Rapid Turnover of c-FLIPshort Is Determined by Its Unique C-terminal Tail

Abstract

Death receptors are cell surface receptors that belong to the tumor necrosis factor receptor superfamily, the most well known members of which are tumor necrosis factor receptor 1, the CD95/Fas receptor, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 1 receptors DR4/TRAIL-R1 and DR5/TRAIL-R2 (for a review, see Ref. 1). Upon ligand-mediated oligomerization of CD95/Fas or TRAIL receptors, the Fas-associated death domain protein (FADD) attaches to the death receptor via homophilic death domain interactions. The death effector domain (DED) of FADD is in turn linked to the apoptotic machinery due to its affinity for the initiator caspases, procaspase-8 and procaspase-10. The close proximity of procaspase-8 molecules results in dimerization of the procaspases, the assembly of which forms an enzymatically active site (2, 3). This enzymatic activity induces serial cleavages converting the zymogen into p10 and p18 fragments, forming the proteolytically active caspase-8 heterotetramer. In responsive cells, activated caspase-8 is then able to activate effector caspases thereby initiating apoptosis (for a review, see Ref. 4). In addition to the initiator caspases, the DED in FADD also interacts with the apoptosis modulator cellular FLICE-inhibitory protein (c-FLIP; Ref. 5), which exists as two splice variants, the long splice form of FLIP (FLIP L ) and the short splice form of FLIP (FLIP S ). Together with the activated death receptor, FADD, and caspase-8 and -10, the FLIP proteins form the core of the death-inducing signaling complex (DISC; Ref. 6), which also contains a number of other proteins, many of which seem to be involved in modulating death receptor signals (for a review, see Ref. 1). The DISC assembly with pro-and antiapoptotic proteins allows for control of death receptor signaling through a number of different routes. Consequently in addition to apoptosis, stimulation of the receptors may also lead to cell survival and proliferation. Modulation of the apoptotic pathway at the level of the activated receptor is required in situations when the extrinsic, but not the intrinsic, apoptosis pathway needs to be inhibited or when localized initiator caspase activity is required for specialized signaling functions. For example, caspase-8 activity is necessary for normal T cell development (7,8) and is probably acquired through complex regulation of anti-and proapoptotic proteins in the DISC. In addition, some malignant cells are able to convert death receptor stimulation into proliferative signals (9, 10). Therefore, active regulation of the DISC proteins, both transcriptional and post-translational, determines the outcome of death receptor stimulation.c-FLIP L and c-FLIP S have been characterized as specific inhibitors of death receptor-mediated apoptosis (for a review, see Ref. 11). c-FLIP L is homologous to caspase-8, consisting of two tandemly repeated DEDs and a catalytically inactive caspase-like domain. Although c-FLIP S shares most of its se-* This work was supported by the Academy of Finlan...

Quercetin sensitizes human hepatoma cells to TRAIL‐induced apoptosis via Sp1‐mediated DR5 up‐regulation and proteasome‐mediated c‐FLIPS down‐regulation

“…We found that quercetin-induced downregulation of c-FLIP is not controlled at the transcriptional level, but rather is mediated via the proteasomal pathway, as shown by our observation that pretreatment with MG132 attenuated the quercetin-induced down-regulation of c-FLIP L and c-FLIP S . Similar to our results, a recent study showed that FLIP S is more prone to ubiquitination and has a considerably shorter half-life than FLIP L [Poukkula et al, 2005]. Very interestingly, we found that overexpression of c-FLIP S, but not c-FLIP L , significantly reduced the apoptosis induced by quercetin/TRAIL treatment.…”

Mutational analyses of c-FLIPR, the only murine short FLIP isoform, reveal requirements for DISC recruitment

“…This suggests that the C-terminal tail influences the antiapoptotic function of mouse c-FLIP R , which is in contrast to human c-FLIP short whose C-terminal deletion did not interfere with apoptosis inhibition. 20 This conclusion is supported by our observation that incubation of cells with high concentrations of CD95L for longer time periods activated caspase-8 in c-FLIP R -DC-but not c-FLIP R -expressing cells, as shown by the appearance of the p18 caspase-8 fragment (Figure 3c). …”

“…The reduced antiapoptotic activity of c-FLIP R -DC is in contrast to C-terminal deletion mutants of human c-FLIP short or MC159, which suppressed death receptor-mediated apoptosis equally as the wild-type proteins. 20,23 Accordingly, an equal incorporation of c-FLIP short and its C-terminal deletion mutant into the TRAIL-DISC has been observed. 20 Although we cannot exclude that the disparity of the mutants in DISC binding might be attributed to different experimental systems, our results suggest that the C-terminus of c-FLIP R mediates DISC interaction.…”

“…20,23 Accordingly, an equal incorporation of c-FLIP short and its C-terminal deletion mutant into the TRAIL-DISC has been observed. 20 Although we cannot exclude that the disparity of the mutants in DISC binding might be attributed to different experimental systems, our results suggest that the C-terminus of c-FLIP R mediates DISC interaction. A contribution of the C-terminus to DISC binding is therefore the first described functional difference between c-FLIP R and c-FLIP short .…”

“…Poukkula et al 20 found two lysine residues (K192, K195), which mediate ubiquitin-dependent degradation of human c-FLIP short . To investigate whether these lysines are also required for degradation of mouse c-FLIP R , we mutated the homolog residues to arginine (K196R/K200R).…”