The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation*

Background: Caspase 8 and c-FLIPL regulate NF-κB activation via RIP1. Results: The caspase 8 prodomain mediates NF-κB activation, and its interaction with the RIP1 death domain is inhibited by c-FLIPL but not c-FLIP(p43). Conclusion: The C-terminal domain of c-FLIPL inhibits the interaction of caspase 8 with the RIP1 death domain and caspase 8-dependent NF-κB activation. Significance: This study provides a novel molecular mechanism by which c-FLIPL cleavage is required for NF-κB activation. Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIPL) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIPL regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIPL, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIPL but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIPL specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.

in response to Fas or antigen receptors, caspase 8 can trigger the activation of the transcription factor nuclear factor B (NF-B) that drives many target genes, including antiapoptotic proteins (19 -22).
Cellular FLICE-inhibitory protein (c-FLIP) is a crucial modulator of caspase 8, existing in the various splicing forms c-FLIP L , c-FLIP S , and c-FLIP R (23,24). The full-length c-FLIP L shows overall homology to caspase 8 and contains two N-terminal DEDs followed by the protease domain that lacks the catalytic cysteine, whereas c-FLIP S and c-FLIP R contain only two DEDs and a short C-terminal tail (25,26). Upon heterodimerization with caspase 8 in the DISC, c-FLIP L is processed at Asp-376 into an N-terminal p43 fragment designated c-FLIP(p43) and modulates caspase 8 catalytic activity (27,28). c-FLIP is known to play a regulatory role in the apoptotic and non-apoptotic signaling pathways (25, 26, 29 -31). c-FLIP L inhibits death receptor-mediated apoptosis by preventing the homodimerization of caspase 8 in the DISC, but it can also exert proapoptotic activity under limited conditions (32). c-FLIP possesses the ability to induce the activation of the NF-B signaling pathway (33)(34)(35)(36)(37)(38)(39), and we have shown previously that c-FLIP(p43) is able to induce NF-B activation by interaction with TNF receptor-associated factor 2 (TRAF2) (36). c-FLIP(p22), another c-FLIP fragment cleaved by caspase 8 at Asp-196, has also been shown to mediate NF-B activation by binding to the inhibitor of NF-B (IB) kinase complex (38). By contrast, it has been shown that c-FLIP L and c-FLIP S inhibit NF-B activation induced by FasL (19,20,40). These seemingly contradictory findings may be explained partly by the notion that c-FLIP L and its isoforms can promote or inhibit Fas-mediated NF-B activation, depending on their expression levels and cellular context (39).
RIP1 is known to be critical to induce NF-B activation in response to death receptors and other stimuli (41,42). We have shown that caspase 8 promotes the interaction between RIP1 and c-FLIP(p43) (37). It has also been demonstrated that c-FLIP(p43) interacts with the IB kinase complex upon Fas stimulation (39). Thus, in addition to the specific interaction between c-FLIP(p43) and TRAF2 (36), these studies collectively indicate the importance of c-FLIP L processing in the activation of the NF-B signaling pathway. In this study, we have further explored the molecular mechanism by which c-FLIP L processing regulates NF-B activation. Our results demonstrate that the C-terminal domain of c-FLIP L inhibits the interaction between the caspase 8 prodomain and the RIP1 DD and, thereby, regulates caspase 8-dependent NF-B activation.
Assay for Apoptosis-Cells were stained with Hoechst 33342 (10 M, Merck Millipore) for the last 30 min of incubation. The stained cells were washed with PBS and observed for nuclear morphology under a fluorescence light microscope (Axiovert 200 M, Carl Zeiss, Jena, Germany). Apoptosis (percent) was calculated as (condensed nuclei / total nuclei) ϫ 100.
