TRUSS, a Novel Tumor Necrosis Factor Receptor 1 Scaffolding Protein That Mediates Activation of the Transcription Factor NF-κB

Abstract

We describe the cloning and characterization of tumor necrosis factor receptor (TNF-R)-associated ubiquitous scaffolding and signaling protein (TRUSS), a novel TNF-R1-interacting protein of 90.7 kDa. TRUSS mRNA was ubiquitously expressed in mouse tissues but was enriched in heart, liver, and testis. Coimmunoprecipitation experiments showed that TRUSS was constitutively associated with unligated TNF-R1 and that the complex was relatively insensitive to stimulation with TNF-α. Deletion mutagenesis of TNF-R1 indicated that TRUSS interacts with both the membrane-proximal region and the death domain of TNF-R1. In addition, the N-terminal region of TRUSS (residues 1 to 440) contains sequences that permit association with the cytoplasmic domain of TNF-R1. Transient overexpression of TRUSS activated NF-κB and increased NF-κB activation in response to ligation of TNF-R1. In contrast, a COOH-terminal-deletion mutant of TRUSS (TRUSS_1-723) was found to inhibit NF-κB activation by TNF-α. Coprecipitation and coimmunoprecipitation assays revealed that TRUSS can interact with TRADD, TRAF2, and components of the IKK complex. These findings suggest that TRUSS may serve as a scaffolding protein that interacts with TNF-R1 signaling proteins and may link TNF-R1 to the activation of IKK.

ACKNOWLEDGMENTS

We thank Murry Wynes, Annemie Van Linden, Vincent Cottin, and Hong-Bing Shu for their invaluable intellectual input and insights into this work and for critically reviewing the manuscript. A. Van Linden and V. Cottin also graciously provided the FLAG-tagged TNF-R1 construct and the constructs encoding the GST-TNF-R1 fusion proteins used in this study. We also thank Linda Remigio for outstanding technical assistance and Hong-Bing Shu for generously providing us with plasmid constructs encoding several TNF-signaling molecules, for extensively helping us with the coprecipitation experiment, and for reviewing the manuscript. Last, we are indebted to Gongyi Zhang for his collaborative interest in this work and for providing us with purified recombinant His-tagged IKKγ.

This work was supported by Public Health Service grants HL55549, HL68628, and HL65326 from the National Heart, Lung and Blood Institute of the National Institutes of Health. Surinder Soond was supported in part by a Nelson Family Fellowship from the National Jewish Medical and Research Center. Jenny Terry was supported in part by The Cancer Research Institute Pre-doctoral Emphasis Pathway in Tumor Immunology.