus Western blotting de- fusions with whole proteins. The anti-poly(ADP-ribose) polymerase tection kit (Amersham Pharmacia Biotech), and then developed on Kodak (PARP) Ab was purchased from Biomol (Plymouth Meeting, PA), and the ﬁlms. anti-Bcl-2 and anti-CD45 Abs from Santa Cruz Biotechnology (Santa

Cruz, CA). The anti-Bap-31 Ab was a gift from G. Shore (Department of Lipid raft extraction Downloaded from Biochemistry, McGill University). HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG used in Western blots were obtained from Jackson Lipid rafts were prepared by sucrose gradient ultracentrifugation of cell 8 ␮ ImmunoResearch Laboratories (West Grove, PA), and N-octylglucoside lysates. Resting Jurkat cells (10 cells) or cells stimulated with 1 g/ml from Sigma-Aldrich (St. Louis, MO). anti-Fas Ab (CH11) for 7 min were washed in ice-cold PBS and lysed in 0.5 ml of cold buffer (1% Triton X-100, 20 mM of MES, and 150 mM of Cell maintenance and transfections NaCl, pH 6.5, containing protease inhibitors (Roche, Basel, Switzerland)). The same procedure was used with 3 ϫ 108 untreated or anti-

Wild-type and Bcl-2-expressing Jurkat T cells (JIB2) were maintained at CD3-stimulated PBMC. The lysates were cleared by centrifugation at http://www.jimmunol.org/ 37¡C and 5% CO2 in RPMI 1640 (Life Technologies, Burlington, Ontario, 14,000 rpm for 30 s at 4¡C, and the supernatants were subjected to sucrose Canada) supplemented with 10% FCS. Bcl-2-inducible expression in Jur- gradient fractionation using ultracentrifugation (100,000 ϫ g,4¡C, 17 h). kat cells was obtained, as follows. The Bcl-2-expressing system was pro- Eleven to 12 fractions of 1 ml were collected, and N-octylglucoside (60 duced by transferring an EcoRI/BglII fragment of the human Bcl-2 cDNA ␮g/ml) was added to dissolve lipids. A total of 10 ␮l of each fraction was from the pIC-Bcl-2 plasmid, kindly provided by J. Adams (Walter and subjected to dot-blot analysis using HRP-conjugated cholera toxin (Sigma- Eliza Hall Institute, Melbourne, Australia), and subcloned into the EcoRI Aldrich) to detect GM1, a positive marker of rafts. A total of 15Ð20 ␮l and BamHI sites of the neo-CMVt vector, a kind gift from J. Hiscott (Lady from each fraction was resolved by SDS-PAGE and subjected to Western Davis Institute, Montreal, Quebec, Canada). The CMVt-Bcl-2 construct blot analysis for the screening of raft-associated proteins using the was transfected by electroporation into a Jurkat clone carrying the CMVt- appropriate Abs. rtTA plasmid. The clones carrying both vectors were selected for puromy- cin and neomycin resistance and subcloned by limiting dilution. The JIB2 by guest on October 2, 2021 clone was selected for its low basal Bcl-2 expression, and high resistance DISC immunoprecipitation to anti-Fas-induced apoptosis following Bcl-2 expression in the presence of Jurkat cells (20 ϫ 106) were left untreated or stimulated with 1 ␮g/ml ␮ 1 g/ml of the tetracycline analog, doxycyclin (DOX; Sigma-Aldrich). anti-Fas Ab (CH-11) for the indicated time points. The stimulation was Fusion protein construction, production, and purification then stopped with ice-cold PBS and cells were lysed, on ice for 30 min, in PBS containing 0.1% Nonidet P-40 detergent and protease inhibitors. Ly- Human caspase-3 cDNA cloned into BamHI-EcoRI sites of the pBSK plas- sates from untreated cells were incubated with 1 ␮g/ml anti-Fas (CH11) on mid has served as a template to introduce a point mutation into caspase-3 ice for 30 min. All the lysates were then centrifuged at 3500 rpm for 10 min catalytic site QA(C163A)RG to generate an inactive dominant-negative at 4¡C. The supernatants were incubated for1hat4¡C with agarose beads variant of caspase-3 (C3DN). This was achieved by a two-round PCR using coupled to goat anti-mouse IgM (Sigma-Aldrich) to immunoprecipitate