Priming Phosphorylation of TANK-Binding Kinase 1 by IκB Kinase β Is Essential in Toll-Like Receptor 3/4 Signaling

TRIF is an essential adaptor for TLR3/4 signaling to activate transcription factor IRF-3. We examined the molecular mechanism of TLR3 signaling, and found that TLR3 stimulation by dsRNA induces phosphorylation of TRIF at Ser210 and is required for IRF-3 recruitment. TBK1 is known to be responsible for IRF-3 phosphorylation and activation. We found that TBK1 is also responsible for phosphorylation of Ser210 in TRIF. Unexpectedly, we discovered that IKKβ plays an essential role in TLR3/4 signaling using a pharmacological inhibitor and gene deletion. Of note, IKKβ is essential in TLR3/4 but not in RIG-I signaling. Mechanistically, IKKβ transiently associates with and induces the phosphorylation of TBK1 upon TLR3 stimulation. These results suggest a phosphorylation cascade of IKKβ and TBK1, where priming phosphorylation of TBK1 by IKKβ is required to surpass the threshold to induce signaling, thereby activating IRF-3.