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c-FLIPL is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis

2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês

10.1093/emboj/cdf356

1460-2075

David Chang,

Ubiquitin and proteasome pathways

Article15 July 2002free access c-FLIPL is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis David W. Chang David W. Chang Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author Zheng Xing Zheng Xing Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Present address: Department of Molecular and Cell Biology, University of California at Berkley, Berkley, CA, 94720 USA Search for more papers by this author Yi Pan Yi Pan Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author Alicia Algeciras-Schimnich Alicia Algeciras-Schimnich The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, 60637 USA Search for more papers by this author Bryan C. Barnhart Bryan C. Barnhart The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, 60637 USA Search for more papers by this author Shoshanit Yaish-Ohad Shoshanit Yaish-Ohad Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author Marcus E. Peter Marcus E. Peter The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, 60637 USA Search for more papers by this author Xiaolu Yang Corresponding Author Xiaolu Yang Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author David W. Chang David W. Chang Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author Zheng Xing Zheng Xing Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Present address: Department of Molecular and Cell Biology, University of California at Berkley, Berkley, CA, 94720 USA Search for more papers by this author Yi Pan Yi Pan Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author Alicia Algeciras-Schimnich Alicia Algeciras-Schimnich The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, 60637 USA Search for more papers by this author Bryan C. Barnhart Bryan C. Barnhart The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, 60637 USA Search for more papers by this author Shoshanit Yaish-Ohad Shoshanit Yaish-Ohad Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author Marcus E. Peter Marcus E. Peter The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, 60637 USA Search for more papers by this author Xiaolu Yang Corresponding Author Xiaolu Yang Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA Search for more papers by this author Author Information David W. Chang1, Zheng Xing1,2, Yi Pan1, Alicia Algeciras-Schimnich3, Bryan C. Barnhart3, Shoshanit Yaish-Ohad1, Marcus E. Peter3 and Xiaolu Yang 1 1Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 USA 2Present address: Department of Molecular and Cell Biology, University of California at Berkley, Berkley, CA, 94720 USA 3The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL, 60637 USA ‡D.W.Chang, Z.Xing and Y.Pan contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3704-3714https://doi.org/10.1093/emboj/cdf356 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Activation of the caspase cascade is a pivotal step in apoptosis and can occur via death adaptor-mediated homo-oligomerization of initiator procaspases. Here we show that c-FLIPL, a protease-deficient caspase homolog widely regarded as an apoptosis inhibitor, is enriched in the CD95 death-inducing signaling complex (DISC) and potently promotes procaspase-8 activation through hetero-dimerization. c-FLIPL exerts its effect through its protease-like domain, which associates efficiently with the procaspase-8 protease domain and induces the enzymatic activity of the zymogen. Ectopic expression of c-FLIPL at physiologically relevant levels enhances procaspase-8 processing in the CD95 DISC and promotes apoptosis, while a decrease of c-FLIPL expression results in inhibition of apoptosis. c-FLIPL acts as an apoptosis inhibitor only at high ectopic expression levels. Thus, c-FLIPL defines a novel type of caspase regulator, distinct from the death adaptors, that can either promote or inhibit apoptosis. Introduction The key mediators of apoptosis are a group of cysteinyl, aspartate-specific proteases known as caspases (Chang and Yang, 2000). Produced as latent precursors or procaspases, these proteases become activated during apoptosis through proteolytic processing at critical aspartate residues. The activation often