two sets of overlapping primers. PCR products were then digested using CH-11 immune complexes. The beads were washed extensively with lysis Ј buffer and subsequently recovered by centrifugation, resuspended in non- KpnI and EcoRI restriction sites, then inserted in frame 3 to the His6 stretch/TAT linker of the pTAT vector, a generous gift from S. Dowdy reducing 2ϫ loading buffer, and boiled for 10 min. The composition of Fas (Howard Hughes Medical Institute, Washington University, St. Louis, complexes in different DISC elements was then analyzed by Western blot MO). After verification of the nucleic acid sequences, TAT-C3DN fusion using the appropriate Abs. protein production was conducted in BL21 pLys S strain (Novagen, Mad- ison, WI). Highly expressing clones were selected and induced to produce fusion proteins using isopropyl ␤-D-thiogalactoside (1 mM). Protein was Results extracted from bacteria by sonication in a denaturing buffer composed of Caspase-3 cleavage is triggered independently of mitochondrion 20 mM of HEPES, 150 mM of NaCl, and8Mofurea (pH 8). Resulting in Fas-mediated apoptosis extracts were cleared by centrifugation for 15 min at 6000 rpm. TAT fusions were purified from cleared extracts by affinity chromatography using A Bcl-2-inducible expression system in Jurkat cells under the con- nickel NTA (Invitrogen, Burlington, Ontario, Canada) and eluted by imi- trol of DOX was generated to determine whether the cleavage of dazole (Sigma-Aldrich). The protein was further subjected to gel filtration caspase-3 occurs before or following mitochondrial events during chromatography on Sephadex G-25, PD-10 columns (Pharmacia/Apbio- Fas-induced apoptosis. Bcl-2-negative (ϪDOX) or Bcl-2-express- tech, Baie d’Urfe«, Quebec, Canada) to remove urea and imidazole, then ϩ stored at 4¡C until needed for functional assays. ing Jurkat cells ( DOX) were subjected to anti-Fas treatment and apoptosis levels, mitochondrial transmembrane potential loss, and Apoptosis assay caspase activation were analyzed using AnV assay, DIOC6 stain- Apoptosis was induced with anti-Fas Abs added for the indicated times. ing, and Western blotting, respectively. Four hours after Fas cross- Cell aliquots (105 cells) from each condition were resuspended and incu- linking, up to 70% of Bcl-2-negative cells underwent apoptosis, bated for 10 min in binding buffer (10 mM of HEPES, 150 mM of NaCl, while only 13% of DOX-treated cells displayed signs of apoptosis 5 mM of KCl, 1 mM of MgCl2, and 1.8 mM of CaCl2/pH 7.4) containing 1 ␮g/ml propidium iodide (Sigma-Aldrich) and 1 ␮g/ml FITC-conjugated (Fig. 1A). In Bcl-2-expressing cells, anti-Fas treatment did not trig- annexin V (AnV; BioLynx, San Antonio, TX). Apoptosis was monitored ger a decrease in DiOC6 incorporation (Fig. 1B), confirming the by FACS using the FL-1 and FL-2 detectors on a FACScan flow cytometer inhibitory effect of Bcl-2 on the mitochondrial response to Fas 2318 CASPASE-3 REQUIRED FOR Fas-MEDIATED CASPASE-8 ACTIVATION

FIGURE 1. Fas-induced processing of caspase-3 into p20 cleavage product occurs despite blockade of mitochondrial events. A, Bcl-2-transfected cells were stimulated with 1 ␮g/ml DOX for 36 h to stimulate Bcl-2 expression. Induced (ϩDOX) and noninduced cells (ϪDOX) were then subjected to anti-Fas treatment for 3 h with 250 ng/ml CH-11 anti- Fas Ab. Apoptosis in each condition was monitored by FACS analysis after AnV staining. B, Cells from the same experiment also served to monitor Fas-mediated mitochondrial depolarization by FACS analysis using the mitochondrial dye DiOC6. C, Pro- tein extracts from each time point in the same experiment were immuno- blotted to monitor Bcl-2 expression

and the cleavage status of caspase-3, Downloaded from caspase-8, caspase-9, and PARP. Re- sults shown are representative of three independent experiments.