Immunoprecipitation-Cells were seeded in 60-mm culture dishes the day before transfection. The cells were transfected with various expression vectors, including FLAGtagged expression vectors (total 2 to 4 g DNA), by the calcium phosphate method. The cells were harvested, washed with PBS, and solubilized in Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, and 2 mM sodium vanadate) containing the protease inhibitor mixture Complete TM (Roche Diagnostics). After repeated centrifugation (15,000 ϫ g, 5 min), postnuclear lysates were precleared with Sepharose 6B (Sigma-Aldrich) for 1 h and then immunoprecipitated with anti-FLAG M2 affinity gel (Sigma-Aldrich) for 3 h. The affinity gel was washed several times with Nonidet P-40 lysis buffer.
Western Blotting-Cells were washed with PBS and solubilized in Triton X-100 lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, and 2 mM dithiothreitol) containing the protease inhibitor mixture Complete TM (Roche Diagnostics). Postnuclear lysates were collected as supernatants by centrifugation (15,000 ϫ g, 5 min). Protein samples (30 g/lane) were separated by SDS-PAGE and transferred onto Hybond-ECL nitrocellulose membranes (GE Healthcare). The membranes were incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots of either ␤-actin or equally transfected proteins for immunoprecipitation were used for loading controls. Protein bands were developed by ECL Western blotting detection reagent (GE Healthcare) and detected by exposure to Hyperfilm TM ECL (GE Healthcare) or ImageQuant LAS 4000 Mini (GE Healthcare). ImageQuant LAS 4000 Mini was used to quantify the intensity of the bands.
Statistical Analysis-Statistical significance was assessed by one-way analysis of variance followed by the Tukey's test for multiple comparisons. Differences of p Ͻ 0.05 were considered to be statistically significant.

Caspase 8 Is Required for FasL-induced NF-B Activation-
HEK293 cells expressed Fas and TNF-R1 as well as caspase 8, FADD, TNF receptor 1-associated death domain protein, RIP1, and TRAF2, whereas HEK293C8L cells expressed caspase 8 at very low levels while expressing the other proteins at levels similar to HEK293 cells (Fig. 1A). c-FLIP L and c-FLIP S were only weakly expressed in HEK293 and HEK293C8L cells ( Fig.  1B and data not shown). Upon FasL stimulation, ϳ35% of HEK293 cells underwent apoptosis within 6 h, whereas HEK293C8L cells did not (Fig. 1C). In HEK293C8L cells, apoptosis was increased in response to FasL stimulation when wildtype caspase 8, but not catalytically inactive caspase 8, was transiently expressed (Fig. 1D). NF-B activation was induced by FasL stimulation in HEK293 cells at levels several times lower than TNF-␣ stimulation (Fig. 1E). In HEK293C8L cells, FasL was unable to induce NF-B activation (Fig. 1E). When catalytically inactive caspase 8 was transiently expressed in HEK293C8L cells, NF-B activation was induced by FasL (Fig.  1F). Thus, these results indicate that caspase 8 is required for FasL-induced NF-B activation in HEK293C8L cells.
