The role of c-FLIP in modulation of CD95-induced apoptosis

Upon stimulation, CD95 (APO-1/Fas) recruits the adapted molecule Fas-associ ated death domain protein (FADD/MORT1 and caspase-8 (FADD-like interleukin- 1 beta-converting enzyme (FLICE)/MACH/MCH5) into the death-inducing signali ng complex (DISC). Recently, a molecule with sequence homology to caspase-8 was identified, termed cellular FLICE-inhibitory protein (c-FLIP). c-FLIP has been controversially reported to possess apoptosis-promoting and -inhib iting functions. Using c-FLIP-specific monoclonal antibodies, we now show t hat c-FLIP is expressed in two isoforms, both of which, like FADD and caspa se-8, are recruited to the CD95 DISC in a stimulation-dependent fashion. In stably transfected BJAB cells, c-FLIP blocks caspase-8 activation at the D ISC and thereby inhibits CD95-mediated apoptosis. During this process, both caspase-8 and c-FLIP undergo cleavage between the p18 and p10 subunits, ge nerating two stable intermediates of 43 kDa that stay bound to the DISC. c- FLIP has been suggested to play a role in protecting activated peripheral T cells from CD95-mediated apoptosis (Irmler, M., Theme, M, Hahne, M., Schne ider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J. (1997) Nature 38 8, 180-195). In contrast to this hypothesis, neither caspase-8 nor c-FLIP w ere cleaved in these cells, ruling out c-FLIP as the main factor regulating DISC activity. Moreover, recruitment of FADD, caspase-8, and c-FLIP to the DISC was strongly reduced in the apoptosis-resistant but readily detectabl e in the apoptosis-sensitive T cells.