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 IKKɛ 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 (Ser396—Ser405) alleviates autoinhibition to allow interaction with CBP (CREB-binding protein) and facilitates phosphorylation at site 1 (Ser385 or Ser386). 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. 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 IKKɛ 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 (Ser396—Ser405) alleviates autoinhibition to allow interaction with CBP (CREB-binding protein) and facilitates phosphorylation at site 1 (Ser385 or Ser386). 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. The transcription factor IRF-3 3The abbreviations used are: IRF, interferon regulatory factor; IBiD, interferon-binding domain; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; IAD, IRF association domain; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; TBK1, Tank-binding kinase 1. is a central component of the innate immune system. It is required for expression of type I interferons α and β (IFN-α and IFN-β), the chemokine RAN-TES (regulated on activation normal T cell expressed and secreted), and many other genes that respond to viral and bacterial infection (1Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 2Hiscott J. Pitha P. Genin P. Nguyen H. Heylbroeck C. Mamane Y. Algarte M. Lin R. J. Interferon Cytokine Res. 1999; 19: 1-13Crossref PubMed Scopus (196) Google Scholar, 3Genin P. Algarte M. Roof P. Lin R. Hiscott J. J. Immunol. 2000; 164: 5352-5361Crossref PubMed Scopus (189) Google Scholar, 4Barnes B. Lubyova B. Pitha P.M. J. Interferon Cytokine Res. 2002; 22: 59-71Crossref PubMed Scopus (273) Google Scholar). In the absence of infection, IRF-3 is in a latent conformation in the cytoplasm (1Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar). Upon infection, the host detects an invading pathogen through pattern recognition receptors such as the toll-like receptors or the cytoplasmic receptors RIG-I and Mda-5. Depending on the microbial pathogen, distinct signaling pathways are triggered, many of which lead to activation of IRF-3. IRF-3 is activated by phosphorylation mediated directly or as part of a signaling cascade by the IκB kinase (IKK)-related kinases, TBK1 and IKKɛ (6Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1366) Google Scholar, 7Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2085) Google Scholar, 8McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (451) Google Scholar, 9Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (464) Google Scholar, 10Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (312) Google Scholar). Phosphorylation of IRF-3 leads to dimerization, translocation to the nucleus, and association with the coactivator CREB-binding protein (CBP) or the closely related p300 (1Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar, 12Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar, 13Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (297) Google Scholar). IRF-3 shares a conserved N-terminal DNA-binding domain with other members of the interferon response factor family of transcription factors (14Panne D. Maniatis T. Harrison S.C. EMBO J. 2004; 23: 4384-4393Crossref PubMed Scopus (133) Google Scholar). It also contains a C-terminal IRF association domain (IAD), which mediates phosphorylation-dependent homo- or hetero-oligomerization with other IRF family members and interaction with CBP/p300 (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar). The crystal structure of the N-terminal DNA-binding domain bound to the interferon-β promoter and the crystal structure of the IAD have been determined (14Panne D. Maniatis T. Harrison S.C. EMBO J. 2004; 23: 4384-4393Crossref PubMed Scopus (133) Google Scholar, 15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar, 16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar). Activation of IRF-3 requires phosphorylation on a cluster of serine and threonine residues in the C-terminal region of the IAD spanning amino acids 385 to 405. Despite considerable efforts, using mutagenesis, phosphopeptide mapping, and phospho-specific antibodies, the exact position and role of phosphorylation sites in the 385-405 region remain controversial. On the one hand, Fujita and co-workers (17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) suggest that phosphorylation at Ser385/Ser386 (site 1) is key to the activation of IRF-3 and that phosphorylation at site 2, spanning residues 396 to 405, has an auxiliary but not essential role. This view is supported by the observation that mutation of Ser385 or Ser386 to Ala abolishes IRF-3 activity (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, 12Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar). In addition, Fujita and co-workers (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar, 17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) used a phosphospecific antibody against Ser386 to show that overexpressed IRF-3, activated by virus or by overexpression of upstream regulators, is phosphorylated on Ser386 and that phosphorylation on Ser386 is observed in the dimeric form of IRF-3. On the other hand, Hiscott and colleagues (11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar) argue that phosphorylation of site 2 residues may also be critical for virus-inducible IRF-3 activation. For instance, substitutions of the site 2 residues Ser396, Ser398, Ser402, Thr404, and Ser405 with the phosphomimetic Asp or Glu (IRF-3 5E) lead to a constitutive phenotype (11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar). Further mutagenesis and use of a phosphospecific antibody has identified Ser396 as a key site 2 residue that is phosphorylated in vivo upon virus infection (18Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Substitution of Ser385 and Ser386 with either Ala or Asp inhibits IRF-3 activation (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar). Taken together, data to a phosphorylation-dependent of IRF-3 activation that requires serine and a cluster of residues Ser396 and of the crystal structure of the IAD by (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar) that phosphorylation of the site 2 residues results in of an C-terminal autoinhibitory with the of Hiscott and colleagues (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). of the autoinhibitory structure by of a through either phosphorylation or mutagenesis, a for interaction with CBP/p300. (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar) also that the in the of the crystal not and that the C-terminal be in dimerization. In (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar) the of the and C-terminal autoinhibitory structure and results to be with the of Fujita and co-workers M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar). suggest that the in the is and that phosphorylation of Ser385/Ser386 results directly in IAD dimerization. a for recognition of CBP/p300 (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar). This model by a crystal structure of the IAD with the interferon-binding domain of CBP B.Y. Liu C. Srinath H. Lam S.S. Correia J.J. Derynck R. Lin K. Full Text Full Text PDF PubMed Scopus Google Scholar). This structure that removal of the autoinhibitory and of the IAD leads to of a that with and that the IAD and form a the role of phosphorylation in IRF-3 as as the role of TBK1 in we purified to full-length IRF-3, the constitutive IRF-3 and the kinase TBK1. as purified IRF-3 is a in and that IRF-3 is a to purified and protein to the phosphorylation sites in IRF-3 is phosphorylated at site 1 at site IRF-3 is phosphorylated at site 1 (Ser385 or as a of constitutive in vivo phosphorylation by an cell TBK1 can directly phosphorylate IRF-3, in a of and dimeric IRF-3, with (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, W. Yoneyama M. T. S. K. H. S. Fujita T. J. 2000; PubMed Scopus Google Scholar). of the phosphorylation sites in a purified IRF-3 phosphorylation of one serine residue at site 1 (Ser385 or and of one residue at site 2 or The purified IRF-3 is not phosphorylated at site is phosphorylated on one serine at site 2 or but mutation of residues and to Ala not in phosphorylation of site 2 also show that CBP requires phosphorylation at site 2, the residues of which are in site 2 is critical for for interaction with CBP, and for phosphorylation at site 1. Phosphorylation at site 1 leads in to IRF-3 dimerization as (17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). and in with cell with and as For of IRF-3 and IRF-3 with at a of infection of in at and by at For of TBK1, with and as of IRF-3 and IRF-3 cell by and in 1 and For TBK1 cell and in 1 and The by and the by at for The cell with for 1 and to a The with of and bound protein using in using a and purified on a in 1 The protein to for IRF-3, for IRF-3 and for TBK1 in a in and at determined by the in and the at using a the amino IRF-3 Phosphorylation with of IRF-3 for at with of TBK1 in a of 1 1 DTT, and 1 IRF-3 and IRF-3 phosphorylated by TBK1 on a in and 1 The using a of protein of and by is the of the the of the determined by of and the by the of a to in 1 and in and at IRF-3 and IRF-3 to in of and 1 to and the by 1 of of to the with and and of the for in The on a For the protein and at the for against and using a The used as a and as for using an with an and data at a of with at a of at used to IRF-3 and 2 and the and IRF-3 and 2 and the at different and determined to have The protein the amino to and through of the of to at using the determined using as an to data using