NF-κB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-1β1. Failure to downregulate NF-κB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice2. A20 is a potent inhibitor of NF-κB signalling, but its mechanism of action is unknown2. Here we show that A20 downregulates NF-κB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family3, removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein (RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex4,5. The carboxy-terminal domain of A20, composed of seven C2/C2 zinc fingers6, then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-κB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.
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The authors would like to thank R. Deshaies, T. Mayor, R. Feldman and members of the Dixit Lab for helpful discussions, T. Mayor and M. Petroski for reagents, D. Yansura for technical assistance, and K. Newton for editorial assistance. We acknowledge the contributions from colleagues whose work has been cited indirectly owing to space limitations. I.E.W. was supported in part by a PSTP fellowship from the University of California at Davis.
The authors declare that they have no competing financial interests.
This document contains supplementary methods, supplementary figures 1-7 and supplementary references. (DOC 1395 kb)
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