OTUD4 Is a Phospho-Activated K63 Deubiquitinase that Regulates MyD88-Dependent Signaling

Ubiquitination is a major mechanism that regulates numerous cellular processes, including autophagy, DNA damage signaling, and inflammation. While hundreds of ubiquitin ligases exist to conjugate ubiquitin onto substrates, approximately 100 deubiquitinases are encoded by the human genome. Thus, deubiquitinases are likely regulated by unidentified mechanisms to target distinct substrates and cellular functions. Here, we demonstrate that the deubiquitinase OTUD4, which nominally encodes a K48-specific deubiquitinase, is phosphorylated near its catalytic domain, activating a latent K63-specific deubiquitinase. Besides phosphorylation, this latter activity requires an adjacent ubiquitin-interacting motif, which increases the affinity of OTUD4 for K63-linked chains. We reveal the Toll-like receptor (TLR)-associated factor MyD88 as a target of this K63 deubiquitinase activity. Consequently, TLR-mediated activation of NF-κB is negatively regulated by OTUD4, and macrophages from Otud4^−/− mice exhibit increased inflammatory signaling upon TLR stimulation. Our results reveal insights into how a deubiquitinase may modulate diverse processes through post-translational modification. OTUD4 nominally encodes a K48-specific deubiquitinase previously shown to counteract proteasomal degradation of DNA repair proteins. Zhao et al. demonstrate that OTUD4 is phosphorylated near its catalytic domain, activating a dormant K63-specific deubiquitinase activity. This enzymatic activity is shown to modulate TLR-dependent signaling by targeting the scaffolding protein MyD88.