Augmented NF-B Activation by c-FLIP L , but Not c-FLIP(p43), Requires Caspase Activity-To examine the effect of c-FLIP on FasL-induced NF-B activation, HEK293 cells were transiently transfected with c-FLIP L or its cleaved form, c-FLIP(p43), and then stimulated with FasL. c-FLIP L and c-FLIP(p43) alone increased NF-B activation at high concentrations in HEK293 cells (Fig. 2, A and B). Nevertheless, FasL was able to augment NF-B activation in HEK293 cells transfected with c-FLIP L or c-FLIP(p43) (Fig. 2, A and B). The stimulation index of FasLinduced NF-B activation became smaller when c-FLIP L or c-FLIP(p43) augmented NF-B activation by themselves (Fig. 2,  A and B). The caspase inhibitor zVAD-fmk blocked NF-B activation induced by c-FLIP L , whereas it only marginally affected that induced by c-FLIP(p43) or RIP1 in HEK293 cells (Fig. 2C). This is consistent with our previous observation with HEK293T cells that expressed much higher amounts of protein products (36). To examine the effect of c-FLIP L processing on NF-B activation induced by FasL, HEK293 cells were transiently transfected with c-FLIP L or c-FLIP(p43) and then stimulated with FasL in the presence or absence of zVAD-fmk. In mocktransfected HEK293 cells, zVAD-fmk did not obviously affect FasL-induced NF-B activation (Fig. 2D). However, interestingly, zVAD-fmk markedly inhibited FasL-induced NF-B activation in HEK293 cells transiently transfected with c-FLIP L (Fig. 2D). By contrast, FasL-induced NF-B activation was relatively insensitive to zVAD-fmk in c-FLIP(p43)-transfected cells (Fig. 2D). In mock-transfected HEK293 cells, FasL stimulation induced the cleavage of caspase 8 from p55/p53 into p43/ p41 and p18 (Fig. 2, E-G). In HEK293 cells transfected with c-FLIP L , FasL stimulation increased the cleavage of c-FLIP L into c-FLIP(p43) (Fig. 2E). zVAD-fmk efficiently inhibited the processing of c-FLIP L and caspase 8 (Fig. 2, E-G). By contrast, the FasL-induced cleavage of caspase 8 into its p43/p41 and p18 fragments was not decreased by expression of c-FLIP L or c-FLIP(p43) (Fig. 2, E-G). This might be due to the fact that the dose of FasL was high and overcame the effect of c-FLIP L or c-FLIP(p43) or that the time point was sufficiently long after FasL stimulation to allow substantial caspase 8 cleavage to proceed even with c-FLIP L or c-FLIP(p43). It might be also possible that c-FLIP L or c-FLIP(p43) was expressed between the levels that promote or inhibit the processing of caspase 8 because it

c-FLIP L Inhibits Interaction between Caspase 8 and RIP1
has been shown that c-FLIP L can act as both an activator and an inhibitor of caspase 8 (44 -46). Overall, these results suggest that augmented NF-B activation by c-FLIP L , but not c-FLIP(p43), requires caspase activity in HEK293 cells.
The C-terminal Region of c-FLIP L Inhibits the Interaction between RIP1 DD and Caspase 8-We further investigated whether c-FLIP L regulates the interaction between RIP1 and caspase 8. Endogenous RIP1 was pulled down efficiently with caspase 8 and caspase 8(p43) but barely with c-FLIP L and c-FLIP(p43) in HEK293C8L cells (37). These data indicate that RIP1 interacts with caspase 8 or caspase 8(p43) more preferentially than c-FLIP L or c-FLIP(p43). Moreover, because caspase 8, but not c-FLIP L , interacted with the RIP1 DD (Fig. 4C), we hypothesized that c-FLIP L may inhibit the interaction between caspase 8 and the RIP1 DD. To address whether c-FLIP L inhibits the binding of the RIP1 DD to caspase 8, RIP1(559 -671) was immunoprecipitated with caspase 8 in the presence of c-FLIP L in HEK293C8L cells. zVAD-fmk was included to prevent the processing of c-FLIP L into c-FLIP(p43). c-FLIP L and c-FLIP(p43) were immunoprecipitated with caspase 8 with similar efficacy (Fig. 6A). The interaction of RIP1(559 -671) with caspase 8 was decreased markedly when c-FLIP L was coexpressed (Fig. 6A). By contrast, c-FLIP(p43) allowed the interaction between caspase 8 and RIP1(559 -671) (Fig. 6A). Thus, these data indicate that the C-terminal region of c-FLIP L inhibits the interaction between the RIP1 DD and caspase 8.
