Inhibition of Death Receptor-mediated Gene Induction by a Cycloheximide-sensitive Factor Occurs at the Level of or Upstream of Fas-associated Death Domain Protein (FADD)

• Published in: The Journal of biological chemistry 2000-08, Vol.275 (32), p.24357-24366
• Main Authors: Wajant, Harald, Haas, Elvira, Schwenzer, Ralph, Mühlenbeck, Frank, Kreuz, Sebastian, Schubert, Gisela, Grell, Matthias, Smith, Craig, Scheurich, Peter
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones - pharmacology; Antibodies, Monoclonal - pharmacology; Apoptosis; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Caspase 8; Caspase 9; Caspases - metabolism; Cell Line; cIAP2 protein; Cycloheximide - pharmacology; Cysteine Proteinase Inhibitors - pharmacology; deletion mutant; fas Receptor - physiology; Fas-Associated Death Domain Protein; FLICE protein; FLIP protein; Gene Expression Regulation - drug effects; Gene Expression Regulation - physiology; HeLa Cells; Humans; Kinetics; NF-^KB protein; NF-kappa B - metabolism; Proteins - metabolism; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor - physiology; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Deletion; TNF Receptor-Associated Factor 1; TRAF1 protein; Transcriptional Activation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000811200

In HeLa cells, induction of apoptosis and nuclear factor κB (NF-κB) activation initiated by TRAIL/Apo2L or the agonistic Apo1/Fas-specific monoclonal antibody anti-APO-1 require the presence of cycloheximide (CHX). Inhibition of caspases prevented TRAIL/anti-APO-1-induced apoptosis, but not NF-κB activation, indicating that both pathways bifurcate upstream of the receptor-proximal caspase-8. Under these conditions, TRAIL and anti-APO-1 up-regulated the expression of the known NF-κB targets interleukin-6, cellular inhibitor of apoptosis 2 (cIAP2), and TRAF1 (TRAF, tumor necrosis factor receptor-associate factor). In the presence of CHX, the stable overexpression of a deletion mutant of the Fas-associated death domain molecule FADD comprising solely the death domain of the molecule but lacking its death effector domain (FADD-(80–208)) led to the same response pattern as TRAIL or anti-APO-1 treatment. Moreover, the ability of death receptors to induce NF-κB activation was drastically reduced in a FADD-deficient Jurkat cell line. TRAIL-, anti-APO-1-, and FADD-(80–208)-initiated gene induction was blocked by a dominant-negative mutant of TRAF2 or the p38 kinase inhibitor SB203580, similar to tumor necrosis factor receptor-1-induced NF-κB activation. CHX treatment rapidly down-regulated endogenous cFLIP protein levels, and overexpression of cellular FLICE inhibitory protein (cFLIP) inhibited death receptor-induced NF-κB activation. Thus, a novel functional role of cFLIP as a negative regulator of gene induction by death receptors became apparent.