ligation. In the presence of Bcl-2, procaspase-9 and PARP degra- plete processing of caspase-8, and progression to cell death are http://www.jimmunol.org/ dation in response to Fas cross-linking were totally inhibited and fully dependent on mitochondrial events. caspase-8 cleavage was almost abrogated (Fig. 1C). However, in three independent experiments, generation of the caspase-3 p20 Caspase-3 is present in lipid rafts before Fas engagement cleavage product still occurred following treatment with anti-Fas Cleavage of caspase-3 in the presence of Bcl-2 suggested that it (Fig. 1C). Therefore, a ﬁrst cleavage step between the large and occurred upstream of the mitochondria and possibly at the plasma small subunits of caspase-3 occurs in the absence of mitochondrial membrane. Therefore, we explored the possibility of association of events, indicating that caspase-3 processing into p20 is triggered caspases-3, caspase-8, and other apoptotic components with the either independently or upstream of the mitochondrial cytochrome plasma membrane, particularly lipid rafts. Lipid rafts were puriﬁed c release. In contrast, removal of the prodomain of caspase-3, com- from lysates of untreated Jurkat cells by ultracentrifugation on a by guest on October 2, 2021

FIGURE 2. Caspase-3 and other apoptotic molecules are associated with lipid rafts in resting Jurkat cells and peripheral blood T lymphocytes. A, Untreated Jurkat cells were lysed in 1% Triton X-100 buffer and subjected to lipid raft isolation by sucrose density-gradient ultracentrifugation, as described in Materials and Methods. The distribution of the lipid raft marker, the ganglioside GM-1 in different sucrose gradient fractions was controlled by slot-blot analysis using an HRP-conjugated cholera toxin. B, The same sucrose fractions analyzed in A were subjected to Western blot analysis to verify the distribution of caspase-3, caspase-7, caspase-8, FLIPL, caspase-9, FADD, and Lamin B between raft and nonraft plasma membrane compartments. Results are representative of three independent experiments. C, Human PBMC were left untreated or stimulated with 1 ␮g/ml OKT3 for 8 or 16 h. Apoptosis levels (percentage of annexin-positive cells) and expression of CD69 and Fas were assessed by ﬂow cytometry. D, Lipid raft proteins (fractions 4Ð6) from resting or activated lymphocytes were extracted and subjected to Western blot analysis for the presence of caspase-3 and p56lck. Results are from one representative experiment among two. The Journal of Immunology 2319

sucrose gradient (19, 30). Fig. 2A shows the distribution of GM-1, a well-established raft-associated molecule (31), in fractions obtained from Jurkat cell extracts. Sucrose fractions 4Ð7 were positive for GM-1, and hence contained proteins from lipid rafts. Sur- prisingly, caspase-3 and -7 were reproducibly (n ϭ 3) detected in the same fractions, indicating that a proportion of the caspase-3 and -7 intracellular pools is constitutively located at the membrane level in lipid rafts, even in the absence of Fas engagement (Fig.

