Caspase-8 plays an important role in the CD95 (APO-1/Fas) signalling pathway. 1–3 It is activated at the CD95 DISC. The mechanism of caspase-8 activation at the DISC is in the focus of intensive studies. It is well established that binding of procaspases-8/a and-8/b to the DISC results in autocatalytic cleavage via a two-step mechanism4, 5 (Figure 1a). The initial cleavage at Asp374 generates two cleavage intermediates p43/p41 and p12. Subsequently, p12 is rapidly converted to p10. Afterwards, an additional cleavage occurs at Asp216, producing the active enzyme subunit p18 and the inactive p26/p24 prodomain. The DED-containing cleavage products p43/p41 and p26/p24 were reported to be present in the DISC in previous biochemical studies. 5, 6 We analysed in more detail all cleavage products of procaspase-8 in the DISC formed upon stimulation of the B-lymphoblastoid cell line SKW6. 4. Cells were treated with LZCD95L for different time points, followed by immunoprecipitation of the DISC components with the agonistic anti-APO-1 and by immunoblotting with a panel of mAbs. The epitopes recognised by these antibodies are shown in Figure 1a. In addition to the established components of the DISC, including full-length procaspase-8 (p55/p53) and its cleavage product p43/p41, we clearly detected the p18 and p10 subunits of procaspase-8 in the DISC (Figure 1b). The presence of p10 and p18 at the DISC was also observed using other T-cell lines Hut78, CEM, H9 as well as primary T cells (data not shown). The detection of these products became possible because we used different conditions for electrophoresis, focusing on lower molecular weight products in comparison to previous studies. In addition, the presence of the p10 subunit was detected using the C5 antibody that had not been used in the analysis of the lower molecular weight procaspase-8 subunits in the CD95 DISC before. The kinetic analysis in Figure 1b demonstrated that p10 and p18 are detectable at the CD95 DISC within 30s after stimulation. The amount of both p10 and p18 at the DISC reached its maximum 1–5min after receptor engagement. After 10 min, the intensity of the bands corresponding to p10 and p18 started to decrease and after 20 min, the intensity of bands was markedly reduced. The kinetic analysis performed with lysates of SKW6. 4 cells (Figure 1b) showed the same main features as reported earlier. 5 P10 and p18 were detected in the cytosol 10 min after the receptor stimulation, and their presence reached a maximum after 20 min, exactly at the time when their amount in the DISC was found to be decreased. To ensure that the p10 and p18 subunits are components of the DISC under different stimulation conditions, SKW6. 4 were stimulated in parallel with LZ-CD95L and anti-APO-1 antibody. We observed the presence of p10 and p18 in the DISC in both cases (data not shown). To exclude the possibility that p10 and p18 subunits are generated during the washing steps, DISC formation and consecutive immunoprecipitation experiments were performed in the presence of the caspase inhibitor zVAD-fmk. Under these conditions, we could also detect active caspase-8 subunits at the DISC (data not shown).

Additionally, we addressed the question of whether the caspase-8 subunits generated at the DISC were functionally active and did not require additional cytosolic processing and can cleave caspase-8 substrates. Thus, in vitro translated,[35S]-labelled procaspase-3 was added to protein-A sepharose containing CD95 immunoprecipitates from unstimulated and stimulated cells. Incubation of protein-A sepharose beads with caspase-3 resulted in the …