TRAF2 Must Bind to Cellular Inhibitors of Apoptosis for Tumor Necrosis Factor (TNF) to Efficiently Activate NF-κB and to Prevent TNF-induced Apoptosis

James E Vince,Delara Pantaki,Rebecca Feltham,Peter D Mace,Stephanie M Cordier,Anna C Schmukle,Angelina J Davidson,Bernard A Callus,Wendy Wei-Lynn Wong,Ian E Gentle,Holly Carter,Erinna F Lee,Henning Walczak,Catherine L Day,David L Vaux,John Silke

doi:10.1074/jbc.m109.072256

Abstract

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-kappaB-inducing kinase stability and suppress constitutive noncanonical NF-kappaB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-kappaB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-kappaB signaling and that efficient activation of NF-kappaB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.

Highlights

The inhibitor of apoptosis (IAP)7 family is composed of baculoviral IAP repeat-containing proteins, several of which
It remains possible that both TRAF2 and cIAP1/2 play similar roles in modulating TNFR1 signaling through gene; TAK1, TGF-␤-activated kinase-1; WT, wild type; GFP, green fluorescent protein; MEF, mouse embryo fibroblast; PI, propidium iodide; DKO, double knock-out; CIM, cIAP1/2-interacting motif; NIK, NF-␬B-inducing kinase
The TRAF2 CIM, but Not the TRAF2 RING Domain, Is tated TNFR1 from TRAF2/TRAF5 DKO cells still bound low Required for Efficient TNF-induced NF-␬B Activation—As levels of cIAP1/2 (Fig. 5), we speculated that the late nuclear described previously [24, 53], TNF-induced NF-␬B activation translocation of p65 in TRAF2/TRAF5 DKO cells observed by in TRAF2 knock-out cells was similar to wild type cells, as immunofluorescence could be caused cIAP1/2 function

Summary

Present address

RIPK1 ubiquitylation, it is unknown whether cIAP1/2 act indirectly by activating the E3 ligase activity of TRAF2, if cIAP1/2 act in combination with TRAF2 to directly ubiquitylate RIPK1, or alternatively, whether cIAP1/2 are the major E3 ligases for RIPK1, and the role of TRAF2 is to recruit them to TNFR1. Both TRAF2 or cIAP1/2 deletion cause constitutive noncanonical NF-␬B activity, which occurs by stabilization of the NF-␬B-inducing kinase NIK, IKK␣ activation, and processing of the noncanonical NF-␬B subunit p100 to the active p52 form. In this study we have identified a CIM in TRAF2, and we used TRAF2 CIM mutants defective in cIAP1/2 binding to determine how cIAP1/2 and TRAF2 coordinately suppress noncanonical NF-␬B activation and regulate signaling from TNFR1

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION