title: Systematic characterization by mass spectrometric analysis of phosphorylation sites in IRF-3 regulatory domain activated by IKK-i.
abstract: Interferon regulatory factor 3 (IRF-3) is a critical transcription factor that regulates innate immune responses against viral infection. Upon infection, IRF-3 is activated through phosphorylation of Ser/Thr residues in its C-terminal domain by the kinases, IKK-i and/or TBK-1. This phosphorylation triggers IRF-3 to interact with the co-activators to form a complex that activates target genes in the nucleus. However, the phosphorylation sites that determine the active/inactive status of IRF-3, estimated using biochemical methods such as mutagenesis and kinase assays, remain controversial. In the present study, phosphorylated IRF-3 189-427 (IRF-3 189C) was prepared by co-expression with IKK-i and was specifically fractionated into 3 major phosphorylation forms using anion-exchange chromatography. Identification of the phosphorylation sites was performed using systematic mass spectrometry approaches as follows: intact molecular mass analysis by nanoESI-MS, MS survey of phosphopeptides, and targeted MS/MS analysis of LC-MS/MS-based proteomics using a high-resolution Orbitrap mass spectrometer. Phosphorylated IRF-3 189C was clearly identified to exist as a mono-phosphoprotein (at Ser-402), and in two di-phosphoprotein forms (at Ser-386, -402 and Ser-396, -402). Thus, we demonstrated that Ser-386, -396 and -402 are directly phosphorylated by IKK-i in the co-expression system. These results will help provide new insights into the IRF-3 activation mechanism.
authors.affiliation: Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, N-12, W-6, Kita-ku, Sapporo 060-0812, Japan. fujii@pharm.hokudai.ac.jp
authors.forename: Kiyonaga
authors.initials: K
authors.lastname: Fujii
authors.forename: Shingo
authors.initials: S
authors.lastname: Nakamura
authors.forename: Kiyohiro
authors.initials: K
authors.lastname: Takahashi
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D050838:Interferon Regulatory Factor-3 / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D010455:Peptides; D010748:Phosphopeptides; D020543:Proteome; D011994:Recombinant Proteins; C496559:CHUK protein, human; D051550:I-kappa B Kinase