occurs sequentially in a cascade, with an initiator procaspase being activated first, which then cleaves and activates executioner procaspases. Active executioner caspases cleave a critical set of cellular proteins to dismantle a cell. Activation of the initiator procaspases is a key regulatory step in apoptosis, and to date all proteins known to promote this activation directly are death adaptors, including the mammalian proteins FADD and Apaf-1 and the Caenorhabditis elegans protein CED-4. During apoptosis, these adaptors bind to the N-terminal prodomain region of procaspases, and facilitate the oligomerization of the C-terminal protease domain. CD95 (APO-1/Fas) is a cell surface death receptor belonging to the TNFR (tumor necrosis factor receptor) superfamily (Peter et al., 1998). Upon binding to the trimeric CD95 ligand or agonistic antibodies, CD95 recruits FADD/MORT1 and procaspase-8 (FLICE/MACH/Mch5) to form the death-inducing signaling complex (DISC), in which procaspase-8 becomes activated (Kischkel et al., 1995; Medema et al., 1997). The precise mechanism of procaspase-8 activation in the DISC is not well understood, although homo-oligomerization of procaspase-8 alone can induce its activation (Martin et al., 1998; Muzio et al., 1998; Yang et al., 1998a). Of particular interest is the role of a procaspase-8-like, protease-deficient protein c-FLIP (Goltsev et al., 1997; Han et al., 1997; Hu et al., 1997; Inohara et al., 1997; Irmler et al., 1997; Shu et al., 1997; Srinivasula et al., 1997; Rasper et al., 1998). c-FLIP is expressed mainly in a long (c-FLIPL) and a short (c-FLIPS) splice form. The latter contains only two tandem repeats of DEDs and inhibits procaspase-8 activation in the DISC (Krueger et al., 2001a). In contrast, c-FLIPL shares extensive homology with procaspase-8, with a C-terminal domain that is highly homologous to the procaspase-8 protease domain yet enzymatically inactive due to the lack of key active site residues. To date, the role of c-FLIPL in apoptosis remains controversial. In most reports, c-FLIPL has been described as anti-apoptotic, largely due to its ability to inhibit apoptosis at high levels of ectopic expression (reviewed in Krueger et al., 2001b). However, in cell lines that have been examined quantitatively, the level of endogenous c-FLIPL is merely 1% of that of endogenous procaspase-8 (Scaffidi et al., 1999). It is unclear what role c-FLIPL plays in these cells. Mice deficient in c-FLIP (which lack both c-FLIPL and c-FLIPS) were generated recently. Embryonic fibroblasts (MEFs) derived from the mice (through an in vitro selection process for cell growth) were shown to be more sensitive to CD95-induced apoptosis than the wild-type MEFs. However, inconsistent with these results, c-FLIP−/− mice showed developmental defects that strikingly resembled those of caspase-8−/− or FADD−/− mice (Yeh et al., 2000). These mice all died between E10.5 and E11.5 with a failure in heart formation and extreme hemorrhage, suggesting a function for c-FLIPL that is similar to that of caspase-8 and FADD. Furthermore, transient overexpression of c-FLIPL could induce as well as inhibit apoptosis, and this pro-apoptotic function required the c-FLIPL protease-like domain (Goltsev et al., 1997; Han et al., 1997; Inohara et al., 1997; Shu et al., 1997). However, it remains undetermined whether c-FLIPL can promote apoptosis at endogenous expression levels and how this might be possible without a protease activity. In the current study, we show that expression of c-FLIPL at levels comparable with the endogenous protein enhances procaspase-8 activation in the DISC and CD95-mediated apoptosis through hetero-dimerization with the protease domain of caspase-8 and enrichment at the DISC, whereas it inhibits apoptosis at high levels of expression. c-FLIPL is therefore a dual-function regulator for caspase-8 activation and apoptosis dependent on its level of expression. Results c-FLIPL potently enhances procaspase-8 activation upon their hetero-dimerization During CD95-mediated apoptosis, both c-FLIPL and procaspase-8 are recruited to the DISC (Scaffidi et al., 1999). To assess the effect of c-FLIPL on procaspase-8 activation, we first mimicked their interaction in the DISC using an inducible dimerization system based on FK506-binding protein (FKBP) and its divalent ligand. We fused the protease domain of