a on a model an component with for and and for the determined through of the of of protein by on a protein phosphorylation by and by to by phosphorylation by of the and of The of by the with of of and Phosphorylation sites by of and in overexpressed in with an N-terminal The protein purified using by and by of purified bound to 1 of in and protein by in the For of of the different IRF-3 with of in a of The at for and a The by a at and a of the The with of and bound protein by in The protein on a of IRF-3, IRF-3 IRF-3 and IRF-3, the IRF-3 the IRF-3 and the kinase TBK1 purified the of a of using a by on a This of IRF-3 or IRF-3 of IRF-3 and of TBK1 of cell the of TBK1 phosphorylation on IRF-3 in we TBK1 with IRF-3 or IRF-3 and the by IRF-3 has the of in purified IRF-3 as a with a of to an of The IRF-3 as a with a of to an of IRF-3 with the kinase TBK1, the protein in the The a of the as IRF-3, and the a of to that of IRF-3 of the an in the of phosphorylation not TBK1 phosphorylation of IRF-3 leads to a with to of the IRF-3 The IRF-3 and to phosphorylation as the protein not of IRF-3 and IRF-3 on IRF-3 and IRF-3 to the and of and at different and different For data data at and the using a For IRF-3 and IRF-3 the that the a For IRF-3, the data a of 2 to the of that IRF-3 is a For IRF-3 we a of 2 the the that IRF-3 is a of the TBK1 Phosphorylation in TBK1 phosphorylation of IRF-3 results in one of which a at the position as dimeric IRF-3 that TBK1 phosphorylation dimerization of IRF-3 in with and proteins to the role of phosphorylation in IRF-3 activation. by IRF-3, IRF-3 IRF-3 and IRF-3 by the dimeric and The and dimeric IRF-3 phosphorylated at a site 1 residue (Ser385 or data not allow to which is, site 1 been phosphorylated by cell kinases to and the protein This constitutive phosphorylation observed in the of the and phosphorylation of IRF-3, of which not the of the cell phosphorylation by TBK1, IRF-3 a at site 2 or data not allow to which The IRF-3 that remained at in 1) not been at site the IRF-3 that at in 1) phosphorylated at sites 1 and the role of phosphorylation at and we residues to (IRF-3 and the TBK1 kinase and The IRF-3 and that dimerization on phosphorylation of Ser385 or Ser386 as in the In addition, we in and dimeric IRF-3 a phosphorylation in one of the site 2 or data that phosphorylation at site 1 leads to dimerization, as the TBK1 phosphorylation-dependent and the IRF-3 have been phosphorylated data also show that phosphorylation at site 1 site 2 a either phosphorylation or the a that a at site 1 either a or a phosphomimetic at site we can that TBK1 at site 2, as site phosphorylated in and the that been to a at site 1 data that in site 2 can be by TBK1, as mutation of residues to Ala not site 2 of IRF-3 and IRF-3 of the C-terminal activation domain of IRF-3 in the and absence of the domain CBP have been determined (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar, 16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar, B.Y. Liu C. Srinath H. Lam S.S. Correia J.J. Derynck R. Lin K. Full Text Full Text PDF PubMed Scopus Google Scholar). show that the of the IRF-3 and of the are The autoinhibitory 1 and be to a the 3 and that with the we that phosphorylation in the which allow interaction with and dimerization. IRF-3 and dimeric IRF-3 by and that IRF-3 is type to a of the IRF-3 been a of type been at that data show that IRF-3 is IRF-3 IRF-3 leads to N-terminal that the the of the protein and that the results C-terminal to and that at residue in a amino acids sites and are autoinhibitory 1 and In of IRF-3 and in with at and that activated IRF-3 has a that of 1 and at and In the IRF-3 sites are not of interaction of 1 and with 3 and (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar). with the of IRF-3 phosphorylation on the interaction with CBP, we using CBP as in IRF-3 on site and on site 1 and site and the constitutive IRF-3 on site 1) with IBiD, the full-length IRF-3 or the N-terminal DNA-binding domain of IRF-3 is, of a at site 2, either through phosphomimetic (IRF-3 5E) or through is to autoinhibition and allow interaction with by phosphorylation of site not required for that a phosphorylation-dependent regulates activation of IRF-3. Phosphorylation of site 1 and site 2 is required for IRF-3 activation. Phosphorylation at site 2 is critical for autoinhibition and interaction with Phosphorylation at site 1 is in essential for IRF-3 dimerization. TBK1 phosphorylated site 2, phosphorylation at site 1 and as by IRF-3 dimerization. suggest that in TBK1 IRF-3 by a two-step in which site 2 is phosphorylated of in the site 2 residues leads to of the autoinhibitory and phosphorylation of site 1 leads to dimerization data not which of the site 1 residues is is to be on (17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and