SYSTEMATIC MUTATIONAL ANALYSIS OF THE DEATH DOMAIN OF THE TUMOR-NECROSIS-FACTOR RECEPTOR 1-ASSOCIATED PROTEIN TRADD

Tumor necrosis factor receptor 1 (TNF-R1) mediates most of the biologi cal properties of TNF including activation of the transcription factor NF-kappa B and programmed cell death, An similar to 80 amino acid reg ion within the intracellular domain of the receptor, termed the death domain, is required for signaling NF-kappa B activation and cytotoxici ty, A TNF-R1-associated protein TRADD has been discovered that interac ts with the death domain of the receptor, Elevated expression of TRADD in cells triggers both NF-kappa B activation and programmed cell deat h pathways, The biological activities of TRADD have been mapped to a 1 11-amino acid region within the carboxyl-terminal half of the protein, This region shows sequence similarity to the death domain of TNF-R1 a nd can self-associate and bind to the TNF-R1 death domain, We have per formed an alanine scanning mutagenesis of TRADD's death domain to expl ore the relationship among its various functional properties, Mutation s affecting the different activities of TRADD do not map to discrete r egions but rather are spread over the entire death domain, suggesting that the death domain is a multifunctional unit, A mutant that separat es cell killing from NF-kappa B activation by the TRADD death domain h as been identified indicating that these two signaling pathways diverg e with TRADD. Additionally, one of the TRADD mutants that fails to act ivate NF-KB was found to act as dominant negative mutant capable of pr eventing induction of NF-kappa B by TNF alpha. Such observations provi de evidence that TRADD performs an obligate role in TNF-induced NF-kap pa B activation.