In HEK293 cells, cFLIP L , or its uncleavable form, c-FLIP L (D376A), induced spontaneous NF-B activation that was only weakly inhibited by RIP1(559 -671) (Fig. 6, B and C). In addition, FasL was able to induce NF-B activation in the pres-ence of c-FLIP L but exerted weaker NF-B activation in the presence of c-FLIP L (D376A) (Fig. 6, B and C). FasL-induced NF-B activation in the presence of c-FLIP L , but not c-FLIP L (D376A), was inhibited by RIP1(559 -671) (Fig. 6C). RIP1(559 -671) also strongly blocked FasL-induced and spontaneous NF-B activation in the presence of c-FLIP(p43) (Fig.  6C). These results support the view that c-FLIP L regulates a RIP1-dependent and a RIP1-independent pathway to NF-B activation, whereas c-FLIP(p43) regulates only a RIP1-dependent pathway, and c-FLIP L (D376A) regulates only a RIP1-independent pathway. Overall, these findings are consistent with a model in which caspase 8 activity is necessary to cleave c-FLIP L to c-FLIP(p43) to promote a RIP1-dependent pathway of NF-B activation, which may be the main c-FLIP pathway at normal physiological levels.

DISCUSSION
Caspase 8 and its modulator, c-FLIP L , were reported to regulate NF-B activation in response to FasL stimulation. We found that FasL-induced NF-B activation is diminished by zVAD-fmk only in the presence of c-FLIP L but not c-FLIP(p43). The caspase 8 prodomain was identified to interact with the RIP1 DD and to be sufficient to mediate NF-B activation induced by FasL or c-FLIP(p43). Moreover, c-FLIP L , but not c-FLIP(p43), inhibited the interaction between the RIP1 DD and caspase 8. Thus, these results reveal that the C-terminal domain of c-FLIP L specifically inhibits the interaction between the caspase 8 prodomain and the RIP1 DD and, thereby, regulates caspase 8-dependent NF-B activation in response to FasL stimulation.
Caspase 8 is essential to induce NF-B activation in response to FasL stimulation (19,20). By contrast, c-FLIP L and c-FLIP S have been reported to inhibit FasL-induced NF-B activation (19,20,40). Recently, it has been shown that subtle differences in concentrations of c-FLIP isoforms determine apoptosis induction as well as NF-B activation in the Fas signaling pathway (32,39). Thus, it is mostly likely that c-FLIP isoforms are able to regulate FasL-induced NF-B activation positively or negatively under various cellular conditions. Even though the amount of c-FLIP isoforms is much smaller than that of caspase 8, they are efficiently recruited to the DISC because of their higher affinity to the DISC compared with caspase 8 (47). Because c-FLIP L and c-FLIP S inhibit the recruitment of caspase 8 to the Fas-DISC (40,48), it is possible that the inhibition of FasL-induced NF-B activation by c-FLIP L or c-FLIP S is ascribed largely to the inhibition of caspase 8 recruitment to the  FEBRUARY 14, 2014 • VOLUME 289 • NUMBER 7 Fas-DISC. Nevertheless, it has been shown that c-FLIP L does not inhibit caspase 8 recruitment but, rather, promotes the formation of the caspase 8(p43)⅐c-FLIP(p43) heterodimers in the Fas-DISC (32,35). Moreover, c-FLIP L was found to increase the recruitment of RIP1 and TRAF2 to the Fas DISC (35). Thus, c-FLIP L is most likely to mediate NF-B activation in response to FasL stimulation only when c-FLIP L does not inhibit the recruitment of caspase 8 to the Fas DISC but inhibits the full activation of caspase 8, which leads to the formation of the active heterotetramer.

c-FLIP L Inhibits Interaction between Caspase 8 and RIP1
We have shown that FasL-induced NF-B activation is regulated by c-FLIP L at the step of caspase 8-dependent cleavage in the DISC. c-FLIP L is processed by caspase 8 into N-terminal c-FLIP(p43) and C-terminal c-FLIP(p12) in the DISC. The caspase 8 complex associated with either c-FLIP L or c-FLIP(p43) exerts enzymatic activity (27,28). It has been shown recently that the enzymatic activity of the caspase 8⅐c-FLIP complex inhibits RIP1/RIP3-dependent necrosis (17). By contrast, the enzymatic activity of the caspase 8⅐c-FLIP(p43) complex is dispensable for FasL-induced NF-B activation because zVAD-fmk did not inhibit FasL-induced NF-B activation in the presence of c-FLIP(p43). However, zVAD-fmk markedly inhibited NF-B activation induced by FasL stimulation only in the presence of c-FLIP L . Consistent with these results, it has been shown previously that zVAD-fmk inhibits caspase 8-dependent NF-B activation induced by overexpression of c-FLIP L (34,36,38). These studies indicate that c-FLIP L processing to c-FLIP(p43) by caspase 8 is indispensable for FasL-induced NF-B activation. In response to FasL stimulation, it is possible that caspase inhibitors such as zVAD-fmk promote NF-B activation by suppressing caspase 8-dependent apoptosis in Fas-susceptible cells expressing no c-FLIP isoforms. However, as shown in this paper, caspase inhibitors are also likely to block NF-B activation by suppressing the cleavage of c-FLIP L into c-FLIP(p43) in Fas-resistant cells expressing a sufficient amount of c-FLIP L .
