The study investigates the mechanism by which the endoplasmic reticulum (ER)-localized protein STING activates the transcription factor IRF3 in response to cytosolic double-stranded DNA (dsDNA). Using an in vitro reconstitution system, the authors demonstrate that a carboxyl-terminal region of STING, containing only 39 amino acids, is sufficient to activate the kinase TBK1 and stimulate IRF3 phosphorylation. They find that STING interacts with both TBK1 and IRF3, and that mutations in STING that disrupt its binding to IRF3 abrogate IRF3 phosphorylation without affecting TBK1 activation. These results suggest that STING functions as a scaffold to specify and promote IRF3 phosphorylation by TBK1, providing a mechanism for the selective activation of IRF3 in certain signaling pathways that activate TBK1. The findings also highlight the potential for developing inhibitors that selectively target IRF3 activation without affecting other TBK1 substrates.The study investigates the mechanism by which the endoplasmic reticulum (ER)-localized protein STING activates the transcription factor IRF3 in response to cytosolic double-stranded DNA (dsDNA). Using an in vitro reconstitution system, the authors demonstrate that a carboxyl-terminal region of STING, containing only 39 amino acids, is sufficient to activate the kinase TBK1 and stimulate IRF3 phosphorylation. They find that STING interacts with both TBK1 and IRF3, and that mutations in STING that disrupt its binding to IRF3 abrogate IRF3 phosphorylation without affecting TBK1 activation. These results suggest that STING functions as a scaffold to specify and promote IRF3 phosphorylation by TBK1, providing a mechanism for the selective activation of IRF3 in certain signaling pathways that activate TBK1. The findings also highlight the potential for developing inhibitors that selectively target IRF3 activation without affecting other TBK1 substrates.