Reconstitution of the Death-Inducing Signaling Complex Reveals a Substrate Switch that Determines CD95-Mediated Death or Survival

Abstract

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

Regulation of CD95/Fas signaling at the DISC

“…Furthermore, in the course of procaspase-8 activation at the DISC procaspase-8 undergoes a substrate specificity switch as has been recently shown by Mac Farlane et al 37 using in vitro reconstituted DISC. Upon initial dimerization procaspase-8 at the DISC has a very restricted substrate range, which is limited to itself and c-FLIP.…”

“…Furthermore, procaspase-8 activity has been suggested to be indispensible for the initiation of non-apoptotic pathways. 37 In line with this, it has been recently reported using transgenic mice that the perturbation of the caspase-8 cleavage site abrogates its pro-apoptotic function without influencing its non-apoptotic function. 39 Another important mechanism of the regulation of procaspase-8 activity, which has been discovered recently, is the protein modification of procaspase-8.…”

“…35 This processing results in the generation of a number of the cleavage products, including p43/p41, p26/ p24, CAP3, p30, p18 and p10 (Figure 2). [34][35][36][37] At the first cleavage step, the N-terminal p43/p41 and the C-terminal p30 cleavage products are generated. Importantly, these cleavage products already possess catalytic activity.…”

New insights into apoptosis signaling by Apo2L/TRAIL

“…Sensitivity to Apo2L/TRAIL seems to be controlled mainly by apical events such as DISC assembly and caspase-8 activation (Wagner et al, 2007;Hughes et al, 2009). Consistent with this notion, a study examining apoptosis stimulation in individual cells within a culture has demonstrated that differences in the extent of caspase-8 activation may be responsible for cell-to-cell variation in Apo2L/TRAIL responsiveness (Spencer et al, 2009).…”

Role of caspase-8 in thymus function

“…FLIP S inhibits caspase-8 activation, whereas the caspase-8/FLIP L heterodimer, which contains unprocessed caspase-8, promotes signaling towards cellular survival and activation rather than apoptosis. 23 Only after the caspase-8 subunit of this heterodimer has been processed does this caspase species become pro-apoptotic. Once activated (i.e., cleaved), caspase-8 can directly cleave and activate caspase-3 via the extrinsic pathway.…”