Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptormediated pathogen detection and as a modulator of IFN levels. Efforts to better understand the biology of this key immunological factor have intensified recently as growing evidence implicates aberrant TBK1 activity in a variety of autoimmune diseases and cancers. Nevertheless, key molecular details of TBK1 regulation and substrate selection remain unanswered. Here, structures of phosphorylated and unphosphorylated human TBK1 kinase and ubiquitin-like domains, combined with biochemical studies, indicate a molecular mechanism of activation via transautophosphorylation. These TBK1 structures are consistent with the tripartite architecture observed recently for the related kinase IKKβ, but domain contributions toward target recognition appear to differ for the two enzymes. In particular, both TBK1 autoactivation and substrate specificity are likely driven by signal-dependent colocalization events. Phosphorylation promotes the dimerization and nuclear translocation of these transcription factors that stimulate production of type I interferons (IFNs) (1,3,5). Recent studies have identified an additional role for TBK1 in the xenophagic elimination of bacteria (6-9) and better-defined how cross-talk within the IKK family regulates innate immune response (10).Under pathological conditions, IKK-mediated pathways can also be activated inappropriately by endogenous signals, contributing to inflammatory disorders and oncogenesis (11,12). Whereas canonical IKKs have long been recognized as bridges between chronic inflammation and cancer, IKK-related kinases more recently have also been implicated in cell transformation and tumor progression (13). TBK1 has been of particular interest, given its identification both as an activator of the oncogenic AKT kinase (14-18) and as an essential factor in KRAS-driven cancers (19).TBK1 activity is regulated by phosphorylation on S172 within the classical kinase activation loop. Serine-to-alanine substitution at this position abolishes TBK1 activity, whereas the phosphomimetic mutation S172E partially restores activity to within ∼200-fold of the wild-type kinase (20). Genetic and pharmacological inhibition studies have indicated that TBK1 can be activated by IKKβ, as well as by apparent autophosphorylation (10). Additional posttranslational modifications of TBK1 lysine residues by K63-linked polyubiquitin chains have been shown to promote production of IFNs in viral infections (21).TBK1 contains a predicted ubiquitin-like domain (ULD) (22) that is located between the N-terminal kinase domain (KD) and the C-terminal scaffolding/dimerization domain (SDD), a domain arrangement that appears to be shared among the IKK family of kinases (3). Deletion or mutation of the ULD in TBK1 or IKKε severely impairs kinase activation and substrate phosphorylation in cells (22,23). Furthermore, the integrity of the ULD in IKKβ is not only required for kinase activity (24) bu...