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• W2072916053 abstract "The ubiquitously expressed latent interferon regulatory factor (IRF) 3 transcription factor is activated in response to virus infection by phosphorylation events that target a cluster of Ser/Thr residues,382GGASSLENTVDLHISNSHPLSLTSDQY408at the C-terminal end of the protein. To delineate the minimal phosphoacceptor sites required for IRF-3 activation, several point mutations were generated and tested for transactivation potential and cAMP-response element-binding protein-binding protein/p300 coactivator association. Expression of the IRF-3 S396D mutant alone was sufficient to induce type I IFN β, IFNα1, RANTES, and the interferon-stimulated gene 561 promoters. Using SDS-PAGE and immunoblotting with a novel phosphospecific antibody, we show for the first time that, in vivo, IRF-3 is phosphorylated on Ser396 following Sendai virus infection, expression of viral nucleocapsid, and double-stranded RNA treatment. These results demonstrate that Ser396 within the C-terminal Ser/Thr cluster is targeted in vivo for phosphorylation following virus infection and plays an essential role in IRF-3 activation. The inability of the phosphospecific antibody to detect Ser396phosphorylation in lipopolysaccharide-treated cells suggests that other major pathways may be involved in IRF-3 activation following Toll-like receptor 4 stimulation. The ubiquitously expressed latent interferon regulatory factor (IRF) 3 transcription factor is activated in response to virus infection by phosphorylation events that target a cluster of Ser/Thr residues,382GGASSLENTVDLHISNSHPLSLTSDQY408at the C-terminal end of the protein. To delineate the minimal phosphoacceptor sites required for IRF-3 activation, several point mutations were generated and tested for transactivation potential and cAMP-response element-binding protein-binding protein/p300 coactivator association. Expression of the IRF-3 S396D mutant alone was sufficient to induce type I IFN β, IFNα1, RANTES, and the interferon-stimulated gene 561 promoters. Using SDS-PAGE and immunoblotting with a novel phosphospecific antibody, we show for the first time that, in vivo, IRF-3 is phosphorylated on Ser396 following Sendai virus infection, expression of viral nucleocapsid, and double-stranded RNA treatment. These results demonstrate that Ser396 within the C-terminal Ser/Thr cluster is targeted in vivo for phosphorylation following virus infection and plays an essential role in IRF-3 activation. The inability of the phosphospecific antibody to detect Ser396phosphorylation in lipopolysaccharide-treated cells suggests that other major pathways may be involved in IRF-3 activation following Toll-like receptor 4 stimulation. Recognition of invading pathogens such as viruses and bacteria by host cells is known to trigger the activation of multiple latent transcription factors, such as NF-κB, AP-1 (ATF-2/c-Jun), and the interferon regulatory factors (IRFs) 1The abbreviations used are: IRF, interferon regulatory factor; IFN, interferon; LPS, lipopolysaccharide; STAT, signal transducers and activators of transcription; ISRE, interferon-stimulated response element(s); ds, double-stranded; CBP, cAMP-response element-binding protein-binding protein; PRD, positive regulatory domain(s); RANTES, regulated on activation normal T cell expressed and secreted; ISG, IFN-stimulated gene; N, nucleocapsid; SeV, Sendai virus; HEK, human embryonic kidney; TLR, Toll-like receptor; WCE, whole cell extracts; HAU, hemagglutination units 1The abbreviations used are: IRF, interferon regulatory factor; IFN, interferon; LPS, lipopolysaccharide; STAT, signal transducers and activators of transcription; ISRE, interferon-stimulated response element(s); ds, double-stranded; CBP, cAMP-response element-binding protein-binding protein; PRD, positive regulatory domain(s); RANTES, regulated on activation normal T cell expressed and secreted; ISG, IFN-stimulated gene; N, nucleocapsid; SeV, Sendai virus; HEK, human embryonic kidney; TLR, Toll-like receptor; WCE, whole cell extracts; HAU, hemagglutination units (1Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Google Scholar, 2Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Google Scholar, 3Servant M.J. Grandvaux N. Hiscott J. Biochem. Pharmacol. 2002; 64: 985-992Google Scholar). Once activated, these transcription factors regulate the expression of a set of genes encoding for immunomodulatory cytokines and chemokines that are involved in the establishment of the antiviral/bacterial state, which limits the spread of infection through innate and adaptive immune mechanisms (1Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Google Scholar, 2Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Google Scholar, 3Servant M.J. Grandvaux N. Hiscott J. Biochem. Pharmacol. 2002; 64: 985-992Google Scholar). The best characterized component of the innate host defense to virus is the family of transcriptionally activated interferon (IFN) proteins, which include type I IFN-α and IFN-β and type II IFN-γ. Type I IFNs are mainly induced in response to infection by various types of RNA and DNA viruses (2Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Google Scholar, 4Sen G.C. Annu. Rev. Microbiol. 2001; 55: 255-281Google Scholar), although the bacterial endotoxin lipopolysaccharide (LPS) induces production of IFN in certain cells, albeit at low levels (5Sing A. Merlin T. Knopf H.