2B). Caspase-8, FLIP-long isoform (FLIPL), and FADD were also found in these raft-rich fractions, before Fas cross-linking. Local- ization of these molecules in lipid-rich compartment was specific because caspase-9 was not found in GM-1-positive fractions (Fig. 2B). Furthermore, Lamin B, a protein exclusively found in the nucleus, did not colocalize with caspase-3, -7, and -8, confirming that localization of these caspases in lipid raft fractions did not result from a contamination by nonraft fractions (fractions 10 and 11) during the process of extraction. Lipid rafts were extracted from peripheral blood T cells to determine whether the localization of caspase-3 in lipid rafts could be observed in a more physiolog- ical system. Caspase-3 was found as a constituent of lipid rafts Downloaded from along with the well-known raft-associated molecule p56lck in untreated, nonapoptotic peripheral lymphocytes (Fig. 2, C and D). Caspase-3 localization in these signaling platforms persisted during at least 16 h of stimulation with an anti-CD3 Ab. These data demonstrate that effector caspases are localized in lipid rafts in the absence of Fas cross-linking, and that caspase-3 colocalizes with FIGURE 3. Fas ligation induces an increase in caspase-3 levels in lipid http://www.jimmunol.org/ p56lck in lipid rafts of normal peripheral T lymphocytes. rafts. A, Lipid raft were extracted from Jurkat cells that were left untreated (Ϫ) or stimulated with anti-Fas Ab (ϩ), and raft fraction was analyzed by Fas engagement increases caspase-3 levels in lipid rafts Western blot for the presence of a raft marker, p56Lck, and the absence of Upon receptor engagement, redistribution of signaling molecules a receptor excluded from rafts, the CD45. B, To analyze the effect of Fas between raft and nonraft compartments is indicative of the in- ligation on redistribution of death-signaling molecules, fractions from the same experiment were analyzed for the presence of FADD, caspase-8, and volvement of these molecules in early steps of receptor’s signal caspase-3 by Western blotting. transduction. Hence, we examined the impact of Fas cross-linking on caspase-3 distribution in lipid rafts of Jurkat cells. Results by guest on October 2, 2021 shown in Fig. 3B consistently showed in three independent exper- precipitated DISC before Fas cross-linking (time 0) excludes the iments that caspase-3 was localized in lipid rafts of untreated cells possibility of a contamination by cytoplasmic proteins at this time and that 7 min following Fas ligation, increased levels of caspase-3 point. Together with data from Figs. 2 and 3, these results dem- were associated to these domains. Fas cross-linking also resulted in onstrate that caspase-3 is associated to Fas DISC at the plasma a strong increase in caspase-8 and FADD levels in the fractions membrane. corresponding to lipid rafts (Fig. 3B). On the contrary, CD45 was not recruited to these fractions either before or after Fas cross- Processing of caspase-3 is an early event in Fas-induced linking (Fig. 3A). Therefore, Fas-induced recruitment of caspase-3 apoptosis in Jurkat T cells to lipid rafts, which are well-established signaling platforms, sug- The localization of caspase-3 in lipid rafts and its association with gests that this caspase plays a role in Fas signaling at the plasma elements of the DISC suggest that this caspase is mobilized in the membrane level. Caspase-3 is an integral component of the Fas DISC Because caspase-3 was found in lipid rafts where the DISC is formed, the presence of this protease in the DISC following Fas aggregation was investigated. Jurkat cells were subjected to a time course treatment with anti-Fas Ab and lysed in a mild lysis buffer, and cell surface cross-linked Fas was further isolated by immunoprecipitation. Interestingly, in two independent experiments, caspase-3 was detected in immune complexes of untreated cells (time 0 in Fig. 4), supporting the constitutive localization of this FIGURE 4. Analysis of the Fas-signaling complex (DISC) in the Jurkat caspase in proximity to Fas and its signaling elements (Fig. 4). cell line. Untreated cells (time 0) were first lysed and then incubated with Caspase-3 levels in the DISC gradually increased upon Fas cross- anti-Fas Ab before Fas immunoprecipitation, as described in Materials and linking with a peak at 20-min postligation. Constitutive association Methods. Stimulated cells were incubated with CH11 for the indicated time points at 37¡C before cell lysis, and cell surface Fas was immunoprecipi- and further recruitment to the DISC were specific to caspase-3 tated using anti-mouse IgG-coupled agarose beads. Immune complexes because a protein endowed with a cytoplasmic distribution such as were resolved by SDS-PAGE and subjected to Western blot analysis for ␬ NF- B (p65) was not detected in the DISC at any time point dur- the presence of FADD, caspase-8, caspase-3, and NF-␬B (p65) using re- ing the kinetic (Fig. 4). The increase in FADD levels, but not spective Abs. An aliquot of the total lysate (TL) was loaded as a positive NF-␬B, confirms that only proteins from the DISC were immuno- control. These data are from one of two independent experiments giving precipitated. Furthermore, the absence of FADD in the immuno- the same results. 2320 CASPASE-3 REQUIRED FOR Fas-MEDIATED CASPASE-8 ACTIVATION