As discussed above, caspase 8 activity is required for c-FLIP L cleavage into c-FLIP(p43) but not for downstream molecular events in the FasL-induced NF-B signaling pathway. We showed previously that c-FLIP(p43), but not c-FLIP L , specifically interacts with TRAF2 (36). It has also been reported that c-FLIP(p43) interacts with the IB kinase complex upon Fas stimulation (39). These studies collectively underscore the importance of c-FLIP L cleavage for FasL-induced NF-B activation. Upon TNF-␣ stimulation, TNF receptor 1 recruits TNF receptor 1-associated death domain protein, which binds TRAF2 and RIP1, and in the membrane-associated complex both proteins undergo polyubiquitination, which recruits and activates the TGF-␤-activated kinase (TAK1) complex and the IB kinase complex (49). In agreement with this study, it has been shown that RIP1 plays an essential role in FasL-induced NF-B activation (20). However, in contrast with TNF receptor 1, the caspase 8⅐c-FLIP(p43) heterodimers might be used as a  FEBRUARY 14, 2014 • VOLUME 289 • NUMBER 7 platform that recruits TRAF2 and RIP1 and induces NF-B activation upon FasL stimulation.

c-FLIP L Inhibits Interaction between Caspase 8 and RIP1
RIP1 interacts with multiple proteins, including death receptors, adaptor proteins, and caspases (41,42). Consistent with previous studies (33,50,51), we have shown that RIP1 interacts with caspase 8. However, partly different from these studies (33, 50), we have found that the RIP1 DD is responsible for interac-tion with the caspase 8 prodomain. Other RIP family members that did not contain a DD (including RIP3) were not immunoprecipitated with caspase 8. In addition, the RIP1 DD was immunoprecipitated with caspase 8 but not c-FLIP L . Unlike transfected RIP1, which has been shown to interact with the c-FLIP L in HEK293T cells overexpressing both proteins (33), endogenous RIP1 was immunoprecipitated efficiently with

c-FLIP L Inhibits Interaction between Caspase 8 and RIP1
caspase 8 and caspase 8(p43) but not c-FLIP L . Thus, it is most likely that RIP1 primarily binds caspase 8 via the DD-DED interaction in the caspase 8⅐c-FLIP(p43) heterodimers.
Ripoptosome is a large, 2-MDa intracellular complex that contains RIP1, FADD, caspase 8, caspase 10, and c-FLIP isoforms and regulates the cell death decision between caspasedependent apoptosis and RIP1-dependent necrosis (52,53). In the absence of cellular inhibitor of apoptosis proteins (c-IAPs), c-FLIP L inhibits the formation of Ripoptosome and blocks apoptosis and necrosis, whereas c-FLIP S promotes Ripoptosome formation and necrosis (52). The interaction of RIP1 with caspase 8 in the Ripoptosome is decreased by c-FLIP L but increased by c-FLIP S (52). This is in line with this study showing that the C-terminal domain of c-FLIP L inhibits the binding of RIP1-DD to caspase 8. However, in contrast to c-FLIP L being cleaved efficiently to c-FLIP(p43) by caspase 8 in the Fas-DISC, it is possible that the caspase 8⅐c-FLIP L heterodimer is not efficiently converted to the caspase 8⅐c-FLIP(p43) heterodimer in the Ripoptosome and that the caspase 8⅐c-FLIP L heterodimer is most likely to either interfere with RIP1 recruitment or mediate RIP1 cleavage and, thereby, prevent RIP1/RIP3-dependent necrosis.
