Abstract
Tumor necrosis factor receptor 1 (TNF-R1) mediates most of the biological properties of TNF including activation of the transcription factor NF-kappaB and programmed cell death. An approximately 80-amino acid region within the intracellular domain of the receptor, termed the death domain, is required for signaling NF-kappaB activation and cytotoxicity. A TNF-R1-associated protein TRADD has been discovered that interacts with the death domain of the receptor. Elevated expression of TRADD in cells triggers both NF-kappaB activation and programmed cell death pathways. The biological activities of TRADD have been mapped to a 111-amino acid region within the carboxyl-terminal half of the protein. This region shows sequence similarity to the death domain of TNF-R1 and can self-associate and bind to the TNF-R1 death domain. We have performed an alanine scanning mutagenesis of TRADD's death domain to explore the relationship among its various functional properties. Mutations affecting the different activities of TRADD do not map to discrete regions but rather are spread over the entire death domain, suggesting that the death domain is a multifunctional unit. A mutant that separates cell killing from NF-kappaB activation by the TRADD death domain has been identified indicating that these two signaling pathways diverge with TRADD. Additionally, one of the TRADD mutants that fails to activate NF-kappaB was found to act as dominant negative mutant capable of preventing induction of NF-kappaB by TNFalpha. Such observations provide evidence that TRADD performs an obligate role in TNF-induced NF-kappaB activation.
Highlights
Icals and activation of protein kinases and sphingomyelinases
In Vitro Binding Properties of TRADD Death Domain Mutants—A derivative of TRADD extending from amino acids 195 to 312 retains all the activities associated with full-length TRADD including multimerization, binding to Tumor necrosis factor receptor 1 (TNF-R1), and induction of NF-B and of programmed cell death [12]
We focused on amino acids 196 –305 for mutagenesis, since the carboxyl-terminal seven amino acids are dispensable for TRADD function [12]
Summary
Icals and activation of protein kinases and sphingomyelinases (reviewed in Ref. 8). it is not clear if these events reflect primary cellular changes in response to receptor occupancy or secondary events that are a consequence of other more immediate postreceptor activation steps triggered by TNF␣. Structure-function analysis of signaling by TNF-R1 has demonstrated that an ϳ80-amino acid region within the cytoplasmic domain is required for initiation of programmed cell death and NF-B activation [16].
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