Interferon regulatory factor 3 is regulated by a dual phosphorylation-dependent switch

The transcription factor interferon regulatory factor 3 (IRF-3) regulates genes in the innate immune response. IRF-3 is activated through phosphorylation by the kinases IKK is an element of and/or TBK1. Phosphorylation results in IRF-3 dimerization and removal of an autoinhibitory structure to allow interaction with the coactivators CBP/p300. The precise role of the different phosphorylation sites has remained controversial. Using purified proteins we show that TBK1 can directly phosphorylate full-length IRF-3 in vitro. Phosphorylation at residues in site 2 (Ser(396) - Ser(405)) alleviates autoinhibition to allow interaction with CBP ((C) under bar CREB-(b) under bar binding (p) under bar protein) and facilitates phosphorylation at site 1 (Ser(385) or Ser(386)). Phosphorylation at site 1 is, in turn, required for IRF-3 dimerization. The data support a two-step phosphorylation model for IRF-3 activation mediated by TBK1.