procaspase-8 to a derivative of FKBP called Fv (Clackson et al., 1998) (Fv-CASP-8, Figure 6A). After 4 h of treatment with a divalent Fv ligand AP20187, in vitro-translated, [35S]methionine-labeled Fv-CASP-8 underwent autoproteolytic processing (Figure 1A, lane 2). The activation of procaspase-8 in this system closely resembled that which occurred in the DISC (Medema et al., 1997), because it not only yielded the same final products but also proceeded with the same order of events (D.W.Chang, Z.Xing, V.L.Capacio, M.E.Peter and X.Yang, submitted for publication). Interestingly, when Fv-FLIP was included in the reaction, the processing of Fv-CASP-8 was markedly enhanced; >50% of Fv-CASP-8 was cleaved after only 2 h of AP20187 treatment (Figure 1A, lanes 7–9), whereas in the absence of Fv-FLIP virtually no zymogen cleavage was observed at this time point (Figure 1A, lanes 1 and 4). AP20187 could induce not only the Fv-FLIP:Fv-CASP-8 hetero-dimerization but also the homo-dimerization of each of these two Fv fusion proteins. To confirm that the increase in procaspase-8 processing was indeed due to the procaspase-8:c-FLIPL hetero-interaction, we took advantage of rapamycin-induced hetero-dimerization of FKBP and FRB (the rapamycin-binding region of the FKBP– rapamycin-associated protein; Chen et al., 1995). As shown in Figure 1B, forced hetero-dimerization of Fv-CASP-8 with FRB–FLIP (Figure 6A) resulted in the processing of procaspase-8. Interestingly, AP20187- induced procaspase-8:c-FLIPL dimerization also led to the cleavage of c-FLIPL, resulting in the release of its small subunit (Figure 1C, lane 2), and this processing was similar to that which occurred in the DISC (see below). Figure 1.c-FLIPL enhances caspase-8 activation upon induced proximity. (A) The c-FLIPL protease-like domain enhances caspase-8 processing. A 1 μl aliquot of in vitro translated, 35S-labeled Fv-CASP-8 was treated with 100 nM AP20187 for the indicated time (lanes 1 and 2), or treated with vehicle (−) or 100 nM AP20187 (+) for 2 h in the presence of the indicated amounts of in vitro translated, non-radioisotope-labeled Fv-FLIP (lanes 3–10). The reaction mixtures were then resolved by SDS–PAGE, and 35S-labeled products were visualized by autoradiography. The deduced domain structures of the marked bands are shown on the left and the order of processing is marked above. Molecular weight standards (in kDa) are shown on the right. (B) Enhancement of caspase-8 processing by c-FLIPL is due to their hetero-dimerization. A 1 μl aliquot of [35S]Fv-CASP-8 was treated with 1 mM rapamycin (Rap.) for 8 h in the presence of 1 μl of unlabeled FRB-FLIP. The products were analyzed as in (A). No processing was observed in the absence of FRB-FLIP (not shown). Domain structures are shown on the left and molecular weight standards on the right. (C) Dimerization induces processing of Fv-FLIP by caspase-8. A mixture of 1 μl of [35S]Fv-FLIP and 1 μl of unlabeled Fv-CASP-8 or Fv-CASP-8(C360S) was treated with or without AP20187 for 2 h and analyzed as in (A). The deduced domain structures are shown on the left. (D) Caspase-8 processing in transfected cells. 293 cells were transfected with the indicated combinations of Fv-CASP-8 (1 μg), Fv-FLIP (1 μg) and pRK5-CrmA (2 μg). At 13 h after transfection, cells were treated with vehicle (−) or 50 nM AP20187 (+) for 5 h. Cell extracts were analyzed by immunoblotting analysis using an anti-FLAG antibody. (E) Fv-FLIP enhances the cell death activity of caspase-8 in mammalian cells. HeLa cells were transfected with the indicated combinations of Fv constructs (in ng) and pRK5-crmA (1.5 μg, lane 6), together with pCMV-lacZ. At 6 h after transfection, cells were incubated with AP20187 (final concentration 125 nM), FK506 (200 nM) and z-DEVD (5 μM) for 10 h and scored for apoptosis. Download figure Download PowerPoint Figure 2.c-FLIPL functions as an activator for CD95-mediated apoptosis. (A) Dosage-dependent effect of c-FLIPL on CD95-mediated apoptosis. HeLa cells seeded in 6-well plates were transfected with the indicated amounts of c-FLIPL/pcDNA3 construct plus the vector DNA to make the total amount of DNA constant. After 16 h, cells were treated with CH11 (75 ng/ml) plus cycloheximide (1 μg/ml) for 6 h and scored for apoptosis. (B) CD95-mediated apoptosis in MCF7-CD95 cells stably expressing c-FLIPL. Left: expression of