Functional Regulation of MyD88-Activated Interferon Regulatory Factor 5 by K63-Linked Polyubiquitination

Abstract

Interferon regulatory factor 5 (IRF-5) plays an important role in the innate antiviral and inflammatory response. Specific IRF-5 haplotypes are associated with dysregulated expression of type I interferons and predisposition to autoimmune disorders. IRF-5 is activated by Toll-like receptor 7 (TLR7) and TLR9 via the MyD88 pathway, where it interacts with both MyD88 and the E3 ubiquitin ligase, TRAF6. To understand the role of these interactions in the regulation of IRF-5, we examined the role of ubiquitination and showed that IRF-5 is subjected to TRAF6-mediated K63-linked ubiquitination, which is important for IRF-5 nuclear translocation and target gene regulation. We show that while the murine IRF-5 and human IRF-5 variant 4 (HuIRF-5v4) and HuIRF-5v5 are ubiquitinated, an IRF-5 bone marrow variant mutant containing an internal deletion of 288 nucleotides is not ubiquitinated. Lysine residues at positions 410 and 411 in a putative TRAF6 consensus binding domain of IRF-5 are the targets of K63-linked ubiquitination. Mutagenesis of these two lysines abolished IRF-5 ubiquitination, nuclear translocation, and the IFNA promoter-inducing activity but not the IRF-5-TRAF6 interaction. Finally, we show that IRAK1 associates with IRF-5 and that this interaction precedes and is required for IRF-5 ubiquitination and activation. Thus, our findings offer a new mechanistic insight into IRF-5 gene induction program through hitherto unknown processes of IRF-5 ubiquitination.

ACKNOWLEDGMENTS

We thank P. Desai for a generous gift of DH5α supercompetent cells and enzymes for cloning, H. Gottlinger and Z. Chen for the ubiquitin plasmids, T. Michiels for the IFNA4 luciferase plasmid, and S. Meir and M. Beilharz for valuable suggestions. We also thank T. Mak and X. Li for TRAF6- and IRAK1-defective cell lines.

The study was supported by Public Health Service grants NIAID R01 AI067632-02A1 and CA19737-22A1, by a pilot grant from the Alliance for Lupus Research to P.M.P., and by NIAID grant AI067497 to K.A.F.