IκB kinase β phosphorylates the K63 deubiquitinase A20 to cause feedback inhibition of the NF-κB pathway

Misregulation of NF-kappa B signaling leads to infectious, inflammatory, or autoimmune disorders. I kappa B kinase beta (IKK beta) is an essential activator of NF-kappa B and is known to phosphorylate the NF-kappa B inhibitor, I kappa B alpha, allowing it to undergo ubiquitin-mediated proteasomal degradation. However, beyond I kappa B alpha, few additional IKK beta substrates have been identified. Here we utilize a peptide library and bioinformatic approach to predict likely substrates of IKK beta. This approach predicted Ser381 of the K63 deubiquitinase A20 as a likely site of IKK beta 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 IKK beta 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-kappa B signaling pathway. Phosphorylation of A20 by IKK beta thus represents part of a novel feedback loop that regulates the duration of NF-kappa B signaling following activation of innate immune signaling pathways.