c-FLIPL, caspase-8, FADD and CD95 in the MCF7-CD95 cells expressing no exogenous c-FLIPL (MCF7-C) or stably expressing low (MCF7-FLIP-Lo) or high levels (MCF7-FLIP-Hi) of c-FLIPL. The exogenous c-FLIPL (HA-c-FLIPL) migrated more slowly than the endogenous c-FLIPL on SDS–PAGE due to the tags. Right: MCF7-CD95 cells were treated with the indicated concentrations of anti-APO-1 plus protein A (5 ng/ml) for 3 h and scored for apoptosis. These cells were similarly sensitive to staurosporine-induced apoptosis (data not shown). (C) Activation of overall caspase-8 in the MCF7-CD95 cells expressing different levels of c-FLIPL. Different MCF7 cells were treated with anti-APO-1 as in (B) for the indicated times, and whole-cell lysates were analyzed by immunoblotting with the anti-caspase-8 mAb C15, which recognized a region in the large subunit. No p18 band was detected in MCF7-FLIP-Hi cells even after a much longer exposure. (D) Endogenous c-FLIPL is required for CD95-mediated apoptosis. HeLa cells cultured in 12-well plates were transfected with the indicated amount of the c-FLIPL antisense plasmid plus 0.15 μg of pEGFP-N3. The total amount of DNA used for transfection was made constant using vector DNA. Top panel: 24 h after transfection, cells were treated with anti-APO-1 (0.4 ng/ml) plus protein A (5 ng/ml) for 4 h, and green fluorescent protein (GFP)-positive cells were scored for apoptosis. Without CD95 stimulation, the background cell death in each condition was approximately the same and 30% of transfected cells. (Figure 1E, columns 4 and 5, open bars). Cell death was increased further by addition of AP20187 (Figure 1E, columns 4 and 5, closed bars) and inhibited by the monomeric FKBP ligand, FK506 (data not shown). In addition, apoptosis was diminished by the caspase inhibitors crmA (Figure 1E, column 6) and zDEVD-fmk (data not shown), and could not be induced by the active site mutant of Fv-CASP-8 [Fv-CASP-8 (C/S), Figure 1E, column 7]. Similar results were observed when 293 cells were used for transfection (data not shown). This experiment also showed that transfection of ∼8-fold more procaspase-8 DNA was required to induce cell death equivalent to that observed with co-transfection of Fv-CASP-8 and Fv-FLIP (Figure 1E, column 1 versus columns 4 and 5). Therefore, c-FLIPL is a potent activator of caspase-8-mediated killing. Ectopic expression of c-FLIPL at physiologically relevant levels enhances CD95-mediated apoptosis To test the effect of c-FLIPL on CD95-dependent and -independent apoptosis, we introduced increasing amounts of c-FLIPL DNA into HeLa cells. The effect of c-FLIPL was dependent on its expression levels, which can be functionally grouped into three distinct concentration ranges. Very small amounts of c-FLIPL sensitized these cells to CD95-mediated apoptosis (region I in Figure 2A). The pro-apoptotic function of c-FLIPL required the caspase-like domain, as a c-FLIPL mutant lacking the large subunit (c-FLIPLΔ, see Figure 6A) did not show this activity (data not shown). These results raised the possibility that the function of endogenous c-FLIPL in HeLa cells is pro-apoptotic. Consistent with published reports, high levels of ectopic c-FLIPL expression led to decreased CD95 sensitivity (region II in Figure 2A), while at very high expression levels c-FLIPL induced apoptosis on its own (region III in Figure 2A). Similar dosage-dependent effects of c-FLIPL were also observed in another CD95-sensitive cell line MCF7-CD95 (data not shown). At very high expression levels achievable by transient expression, c-FLIPL may associate with and activate procaspase-8 outside of the DISC through both their DED domains and their protease/protease-like domain, which explains c-FLIPL-enhanced apoptosis independent of the DISC. Figure 3.Processing of procaspase-8 and c-FLIPL during CD95-mediated apoptosis. (A) Processing of caspase-8 and c-FLIPL in MCF7-CD95 cells. MCF7-CD95 cells were treated with anti-APO-1 (0.5 μg/ml) plus protein A (10 ng/ml) for the indicated times. Whole-cell lysates were analyzed by immunoblotting with anti-caspase-8 (top and middle) or anti-c-FLIP (bottom) antibody. Molecular weight standards are shown on the right. (B) Processing of c-FLIPL requires caspase-8 activity. Wild-type or caspase-8 deficient Jurkat cells were treated with anti-APO-1 and the cell lysates were analyzed as in (A). The small