TNFα-mediated I kappa kinase phosphorylation of ITCH impairs ubiquitin ligase activity (IRM11P.625)

Abstract The I Kappa Kinase (IKK) signalosome serves as a central hub for the relay of inflammatory signaling. Once activated, the kinase components of this hub, IKKα and IKKβ, can phosphorylate substrates outside of the known NF-κB inflammatory cascade. Additionally, two other closely related kinases, IKKε and TBK1, share kinase homology with IKKα and IKKβ, but phosphorylate substrates related to IFN regulatory factor (IRF) activation. Despite their well-known roles in NF-κB and IRF activation, we and others have found substrates outside of these signaling cascades. Given this, we have devised a proteomic and bioinformatic approach to identify novel IKK substrates. In the present work, we show IKK-mediated phosphorylation of ITCH, an E3 ubiquitin ligase implicated in the maintenance of mucosal immunologic homeostasis. We identify the site of phosphorylation, and demonstrate the inactivating effect of IKK phosphorylation on ubiquitin ligase activity. Given that mice null for ITCH develop pulmonary interstitial fibrosis and gastritis and given that TNF strongly activates the IKKs and this phosphorylation event, we bred ITCH-/- mice with TNFR1-/- mice to determine if loss of TNF activation of IKKα/β could reverse this phenotype. Both mortality and lung inflammation were improved in TNFR1-/-ITCH-/- when compared with ITCH-/- mice. These results show that bioinformatic and proteomic studies can be coupled with mucosal immunology for novel insights.