Sensing of pathogenic microbes and tissue damage by the innate immune system triggers immune cells to secrete cytokines that promote host defense. Viral RNA, cytosolic DNA, and the bacterial cell wall component lipopolysaccharide activate signaling cascades through a number of pattern recognition receptor (PRR)–adaptor protein pairs, including RIG-I–MAVS, cGAS-STING, and TLR3/4-TRIF (TLR3/4, Toll-like receptors 3 and 4). Activation of these signaling modules results in the production of type I interferons (IFNs), a family of cytokines that are essential for host protection. The adaptor proteins MAVS, STING, and TRIF each activate the downstream protein kinase TBK1, which then phosphorylates the transcription factor interferon regulatory factor 3 (IRF3), which drives type I IFN production. Although much progress has been made in our understanding of PRR and adaptor protein activation, the mechanism by which the adaptor proteins activate TBK1 and IRF3 remains unclear.

Other signaling pathways besides the RIG-I–MAVS, cGAS-STING, and TLR3/4-TRIF pathways activate TBK1. However, IRF3 phosphorylation by TBK1 is observed only in the IFN-producing pathways that use MAVS, STING, or TRIF as the adaptor protein. The discrepant activation of TBK1 and IRF3 implies the existence of a kinase-substrate specification mechanism exclusive to the IFN-producing pathways. Specification of TBK1-mediated IRF3 activation is essential for the tight regulation of IFN production, which would otherwise lead to autoimmune diseases.

Using biochemical and mouse cell– and human cell–based assays, we found that both MAVS and STING interacted with IRF3 in a phosphorylation-dependent manner. We show that both MAVS and STING are phosphorylated in response to stimulation at their respective C-terminal consensus motif, pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site). This phosphorylation event then recruits IRF3 to the active adaptor protein and is essential for IRF3 activation. Point mutations that impair the phosphorylation of MAVS or STING at their consensus motif abrogated IRF3 binding and subsequent IFN induction.

We found that MAVS is phosphorylated by the kinases TBK1 and IKK, whereas STING is phosphorylated by TBK1. Phosphorylated MAVS and STING subsequently bind to conserved, positively charged surfaces of IRF3, thereby recruiting IRF3 for its phosphorylation and activation by TBK1. Point mutations at IRF3’s positively charged surfaces abrogated IRF3 binding to MAVS and STING and subsequent IRF3 phosphorylation and activation. We further show that TRIF-mediated activation of IRF3 depends on TRIF phosphorylation at the pLxIS motif commonly found in MAVS, STING, and IRF3. These results reveal that phosphorylation of innate immune adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate type I IFN production.

We uncovered a general mechanism of IRF3 activation by the innate immune adaptor proteins MAVS, STING, and TRIF, which functions in three distinct pattern recognition pathways. Following its activation, each adaptor protein recruits and activates downstream kinase TBK1, which phosphorylates the cognate upstream adaptor protein at a consensus motif. Phosphorylated MAVS, STING, or TRIF in turn recruits IRF3 through its conserved, positively charged phospho-binding domain, allowing IRF3 phosphorylation by TBK1. Phosphorylated IRF3 subsequently dissociates from the adaptor protein and dimerizes though the same phospho-binding domain before translocating into the …