amount of c-FLIPL processing after prolonged treatment was probably caused by caspase-10, which is also recruited to the DISC (Figure 5C). Download figure Download PowerPoint To further analyze the effect of c-FLIPL on CD95-mediated apoptosis, we generated MCF7-CD95 cells that stably expressed exogenous c-FLIPL. In the clones that expressed exogenous c-FLIPL at levels comparable with the endogenous protein, sensitivity to CD95-mediated apoptosis increased >2-fold, whereas, in clones that had a much higher expression level of c-FLIPL, apoptosis was almost completely blocked (Figure 2B). The effect of c-FLIPL on apoptosis correlated with overall procaspase-8 activation in these cells (Figure 2C). In the cells expressing low levels of exogenous c-FLIPL, overall processing of procaspase-8 was enhanced compared with control cells. In contrast, in cells expressing high levels of c-FLIPL, no mature caspase-8 subunit p18 was detected even though generation of the processing intermediates p43/p41 was evident (Figure 2C). This latter result confirmed a recent report that c-FLIPL at high expression levels blocks the full activation of procaspase-8 (Krueger et al., 2001a). To eliminate effects that could be caused by clonal variation, we also constructed MCF7-CD95 cells that express exogenous c-FLIPL under the control of an inducible metallothionein promotor. Again, maximal apoptosis sensitivity in these inducible cells was observed when the exogenous c-FLIPL level was near that of the endogenous protein, whereas c-FLIPL inhibited apoptosis at higher concentrations (data not shown). Taken together, our results suggest that in both HeLa and MCF7 cells, c-FLIPL at endogenous levels promotes CD95-mediated apoptosis. To test directly for a pro-apoptotic role for endogenous c-FLIPL, we downregulated endogenous c-FLIPL in HeLa cells using an antisense c-FLIPL construct. A 77% reduction in the expression level of endogenous c-FLIPL with no change in expression of caspase-8 resulted in a decrease of CD95 apoptosis sensitivity by ∼50% (Figure 2D). This inhibition occurred in spite of a concurrent reduction of the anti-apoptotic splice variant c-FLIPS (data not shown). This result indicates that in HeLa cells, c-FLIPL is required for maximal apoptosis induced by CD95. Processing of c-FLIPL depends on caspase-8 and occurs very early during CD95-mediated apoptosis In the DISC of most cells, only the processed p43 fragment of c-FLIPL can be detected, suggesting that most of the processed c-FLIPL is cleaved at the DISC. A comparison of the cleavage kinetics between c-FLIPL and procaspase-8 revealed that in MCF7-CD95 cells, processing of c-FLIPL preceded that of procaspase-8 during CD95-mediated apoptosis (Figure 3A). In contrast, c-FLIPS was not cleaved (Figure 3A). The cleavage of c-FLIPL was dependent mainly on caspase-8 because it was severely delayed in Jurkat cells lacking functional caspase-8 (Juo et al., 1998; Figure 3B). Taken together, these results are consistent with the notion that c-FLIPL is involved in procaspase-8 activation in the DISC at the initiation step of CD95-mediated apoptosis. Figure 4.c-FLIPL enhances caspase-8 activation in the DISC. (A) Effects of c-FLIPL on caspase-8 processing in the DISC in MCF7-CD95 cells inducibly expressing c-FLIPL. MCF7-FLIP-In cells were treated with the indicated concentration of ZnCl2 for 5 h. Cells were then stimulated with anti-APO-1 (+) or left untreated (−), and DISC complexes were isolated. Cell lysates and DISC were subjected to immunoblotting analysis using anti-c-FLIP (top two panels) or anti-caspase-8 antibody (bottom two panels). **IgG heavy chain. (B) Caspase-8 processing in MCF7 transfectants stably expressing c-FLIPL. Different MCF7 cells were stimulated with anti-APO-1 or left untreated. Cell lysates and DISC were analyzed by immunoblotting with anti-c-FLIP (top panels) or anti-caspase-8 (bottom panels) antibody. (C) c-FLIPL increases caspase-8 activity in the DISC. A total of 2 × 107 MCF7-c-FLIP-In cells were treated with ZnCl2 for 2 h, washed and kept without ZnCl2 for another 4 h at 37°C. The DISC was isolated and the caspase-8 activity in the DISC was assayed using IETD-AFC. The data shown are representative of three independent experiments. Download figure Download PowerPoint c-FLIPL enhances procaspase-8 activation in the DISC To study directly c-FLIPL's activity in