To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.

APO‐1 (Fas/CD95), a member of the tumor necrosis factor receptor superfamily, induces apoptosis upon receptor oligomerization. In a search to identify intracellular signaling molecules coupling to oligomerized APO‐1, several cytotoxicity‐dependent APO‐1‐associated proteins (CAP) were immunoprecipitated from the apoptosis‐sensitive human leukemic T cell line HUT78 and the lymphoblastoid B cell line SKW6.4. CAP1–3 (27–29 kDa) and CAP4 (55 kDa), instantly detectable after the crosslinking of APO‐1, were associated only with aggregated (the signaling form of APO‐1) and not with monomeric APO‐1. CAP1 and CAP2 were identified as serine phosphorylated MORT1/FADD. The association of CAP1–4 with APO‐1 was not observed with C‐terminally truncated non‐signaling APO‐1. In addition, CAP1 and CAP2 did not associate with an APO‐1 cytoplasmic tail carrying the lprcg amino acid replacement. Moreover, no APO‐1‐CAP association was found in the APO‐1+, anti‐APO‐1‐resistant pre‐B cell line Boe. Our data suggest that in vivo CAP1–4 are the APO‐1 apoptosis‐transducing molecules.

Upon activation, the apoptosis‐inducing cell membrane receptor CD95 (APO‐1/Fas) recruits a set of intracellular signaling proteins (CAP1‐4) into a death‐inducing signaling complex (DISC). In the DISC, CAP1 and CAP2 represent FADD/MORT1. CAP4 was identified recently as an ICE‐like protease, FLICE, with two death effector domains (DED). Here we show that FLICE binds to FADD through its N‐terminal DED. This is an obligatory step in CD95 signaling detected in the DISC of all CD95‐sensitive cells tested. Upon prolonged triggering of CD95 with agonistic antibodies all cytosolic FLICE gets proteolytically activated. Physiological FLICE cleavage requires association with the DISC and occurs by a two‐step mechanism. Initial cleavage generates a p43 and a p12 fragment further processed to a p10 fragment. Subsequent cleavage of the receptor‐bound p43 results in formation of the prodomain p26 and the release of the active site‐containing fragment p18. Activation of FLICE is blocked by the peptide inhibitors zVAD‐fmk, zDEVD‐fmk and zIETD‐fmk, but not by crmA or Ac‐YVAD‐CHO. Taken together, our data indicate that FLICE is the first in a cascade of ICE‐like proteases activated by CD95 and that this activation requires a functional CD95 DISC.

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligand's signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.