FIGURE 5. Caspase-3 cleavage pro- ducing the p20 species occurs early during Fas-induced apoptosis. A, Cells were treated with 250 ng/ml CH11 for indicated times, and apoptosis was monitored by FACS analysis using AnV/propidium iodide double staining. AnVϪ cells were counted as viable, whereas AnVϩ cells were counted as apoptotic. B, Cell extracts from each time point in the same experiment were subjected to Western blot analysis using anti-caspase-3, anti-caspase-3, -caspase-8, -PARP, and -DFF Abs (nonspeciﬁc bands recognized by the anti-caspase-8 antiserum are indicated by an asterisk). C, Two hours following anti-Fas treatment, Jurkat cells were stained with AnV, and positive and negative cells were puriﬁed using a cell sorter. Downloaded from Protein extracts from each population were analyzed by Western blot using anti- caspase-3 or -caspase-8 Abs. http://www.jimmunol.org/ early steps of Fas signaling. To verify this hypothesis, we analyzed samples from the same experiment clearly shows that Fas-medi- the activation status of caspase-3 and -8 in response to Fas ligation ated caspase-8 processing was inhibited by TAT-cas3c/s (Fig. 6C). on Jurkat T cells. The 20-kDa cleavage product (p20) of caspase-3 Together with the inhibition of caspase-8 (but not caspase-3) was detected in lysates from Jurkat cells 60 min following Fas cleavage by Bcl-2, these results demonstrate that caspase-3 activity cross-linking, even before phosphatidylserine expression (Fig. 5, A is essential for complete caspase-8 activation during Fas-induced and B). At this time point, two caspase-3 substrates, DFF and cell death. PARP, were already partially cleaved. Caspase-8 p45, p43, and p18 cleavage products appeared only 120 min after anti-Fas treat-

Discussion by guest on October 2, 2021 ment. To further confirm this early processing of caspase-3, Jurkat Some members of the TNFR family, including nerve growth fac- Ϫ cells were treated with anti-Fas Ab for 2 h, and AnV (viable) or tor/p75, CD40, TNFR, and Fas, are associated with lipid rafts (33Ð ϩ AnV (apoptotic) cells were sorted by flow cytometry. Results 35). Experiments performed using murine thymocytes have shown shown in Fig. 5C clearly demonstrate that caspase-3 processing that caspase-8 and FADD are also recruited to lipid rafts following Ϫ into p20 occurs in Fas-treated viable cells (AnV ), whereas Fas engagement and that disruption of these structures by choles- ϩ caspase-8 cleavage was only observed in AnV apoptotic cells. terol depletion abolishes Fas-triggered DISC formation and cell These results demonstrate that the processing of caspase-3 into its death (18). Furthermore, in a different study, a small amount of p20 form precedes caspase-8 detectable cleavage during Fas-in- caspase-8 and FADD was detected in the DISC before Fas en- duced apoptosis, which corroborates the previous observation that gagement in murine peripheral T lymphocytes (36). Our data show caspase-3 (but not caspase-8) cleavage still occurs in the presence that significant amounts of caspase-3, caspase-7, caspase-8, and of Bcl-2 in anti-Fas-treated Jurkat cells (Fig. 1). FADD are associated with rafts before Fas cross-linking in Jurkat cells. Coprecipitation of caspase-3 with Fas also demonstrated that Role of caspase-3 activity in caspase-8 processing resulting it is a DISC component. These findings are consistent with the from Fas ligation presence of preassembled Fas trimers at the cell surface, in the The strong inhibition of caspase-8, but not caspase-3 processing by absence of Fas ligand, through domains called preligand assembly Bcl-2 prompted us to analyze the effect of caspase-3 inhibition on domains (37, 38). The colocalization of caspase-3 and -8 with lipid Fas-induced caspase-8 activation. We took advantage of the rafts and the fact that caspase-8 processing was blocked by Bcl-2, widely used protein transduction system using the HIV-1 TAT whereas caspase-3 cleavage was still partially processed, sug- peptide that has the ability to enter treated cells by passive diffu- gested a role of caspase-3 in the amplification of caspase-8 acti- sion through plasma membrane (32). A chimeric protein consisting vation during Fas signaling. Indeed, using a caspase-3 dominant- of the TAT peptide and a catalytically inactive variant of caspase-3 negative form (Fig. 6), we have shown that caspase-3 activity is (TAT-cas3c/s) was used to specifically block caspase-3 activity necessary for complete caspase-8 processing, confirming the active and study the effect of this inhibition on caspase-8 activation in participation of caspase-3 in the amplification of Fas-mediated response to Fas ligation. Fig. 6A shows that 100% of Jurkat cells caspase-8 activation. Therefore, this is the first evidence showing treated with a FITC-conjugated chimera became FITC positive, the recruitment of an effector caspase to the signaling complex of demonstrating the high efficiency of the transduction. Pretreatment a death receptor. of Jurkat cells with TAT-cas3c/s resulted in a 65% inhibition of The molecular basis for such recruitment is not yet clear because apoptosis (Fig. 6B), whereas Jurkat cells treated with an irrelevant the short prodomain of caspase-3 does not carry the homotypic fusion protein TAT-p16 remained sensitive to Fas-mediated killing DED motif found in caspase-8. However, other groups have re- (data not shown). Western blot analysis of caspase-8-carried on ported the simultaneous association of caspase-3 and -8 with FLIP The Journal of Immunology 2321

