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/∈ 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. 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/∈ 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. Immediately after infection by bacteria or viruses, a series of host responses known as innate immunity takes place. Production of type I interferon (IFN-α/β) 1The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; IKK, IκB kinase; TBK, TANK binding kinase; CBP, CREB binding protein; CREB, cAMP-response element-binding protein; NDV, Newcastle disease virus; LPS, lipopolysaccharide; TRIF, TIR domain-containing adapter-inducing IFN-β; TICAM-1, TIR-containing adapter molecule.1The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; IKK, IκB kinase; TBK, TANK binding kinase; CBP, CREB binding protein; CREB, cAMP-response element-binding protein; NDV, Newcastle disease virus; LPS, lipopolysaccharide; TRIF, TIR domain-containing adapter-inducing IFN-β; TICAM-1, TIR-containing adapter molecule. is induced by these pathogens and plays an important role in primary defense as well as subsequent adaptive immunity (1De Maeyer E. De Maeyer-Guignard J. Int. Rev. Immunol. 1998; 17: 53-73Crossref PubMed Scopus (93) Google Scholar, 2Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (2111) Google Scholar, 3Sen G.C. Lengyel P. J. Biol. Chem. 1992; 267: 5017-5020Abstract Full PubMed Google genes are is induced at the transcription IRF-3 and a critical role in this Full Full PubMed Scopus Google IRF-3 is in its in the cytoplasm. by the kinases are activated and in the phosphorylation of IRF-3 specific residues E. J. 1998; 17: PubMed Scopus Google Scholar, 1998; Full Full PubMed Scopus Google Scholar, Biol. 1998; PubMed Scopus Google Scholar, E. 1998; PubMed Scopus Google Scholar, J. Biol. 1998; PubMed Scopus Google known as and to as IRF-3 kinases E. Immunol. PubMed Scopus Google Scholar, J. PubMed Scopus Google phosphorylated IRF-3 J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google and with coactivators and a of IRF-3 results E. J. 1998; 17: PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google is an to specifically the target and in the E. J. 1998; 17: PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google IRF-3 plays a role in the primary of In is and its is secondarily induced by Full Full PubMed Scopus Google to specifically phosphorylated to presumably by the kinases 1998; Full Full PubMed Scopus Google Scholar, J. 1998; 17: PubMed Google Scholar, 1998; PubMed Scopus Google plays a role in the of the primary of that it is the of IRF-3. the is for is IRF-3 and J. 1998; 17: PubMed Google Scholar, 1998; PubMed Scopus Google phosphorylation of IRF-3 is the of E. J. 1998; 17: PubMed Scopus Google Scholar, J. Biol. 1998; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google However, identified critical mutation of Ser-385 or -386 to the of IRF-3 E. J. 1998; 17: PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google the of Ser/Thr residues at and with in a a with J. PubMed Scopus Google and J. PubMed Scopus Google These results strongly suggest that the is the critical phosphorylation target and the have an However, and J. Biol. PubMed Scopus Google have that the is constitutively phosphorylated and the is the target for that is phosphorylated by kinase in J. J. Biol. Chem. Full Full PubMed Scopus Google and is the target of and in J. PubMed Scopus Google and have of with at the and These constitutively with to the and the J. J. Biol. Chem. Full Full PubMed Scopus Google that the of the the in an active IRF-3 J. Biol. PubMed Scopus Google to an in the as a and binding and to the is by the phosphorylation of the to the resulting in a J. Biol. PubMed Scopus Google the is with the mutant IRF-3 as constitutively active J. Biol. PubMed Scopus Google active after viral J. PubMed Scopus Google 5D mutant active as in J. Biol. PubMed Scopus Google it active in J. PubMed Scopus Google and as an in and and These suggest that the observed of 5D is an and 5D is in the study, we the phosphorylation of Ser-386 a antibody and that viral infection specifically induced the phosphorylation of this we that and specifically induce phosphorylation of Ser-386 with a dimerization of IRF-3. various we that the specific phosphorylation of Ser-386 is the determinant for the subsequent activation of and of in with and in with of and Newcastle disease infection as E. J. 1998; 17: PubMed Scopus Google of as E. J. 1998; 17: PubMed Scopus Google as 2001; PubMed Scopus Google of series E. J. 1998; 17: PubMed Scopus Google by the PubMed Scopus Google and J. Immunol. PubMed Scopus Google by and antibody by IRF-3 antibody E. J. 1998; 17: PubMed Scopus Google with the and is by by a with the as a by by the with of at for at for for the with the J. J. Biol. Chem. 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Production of type I interferon (IFN-α/β) 1The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; IKK, IκB kinase; TBK, TANK binding kinase; CBP, CREB binding protein; CREB, cAMP-response element-binding protein; NDV, Newcastle disease virus; LPS, lipopolysaccharide; TRIF, TIR domain-containing adapter-inducing IFN-β; TICAM-1, TIR-containing adapter molecule.1The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; IKK, IκB kinase; TBK, TANK binding kinase; CBP, CREB binding protein; CREB, cAMP-response element-binding protein; NDV, Newcastle disease virus; LPS, lipopolysaccharide; TRIF, TIR domain-containing adapter-inducing IFN-β; TICAM-1, TIR-containing adapter molecule. is induced by these pathogens and plays an important role in primary defense as well as subsequent adaptive immunity (1De Maeyer E. De Maeyer-Guignard J. Int. Rev. Immunol. 1998; 17: 53-73Crossref PubMed Scopus (93) Google Scholar, 2Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (2111) Google Scholar, 3Sen G.C. Lengyel P. J. Biol. Chem. 1992; 267: 5017-5020Abstract Full PubMed Google genes are is induced at the transcription IRF-3 and a critical role in this Full Full PubMed Scopus Google IRF-3 is in its in the cytoplasm. by the kinases are activated and in the phosphorylation of IRF-3 specific residues E. J. 1998; 17: PubMed Scopus Google Scholar, 1998; Full Full PubMed Scopus Google Scholar, Biol. 1998; PubMed Scopus Google Scholar, E. 1998; PubMed Scopus Google Scholar, J. Biol. 1998; PubMed Scopus Google known as and to as IRF-3 kinases E. Immunol. PubMed Scopus Google Scholar, J. PubMed Scopus Google phosphorylated IRF-3 J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google and with coactivators and a of IRF-3 results E. J. 1998; 17: PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google is an to specifically the target and in the E. J. 1998; 17: PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Full PubMed Scopus Google Scholar, J. Biol. Chem. Full Full PubMed Scopus Google IRF-3 plays a role in the primary of In is and its is secondarily induced by Full Full PubMed Scopus Google to specifically phosphorylated to presumably by the kinases 1998; Full Full PubMed Scopus Google Scholar, J. 1998; 17: PubMed Google Scholar, 1998; PubMed Scopus Google plays a role in the of the primary of that it is the of IRF-3. the is for is IRF-3 and J. 1998; 17: PubMed Google Scholar, 1998; PubMed Scopus Google phosphorylation of IRF-3 is the of E. J. 1998; 17: PubMed Scopus Google Scholar, J. Biol. 1998; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google However, identified critical mutation of Ser-385 or -386 to the of IRF-3 E. J. 1998; 17: PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. 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