Overexpression of c-FLIP L or its uncleavable form, c-FLIP L (D376A), induced spontaneous NF-B activation that was not inhibited by RIP1(559 -671). By contrast, FasL-induced NF-B activation in the presence of c-FLIP L or c-FLIP(p43) was inhibited by RIP1(559 -671). Thus, it is possible that c-FLIP L regulates a RIP1-dependent as well as a RIP1-independent NF-B pathway, whereas c-FLIP(p43) regulates only a RIP1-dependent NF-B pathway, and c-FLIP(D376A) regulates only a RIP1-independent NF-B pathway. We have reported previously that a dominant-negative TRAF2 inhibits NF-B activation induced by c-FLIP(p43) but not that induced by c-FLIP L (D376A) (36). Moreover, we have shown that TRAF2 interacts specifically with c-FLIP(p43) but not c-FLIP L (36). Thus, it is most likely that c-FLIP(p43) mediates NF-B activation in a TRAF2/RIP1dependent manner, whereas c-FLIP(D376A) mediates NF-B activation in a TRAF2/RIP1-independent manner. The molecular mechanism of the NF-B signaling pathway induced by c-FLIP L (D376A) remains to be clarified.
How does c-FLIP L or c-FLIP(p43) regulate the interaction of caspase-8 and RIP1? In this study, we have shown that the C-terminal domain of c-FLIP L inhibits the interaction between caspase 8 and the RIP1 DD. We propose a working model for NF-B activation regulated by caspase 8 and c-FLIP L (Fig. 7). When c-FLIP isoforms are not sufficiently expressed, caspase 8 undergoes homodimerization and autoprocessing in response to FasL stimulation, leading to the formation of active heterotetramers and the induction of apoptosis. In the presence of c-FLIP L , caspase 8 is able to heterodimerize predominantly with c-FLIP L in the Fas-DISC and, subsequently, cleaves c-FLIP L at Asp-376 to produce N-terminal c-FLIP(p43) and C-terminal c-FLIP(p12). The C-terminal domain of c-FLIP L has an ability to inhibit the interaction between the caspase 8 DED and RIP1 DD by a currently unknown mechanism. The removal of c-FLIP(p12) allows caspase 8 to interact with RIP1. In addition, TRAF2 is specifically interacted with c-FLIP(p43) (36). The caspase 8⅐c-FLIP(p43) heterodimer subsequently promotes NF-B activation via RIP1 and TRAF2 in a catalytic

c-FLIP L Inhibits Interaction between Caspase 8 and RIP1
FEBRUARY 14, 2014 • VOLUME 289 • NUMBER 7 activity-independent manner. It has been proposed that the caspase 8⅐c-FLIP L heterodimer inhibits RIP1/RIP3 activity to prevent necrosis in a catalytic activity-dependent manner (54). In several cell lines, the full-length RIP1 was detectable in the Fas DISC and associated with the caspase 8⅐c-FLIP(p43) complex (35,37,51). Therefore, it is possible that RIP1 is not necessarily cleaved in the Fas DISC and plays an essential role in the promotion of NF-B activation within the caspase 8⅐c-FLIP(p43) complex.
In conclusion, we have shown that the C-terminal domain of c-FLIP L blocks FasL-induced NF-B activation by inhibiting the interaction between RIP1 and caspase 8. Thus far, it is well known that c-FLIP L modulates the recruitment of caspase 8 to the DISC as well as the catalytic activity of caspase 8 by heterodimerization. Our current findings provide a novel molecular mechanism by which the caspase 8-dependent c-FLIP L cleavage regulates FasL-induced NF-B activation and may partly explain previously controversial studies on a physiological role of c-FLIP L for FasL-induced NF-B activation.