FIGURE 6. Caspase-8 processing in response to Fas-induced apoptosis is inhibited by a dominant-negative form of caspase-3. A, TAT-C3c/s fusion protein was labeled with FITC and incubated with Jurkat cells at a concentration of 0.7 ␮M for 1 h. The transduction capacity of FITC-conjugated TAT- C3c/s was monitored by FACS analysis. B, Jurkat cells treated or not with 0.7 ␮Mof TAT-C3c/s fusion protein were either cul- tured alone or stimulated for the indicated times with anti-Fas Ab. Apoptosis in each condition was monitored by ﬂow cytometry using AnV staining. C, Protein extracts from the same experiment described in A were im- munoblotted with an anti-caspase-8 Ab. Downloaded from

(39), suggesting that this protein might recruit caspase-3 to the shown that once recruited to the DISC, FLIPL is an activator of http://www.jimmunol.org/ DISC. Supporting this hypothesis, we have observed the presence caspase-8 and can promote apoptosis (42, 43). Therefore, FLIPL of FLIPL in lipid rafts of untreated Jurkat cells (Fig. 2). FLIPL is could exert its proapoptotic activity through the recruitment of a catalytically inactive caspase-8-like molecule that was initially caspase-3 in the DISC. How a caspase lacking a DED or a caspase proposed in several studies to inhibit Fas-mediated apoptosis by recruitment domain homotypic motif such as caspase-3 becomes interfering with caspase-8 activation following their concomitant cleaved into p20 following Fas ligation remains unclear. This ac- recruitment to the DISC (40, 41). However, it has recently been tivation could be mediated by autoprocessing as a consequence of by guest on October 2, 2021