M. McWhirter and T. is that TBK1 IRF-3 by phosphorylation of the C-terminal residues S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (451) Google Scholar, B.R. S. W. Grandvaux N. H. Yamamoto M. Akira S. Yeh W.C. Lin R. Hiscott J. J. 2004; PubMed Scopus Google TBK1 is the kinase required for IRF-3 phosphorylation at sites 1 and 2 in phosphorylation at site 2 is as as data that site 1 can be phosphorylated in or phosphomimetic are in site 2 (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar, 17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). we suggest that of autoinhibition by TBK1 phosphorylation of site 2 leads to a structure that is phosphorylated at site 1 by TBK1 or by The of a kinase be with the observation that TBK1 is not for IRF-3 activation by 3 in vivo and that a signaling mediated by kinase or by protein kinase may be required K.L. S. S. Nat. Struct. Mol. Biol. 2004; PubMed Scopus Google Scholar, J. Nguyen M. M. F. E. J. Biol. Chem. Google Scholar). have for either of show that phosphorylation of site 2 by TBK1 results in IRF-3 that is to of but not dimerization, is and for interaction with This is with the crystal structure of IRF-3 in with the of the crystal structure and a IRF-3 and CBP B.Y. Liu C. Srinath H. Lam S.S. Correia J.J. Derynck R. Lin K. Full Text Full Text PDF PubMed Scopus Google Scholar). not be the of IRF-3 virus infection is with other such as IRF-3 or to form homo- or as (1Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). results are with the observation that a is (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar). data that a or a is to CBP in a virus-inducible (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). M. McWhirter and T. be but be to CBP phosphorylation of the site 2 It is that an IRF-3 to CBP in vivo and that IRF-3 dimerization through phosphorylation of site 1 also observed that the phosphorylated IRF-3 to the phosphorylated IRF-3 not the that dimeric IRF are required for to CBP and to in is the different site 2 as by the of phosphomimetic or For mutation of Ser396 or to Ala the virus of IRF-3, of phosphomimetic Asp at residues is to a constitutive phenotype (11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar, M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). of Ser402, Thr404, or Ser405 with Ala virus with Asp a constitutive phenotype (11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar, M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). TBK1 an site 2 residue and Ser405 have been to but data not which of the site 2 residues is The crystal structure of latent IRF-3 has that the site 2 residues are part of the autoinhibitory structure and that of are in a (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar, 16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar). activation with the is that phosphorylation of the autoinhibitory and of 3 and for interaction with the of CBP (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar, B.Y. Liu C. Srinath H. Lam S.S. Correia J.J. Derynck R. Lin K. Full Text Full Text PDF PubMed Scopus Google Scholar). In different different residues in site 2 to be the for activation. at site 2 is for a and as different signaling pathways different of protein The of the residue site 2 that is the phosphorylation on the and cell used in an as the of for phosphorylation of site 1. For Ser396 is phosphorylated in in response to with virus and but not in response to with (18Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The IRF is in but in (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, W. Yoneyama M. T. S. K. H. S. Fujita T. J. 2000; PubMed Scopus Google Scholar). that constitutive phosphorylation of site 1 in the of the mutation is of different constitutive of the kinase have in the that the residues phosphorylated by TBK1 in are and residues are in the and part of the of the autoinhibitory structure (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar). an for by to the of Ser405 has with and Phosphorylation of either residue or is to of the autoinhibitory structure and of the of and for interaction with the of In kinase using a IRF-3 residues that is the TBK1 but the a or of the autoinhibitory and may not a In phosphorylation of Ser396 to be the Substitution of Ser396 with Asp a form of IRF-3, and mutation to Ala the virus of genes (18Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). is in the It a with the of and with the of that phosphorylation of Ser396, of or the structure (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar). In the structure of the autoinhibitory region has that of of the site 2 residues the of the site and phosphorylation of site the region to have in response to a of and a of tenOever of for of the of at for and of the for N-terminal