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,vith 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.
