Exploring phosphorylation of the transcription factor Interferon Regulatory Factor 3 in vivo and in vitro

Immune responses to viral infections depend on type I interferons (IFN-a and IFN-b). The synthesis of IFN-b has been extensively studied in recent years, both because of its overall importance in the innate immune system and as a model system for gene activation and regulation. The key events in IFN-b production involve recognition of viral nucleic acids through either cytosolic RNA helicases (RIG-I, MDA5) or transmembrane Toll-like receptors (TLR3, TLR7, TLR9). The signalling pathways initiated by these receptors converge at the level of TNF-Receptor Associated Factor 3 (TRAF3) and culminate in activation of the IkappaB kinase (IKK)-related kinase TANK Binding Kinase 1 (TBK1), which has been shown to phosphorylate and activate the transcription factor Interferon Regulatory Factor 3 (IRF3). The phosphorylation status of IRF-3 in vivo in response to viruses has not been reported. We have investigated the phosphorylation of IRF3 by using mass spectroscopy. With highly specific phosphogroup-binding columns and MALDI-TOF we have found several phosphorylated residues in IRF3 not hitherto reported. Initially, we investigated the phosphorylation pattern of IRF-3 that was phosphorylated in vitro with TBK1. Since in-vitro phosphorylation may be prone to excess phosphorylation at non-relevant residues, we also investigated the phosphorylation status of IRF3 that was immunoprecipitated from transiently transfected HEK293 cells infected with Sendai Virus, a single-stranded RNA virus. This confirmed that phosphorylation of IRF-3 occurs on at least three residues that have not previously been reported to be regulatory phosphoacceptors. Mutational analysis of these novel phosphorylation residues suggests that at least one of these residues is able to activate IRF3 in the absence of viral infection. The specific function of these phosphoacceptor residues are now being further examined in vivo.