FIGURE 7. The preassembly model. In contrast to the type I/II models, the preassembly model proposes the preassociation of caspase-3, caspase-8, and other signaling molecules with the plasma membrane. Upon Fas ligation, higher levels of these molecules are recruited and stabilized in lipid-rich compartments, leading to the formation of a signaling core, the DISC, already containing effector caspases. However, caspase-3 autoprocessing is inhibited by XIAP, and the release of Smac from the mitochondria allows caspase-3 to disso- ciate from XIAP, remove its own prodomain, and trigger caspase-8 processing. This results in the execution of apoptosis through the cleavage of caspase substrates. Therefore, caspase-3 contributes to the amplification of caspase-8 activation. 2322 CASPASE-3 REQUIRED FOR Fas-MEDIATED CASPASE-8 ACTIVATION caspase-3 oligomerization in the rafts, in an induced proximity 6. Kumar, S. 1999. Mechanisms mediating caspase activation in cell death. Cell manner as originally proposed for the activation of caspase-8 (44). Death Differ. 6:1060. 7. Rathmell, J. C., and C. B. Thompson. 1999. The central effectors of cell death in Previous reports have suggested that during Fas-mediated apo- the immune system. Annu. Rev. Immunol. 17:781. ptosis in type II cells, caspase-3 activation is triggered downstream 8. Shi, Y. 2002. Mechanisms of caspase activation and inhibition during apoptosis. of mitochondrial events (25, 26, 45). This suggested that caspase-3 Mol. Cell 9:459. 9. Woo, M., R. Hakem, M. S. Soengas, G. S. Duncan, A. Shahinian, D. Kagi, was only required for the execution of death signals triggered by A. Hakem, M. McCurrach, W. Khoo, S. A. Kaufman, et al. 1998. Essential caspase-8 at the site of initiation. In this study, we show that in- contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. hibiting the intrinsic pathway by Bcl-2 overexpression leads to a Genes Dev. 12:806. 10. Enari, M., H. Hug, and S. Nagata. 1995. Involvement of an ICE-like protease in drastic inhibition of Fas-mediated caspase-8 and -9 activation, but Fas-mediated apoptosis. Nature 375:78. does not prevent caspase-3 from being processed to its p20 form. 11. Tewari, M., L. T. Quan, K. O’Rourke, S. Desnoyers, Z. Zeng, D. R. Beidler, Our results confirm a previous study also showing the generation G. G. Poirier, G. S. Salvesen, and V. M. Dixit. 1995. Yama/CPP32 ␤, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death of the p20 cleavage product of caspase-3 in Bcl-2 stable transfec- substrate poly(ADP-ribose) polymerase. Cell 81:801. tants (46). These results imply that processing of caspase-3 into 12. Varfolomeev, E. E., M. Schuchmann, V. Luria, N. Chiannilkulchai, p20 and p12 is independent of the mitochondrial potential disrup- J. S. Beckmann, I. L. Mett, D. Rebrikov, V. M. Brodianski, O. C. Kemper, O. Kollet, et al. 1998. Targeted disruption of the mouse caspase 8 gene ablates tion and caspase-9 activity, whereas removal of caspase-3 prodo- cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal main (which results in the appearance of the p17), complete prenatally. Immunity 9:267. caspase-8 activation, and execution of apoptosis depend on these 13. Hisahara, S., T. Araki, F. Sugiyama, K. Yagami, M. Suzuki, K. Abe, K. Yamamura, J. Miyazaki, T. Momoi, T. Saruta, et al. 2000. Targeted expression events. of baculovirus p35 caspase inhibitor in oligodendrocytes protects mice against It was previously shown that in the presence of Bcl-2, the p20 autoimmune-mediated demyelination. EMBO J. 19:341. form of caspase-3 remained associated to XIAP, because Bcl-2 14. Boldin, M. P., T. M. Goncharov, Y. V. Goltsev, and D. Wallach. 1996. Involve- Downloaded from ment of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and inhibits the release of Smac from the mitochondria into the cytosol TNF receptor-induced cell death. Cell 85:803. (46). We have demonstrated in this study that caspase-3 activity 15. Fernandes-Alnemri, T., R. C. Armstrong, J. Krebs, S. M. Srinivasula, L. Wang, was required for complete caspase-8 processing following Fas en- F. Bullrich, L. C. Fritz, J. A. Trapani, K. J. Tomaselli, G. Litwack, and E. S. Alnemri. 1996. In vitro activation of CPP32 and Mch3 by Mch4, a novel gagement. This result suggests that before Smac release into the human apoptotic cysteine protease containing two FADD-like domains. Proc. cytosol, which is inhibited by Bcl-2, caspase-3 is associated to Natl. Acad. Sci. USA 93:7464. XIAP and cannot remove its prodomain or trigger the complete 16. Muzio, M., A. M. Chinnaiyan, F. C. Kischkel, K. O’Rourke, A. Shevchenko, http://www.jimmunol.org/ J. Ni, C. Scaffidi, J. D. Bretz, M. Zhang, R. Gentz, et al. 1996. FLICE, a novel cleavage of caspase-8. Interestingly, in Jurkat cytosolic extracts, FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/ caspase-8 activation induced by addition of cytochrome c was APO-1) death-inducing signaling complex. Cell 85:817. completely abrogated when caspase-3 was depleted (47). This ob- 17. Kischkel, F. C., S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, P. H. Krammer, and M. E. Peter. 1995. Cytotoxicity-dependent APO-1 (Fas/ servation corroborates our results obtained in intact Jurkat cells CD95)-associated proteins form a death-inducing signaling complex (DISC) with and confirms the requirement for caspase-3 activity in caspase-8 the receptor. EMBO J. 14:5579. activation. 18. Hueber, A. O., A. M. Bernard, Z. Herincs, A. Couzinet, and H. T. He. 2002. An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell The results presented in this work are consistent with a model in death in mouse thymocytes. EMBO Rep. 3:190. which critical components of Fas signaling are preassembled in the 19. Brown, D. A., and E. London. 1998. Functions of lipid rafts in biological mem- by guest on October 2, 2021 plasma membrane, notably in the lipid-rich compartment, as sum- branes. Annu. Rev. Cell. Dev. Biol. 14:111. 20. Janes, P. W., S. C. Ley, and A. I. Magee. 1999. Aggregation of lipid rafts ac- marized in Fig. 7. Upon Fas cross-linking, the association of companies signaling via the T cell antigen receptor. J. Cell Biol. 147:447. FADD, caspase-8, and caspase-3 in the DISC increases and leads 21. Drevot, P., C. Langlet, X. J. Guo, A. M. Bernard, O. Colard, J. P. Chauvin, to caspase-3 processing into its 20- and 12-kDa subunits. The par- R. Lasserre, and H. T. He. 2002. 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