Dominant-negative FADD inhibits TNFR60-, Fas/Apo1- and TRAIL-R/Apo2-mediated cell death but not gene induction

Fas/Apo1 and other cytotoxic receptors of the tumor necrosis factor receptor (TNFR) family contain a cytoplasmic death domain (Db) [1-11] that activates the apoptotic process by interacting with the DD-containing adaptor proteins TNFR-associated Db protein (TRADD) [12,13] and pas-associated DD protein (FADD/MORT1) [14,15], leading to the activation of cysteine proteases of the caspase family [16], Stimulation of Fas/Apo1 reads to the formation of a receptor-bound death-inducing signaling complex (DISC), consisting of FADD and two different forms of caspase-8 [17-19]. Transient expression of a dominant-negative mutant of FADD impairs TNFR60-mediated and Fas/Apo1-mediated apoptosis [13,20], but has no effect on TNF-related apoptosis-inducing ligand (TRAIL/Apo2L)-induced cell death [7-10,21], To study the function of FADD in DD-receptor signaling in more detail, we established HeLa cells that stably expressed a green fluorescent protein (GFP)-tagged dominant-negative mutant of FADD, GFP-Delta FADD. Interestingly, expression of this mutant inhibited cell death induced by TNFR60, Fas/Apo1 and TRAIL-R/Apo2. In addition, GFP-Delta FADD did not interfere with TNF-mediated gene induction or with activation of NF-kappa B or Jun N-terminal kinase (JNK), demonstrating that FADD is part of the TNFR60-initiated apoptotic pathway but does not play a role in TNFR60-mediated gene induction. Fas/Apo1-mediated activation of JNK was unaffected by the expression of GFP-Delta FADD, suggesting that in Fas/Apo1 signaling the apoptotic pathway and the activation of JNK diverge at a level proximal to the receptor, upstream of or parallel to FADD.