IκB Kinase β Phosphorylates the K63 Deubiquitinase A20 To Cause Feedback Inhibition of the NF-κB Pathway

Abstract

Misregulation of NF-κB signaling leads to infectious, inflammatory, or autoimmune disorders. IκB kinase β (IKKβ) is an essential activator of NF-κB and is known to phosphorylate the NF-κB inhibitor, IκBα, allowing it to undergo ubiquitin-mediated proteasomal degradation. However, beyond IκBα, few additional IKKβ substrates have been identified. Here we utilize a peptide library and bioinformatic approach to predict likely substrates of IKKβ. This approach predicted Ser381 of the K63 deubiquitinase A20 as a likely site of IKKβ phosphorylation. While A20 is a known negative regulator of innate immune signaling pathways, the mechanisms regulating the activity of A20 are poorly understood. We show that IKKβ phosphorylates A20 in vitro and in vivo at serine 381, and we further show that this phosphorylation event increases the ability of A20 to inhibit the NF-κB signaling pathway. Phosphorylation of A20 by IKKβ thus represents part of a novel feedback loop that regulates the duration of NF-κB signaling following activation of innate immune signaling pathways.

We thank Alex Toker for providing the Flag-A20 construct, and we thank Michael Karin (UCSD) and Tak Mak (Toronto) for supplying NEMO-null MEFs. We thank members of the Cantley lab for technical assistance and critical comments.

This work was supported by grant 1K08 AI53819-01A1 (D.W.A.), by a Burroughs-Wellcome Career Award for Biomedical Scientists (D.W.A.), by the Cleveland Foundation (D.W.A.), and by NIH grants R01 GM56203 (L.C.C.) and R01 DK071939 (A.M.).