Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation.
Interferon regulatory factor (IRF)-3 is a critical transcription factor regulating innate immune responses against viral and bacterial infections. Signals activated by various pathogens are integrated by IRF-3 kinase, resulting in the specific phosphorylation of IRF-3 in the cytoplasm. This phosphorylation induces dimerization and association with the coactivators CREB-binding protein/p300, and the resultant complex activates the target genes in the nucleus. However, the phosphorylation sites that determine the active/inactive status of IRF-3 have been a source of controversy. In this study, we generated an antibody that specifically detects the phosphorylation of Ser-386 and used it as a probe. We found: 1) viral infection specifically induces phosphorylation of the Ser-386; 2) recently identified IRF-3 kinases (IKK-i/epsilon and TBK-1) phosphorylate Ser-386 and induce its dimerization; 3) phosphorylation of Ser-386 is exclusively observed with the dimer; 4) mutation at Ser-386 abolishes the dimerization potential; 5) a constitutively active 5D mutant designed to mimic phosphorylation of Ser/Thr residues other than Ser-385 and -386 is secondarily phosphorylated at Ser-386, presumably by an irrelevant kinase. These results strongly suggest that Ser-386 is the target of the IRF-3 kinase and critical determinant for the activation of IRF-3.
Mori
Mitsuaki
M
Department of Tumor Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Japan.
Yoneyama
Mitsutoshi
M
Ito
Takashi
T
Takahashi
Kiyohiro
K
Inagaki
Fuyuhiko
F
Fujita
Takashi
T
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D051550:I-kappa B Kinase; D015151:Immunoblotting; D050838:Interferon Regulatory Factor-3; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry*; D014162:Transfection
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; C494233:Irf3 protein, mouse; D014157:Transcription Factors; C020723:citrate-binding transport protein; D012694:Serine; C498588:Tbk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human; D051550:I-kappa B Kinase