Lys63-Linked Polyubiquitination of IRAK-1 Is Required for Interleukin-1 Receptor- and Toll-Like Receptor-Mediated NF-κB Activation

Abstract

Stimulation through the interleukin-1 receptor (IL-1R) and some Toll-like receptors (TLRs) induces ubiquitination of TRAF6 and IRAK-1, signaling components required for NF-κB and mitogen-activated protein kinase activation. Here we show that although TRAF6 and IRAK-1 acquired Lys63 (K63)-linked polyubiquitin chains upon IL-1 stimulation, only ubiquitinated IRAK-1 bound NEMO, the regulatory subunit of IκB kinase (IKK). The sites of IRAK-1 ubiquitination were mapped to Lys134 and Lys180, and arginine substitution of these residues impaired IL-1R/TLR-mediated IRAK-1 ubiquitination, NEMO binding, and NF-κB activation. K63-linked ubiquitination of IRAK-1 required enzymatically active TRAF6, indicating that it is the physiologically relevant E3. Thus, K63-linked polyubiquitination of proximal signaling proteins is a common mechanism used by diverse innate immune receptors for recruiting IKK and activating NF-κB.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Bei Dong for subcloning and mutagenesis, Srinivasa Srinivasula for helpful discussions and critical reading of the manuscript, Liwu Li for supplying the human IRAK-1 cDNA, Ted Dawson for pRK5-HA-tagged ubiquitin and pRK5-HA-tagged K63-only ubiquitin, Xiaoxia Li for 293/IL-1R/TLR4 cells, M. Schmidt-Supprian for NEMO-deficient MEFs, and Jun-ichiro Inoue for TRAF6-deficient MEFs.

This work was supported by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, National Cancer Institute.