Caspase-8 is a cysteine protease activated by membranebound receptors at the cytosolic face of the cell membrane, initiating the extrinsic pathway of apoptosis. Caspase-8 activation relies on recruitment of inactive monomeric zymogens to activated receptor complexes, where they produce a fully active enzyme composed of two catalytic domains. Although in vitro studies using drug-mediated affinity systems or kosmotropic salts to drive dimerization have indicated that uncleaved caspase-8 can be readily activated by dimerization alone, in vivo results using mouse models have reached the opposite conclusion. Furthermore, in addition to interdomain autoprocessing, caspase-8 can be cleaved by activated executioner caspases, and reports of whether this cleavage event can lead to activation of caspase-8 have been conflicting. Here, we address these questions by carrying out studies of the activation characteristics of caspase-8 mutants bearing prohibitive mutations at the interdomain cleavage sites both in vitro and in cell lines lacking endogenous caspase-8, and we find that elimination of these cleavage sites precludes caspase-8 activation by prodomain-driven dimerization. We then further explore the consequences of interdomain cleavage of caspase-8 by adapting the tobacco etch virus protease to create a system in which both the cleavage and the dimerization of caspase-8 can be independently controlled in living cells. We find that unlike the executioner caspases, which are readily activated by interdomain cleavage alone, neither dimerization nor cleavage of caspase-8 alone is sufficient to activate caspase-8 or induce apoptosis and that only the coordinated dimerization and cleavage of the zymogen produce efficient activation in vitro and apoptosis in cellular systems.Caspases are a family of cysteine proteases that can be broadly divided by function into the inflammatory caspases and the apoptotic caspases (reviewed in Refs. 1, 2). Although the former group, which includes caspase-1, -4, and -5, is involved in the innate immune response through the cleavage-induced maturation of pro-inflammatory cytokines, the majority of the mammalian caspases are involved in the initiation and execution of apoptosis.The apoptotic caspases, caspase-2, -3, and -6 -10, can be further divided based on protein architecture and mode of activation. Caspase-2 and -8 -10 are termed the "initiator" or "apical" caspases and are composed of a long N-terminal prodomain containing protein-protein interaction motifs and a C-terminal catalytic domain, often composed of an ϳ20-kDa large subunit containing the active site cysteine and an ϳ10-kDa small subunit (3). The initiator caspases are present in the cytosol of nonapoptotic cells as inactive monomers and are activated by recruitment to large protein complexes where they are induced to dimerize by protein-protein interactions with their prodomains. Once dimerized, initiator caspases undergo a series of autocleavage events at aspartic acid residues in their interdomain linker regions (4 -6). Casp...

In the original version of this manuscript, we inadvertently included the incorrect blot for HSP90, a control for equal protein loading, in supplemental Fig. S3. The HSP90 signal shown in supplemental Fig. S3 was taken from the blot shown in Fig. 3B. In both of these experiments, primary antibodies against caspase-8 and HSP90 were used concurrently, so signals for both caspase-8 and the loading control HSP90 are visible on each blot. Below is the corrected version of Fig. S3 with the correct location of the HSP90 loading control shown. These changes do not affect the interpretation or conclusions of this work.