the DISC in living cells, we analyzed the activation of procaspase-8 at the DISC in cells expressing exogenous c-FLIPL either stably or inducibly under the control of the metallothionein promotor. Similar to the Fv-mediated procaspase-8 activation in vitro (Figure 1A), the activation of procaspase-8 in the DISC occurs in two proteolytic steps, with the first cleavage separating the large and small subunits and the second step separating the large subunit and the DISC-binding prodomain. This second step leads to the release of the mature caspase into the cytosol. When higher levels of exogenous c-FLIPL were expressed in the inducible cells, more c-FLIPL was recruited subsequently to the DISC, and concurrently more procaspase-8 was partially processed (Figure 4A, lanes 3 and 4 versus lane 2). Similar results were observed in the c-FLIPL stable transfectants; in cells stably expressing high levels of c-FLIPL, the DISC complex predominantly contained partially processed caspase-8 (Figure 4B). Figure 5.Specific effect of c-FLIPL on caspase-8 and -10 and identification of caspase-10 as a DISC component. (A and B) Fv-FLIP enhances caspase-10 but not caspase-9 processing. A 1 μl aliquot of [35S]Fv-CASP-9 (A) or [35S]Fv-CASP-10 (B) was treated with 100 nM AP20187, in the presence or absence of 1 μl of unlabeled Fv-FLIP. The reaction mix was analyzed as in Figure 1A. Domain structures are shown on the left, and molecular weight standards on the right. (C) Identification of caspase-10 as a DISC component. CD95 was immunoprecipitated from stimulated (DISC) or unstimulated (Ctr) SKW6.4 (S) and H9 (H) cells. CD95 and the associated proteins were analyzed by immunoblotting using appropriate monoclonal antibodies. Download figure Download PowerPoint To confirm that the processed caspase-8 was enzymatically active, we directly assayed the activity of caspase-8 in the DISC using a fluorogenic caspase-8 substrate IETD-AFC. Caspase-8 activity in the DISC was enhanced in cells expressing exogenous c-FLIPL, with the highest activity observed when exogenous c-FLIPL was expressed at a level similar to the endogenous c-FLIPL (Figure 4C, lane 3). However, even high expression levels of c-FLIPL did not completely block the caspase activity of the DISC, and the activity of the c-FLIPL-containing DISC was still higher when compared with the DISC of cells expressing only endogenous c-FLIPL (Figure 4C, lane 4 versus lane 1). This discrepancy was probably due to c-FLIPL's ability to enhance the protease activity of the zymogen form of caspase-8 (see below). Furthermore, in cells stably expressing high levels of c-FLIPL, the amount of caspase-8 in the DISC was only reduced by ∼50% (Figure 4B, lane 5 versus lane 1). This modest decrease in the steady-state level of caspase-8 in the DISC did not appear to account for the nearly complete inhibition of apoptosis in these cells (Figure 2B), but rather the lack of generation of mature caspase-8 subunits in these cells upon CD95 treatment did (Figure 2C), consistent with previous studies by us and others (Scaffidi et al., 1999; Krueger et al., 2001a). Therefore, high levels of c-FLIPL, although promoting the first cleavage of procaspase-8 in the DISC, prevent recruitment of additional cytosolic procaspase-8 to the DISC, thereby inhibiting apoptosis. In contrast, low levels of c-FLIPL expression enhance procaspase-8 activation in the DISC and allow processed caspase-8 to be released from the DISC, resulting in enhanced activation of cytosolic procaspase-8 and apoptosis (Figures 2 and 4). c-FLIPL is a specific activator for procaspases engaged by death receptors To determine whether c-FLIPL affects the activation of other initiator procaspases, we tested its effect on the processing of procaspase-9 and -10. Procaspase-9 is linked to mitochondria-mediated apoptosis, while procaspase-10 is another tandem DED-containing caspase implicated in death receptor signaling. Similar to procaspase-8, dimerization of either procaspase-9 or -10 led to their self-processing (Figure 5A and B; D.W.Chang, Z.Xing, V.L.Capacio, M.E.Peter and X.Yang, submitted for publication). However, c-FLIPL enhanced procaspase-10 but not procaspase-9 activation. These data suggested that caspase-10 might be involved in CD95-mediated apoptosis as a DISC component. We indeed found procaspase-10, together with c-FLIPL, to be part of the endo