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Mol. Cell. Biol. doi:10.1128/MCB.00785-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-mediated Transactivation by the TNFR Family Member LMP1

Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology and Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599. Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030

^* To whom correspondence should be addressed. Email: joseph_pagano{at}med.unc.edu.

	Abstract

We have recently shown that Interferon Regulatory Factor 7 (IRF7) is activated by Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1), a member of the TNFR superfamily, through RIP-dependent K63-linked ubiquitination (Huye et al. 2007, Mol. Cell. Biol., 27: 2910-2918). In this study, with the use of siRNA and TRAF6 knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In EBV latently infected Raji cells, which express high levels of LMP1 and IRF7 endogenously, expression of an shTRAF6 construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6-/- MEFs, reconstitution with TRAF6 expression, but not with TRAF6(C70A) which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase. Finally, we show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites 444, 446, and 452 of human IRF7 variant A are essential for transactivation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IKK. In addition, K63-linked ubiquitination of IRF7 is independent of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and transactivation of a transcription factor.