-P. Nielsen P.J. Loppnow H. Galanos C. Freudenberg M.A. Infect. Immunity. 2000; 68: 1600-1607Google Scholar, 6Toshchakov V. Jones B.W. Prerera P.Y. Thomas K.W. Cody M.J. Zhang S. Williams B.R.G. Major J. Hamilton T.A. Fenton M.J. Vogel S.N. Nat. Immunology. 2002; 3: 392-398Google Scholar). Once produced, these secreted proteins act in a paracrine fashion to induce gene expression in target cells in the adjacent microenvironment through engagement of cell surface IFN receptors and the JAK-STAT signaling pathway. The ISGF3 complex (ISGF3γ/IRF-9-STAT1-STAT2) binds to interferon-stimulated response elements (ISRE) found in numerous IFN-induced genes, such as 2′-5′ oligoadenylate synthase and the double-stranded (ds) RNA-activated kinase, resulting in the induction of proteins that impair viral gene expression and replication (2Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Google Scholar, 4Sen G.C. Annu. Rev. Microbiol. 2001; 55: 255-281Google Scholar). IRF-3 is part of the IRF family of transcription factors that includes nine members with distinct roles in host defense against pathogens, immunomodulation, and growth control (for review see Refs. 3Servant M.J. Grandvaux N. Hiscott J. Biochem. Pharmacol. 2002; 64: 985-992Google Scholar, 7Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene (Amst.). 1999; 237: 1-14Google Scholar, and8Servant M.J. ten Oever B. Lin R. J. Interferon Cytokine Res. 2002; 22: 49-58Google Scholar). Previous studies have demonstrated that the C-terminal region of IRF-3, which comprises a cluster of phosphoacceptor sites,382GGASSLENTVDLHISNSHPLSLTSDQY408, is phosphorylated as a consequence of virus infection (9Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Google Scholar, 10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar). Radioactive orthophosphate labeling, peptide mapping, phosphoamino acid analysis, and decreased mobility in SDS-PAGE have shown that IRF-3 is a phosphoprotein that is further inducibly phosphorylated upon virus infection (10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar, 11Yoneyama M. Suhara W. Fukuhara Y. Fujita T. J. Interferon Cytokine Res. 1997; 17: S53Google Scholar, 12Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Google Scholar, 13Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Google Scholar, 14Smith E.J. Marie I. Prakash A. Garcia-Sastre A. Levy D.E. J. Biol. Chem. 2001; 276: 8951-8957Google Scholar, 15Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Google Scholar). The generation of point mutated forms of IRF-3 suggests that Ser385 and Ser386 (9Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Google Scholar), as well as Ser396, Ser398, Ser402, Ser405, and Thr404 (10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar) are phosphorylated following virus infection and are involved in IRF-3 activation. Indeed, C-terminal phosphorylation is thought to produce a change in protein conformation that reveals the IRF association domain and the DNA-binding domain, thus promoting dimerization and binding to IRF-3 5′-GAAA(C/G)(C/G)GAAN(T/C)-3′ consensus DNA-binding site (9Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Google Scholar, 10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar,16Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Google Scholar). In addition, IRF-3 C-terminal phosphorylation is required for association with the histone acetyltransferase nuclear proteins CBP and p300 (9Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Google Scholar, 10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar, 16Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Google Scholar) causing IRF-3, which normally shuttles into and out of the nucleus, to become predominantly nuclear (9Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Google Scholar, 10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar, 17Kumar K.P. McBride K.M. Weaver B.K. Dingwall C. Reich N.C. Mol. Cell. Biol. 2000; 20: 4159-4168Google Scholar). The activated form of IRF-3, bound to CBP, induces transcription through distinct positive regulatory domains (PRD) in the type I IFN promoters and through select ISRE sites found in other genes such as the chemokine RANTES, the cytokine interleukin-15, and IFN-stimulated gene (ISG) 56 (10–12, 15, 18–22). Finally, phosphorylation of IRF-3 is thought to induce its degradation by a proteasome-mediated mechanism (10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar, 23Ronco L.V. Karpova A.Y. Vidal M. Howley P.M. Genes Dev. 1998; 12: 2061-2072Google Scholar). In addition to virus infection, other stimuli such as LPS and poly(I-C) have been shown to induce IRF-3 activation (9Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Google Scholar, 12Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Google Scholar, 15Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Google Scholar, 24Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Mori W. Shiota K. Okabe Y. Namiki H. Fujita T. Genes Cells. 2001; 6: 375-388Google Scholar, 25Navarro L. David M. J. Biol. Chem. 1999; 274: 35535-35538Google Scholar, 26Kawai T. Takeuchi O. Fujita T. Inoue J. Muhlradt P.F. Sato S. Hoshino K. Akira S. J. Immunol. 2001; 167: 5887-5894Google Scholar, 27Malakhova O.A. Malakhov M.P. Hetherington C.J. Zhang D.E. J. Biol. Chem. 2002; 277: 14703-14711Google Scholar, 28Shinobu N. Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Fukuhara Y. Amano F. Fujita T. FEBS Lett. 2002; 517: 251-256Google Scholar). However, no phosphorylation of IRF-3 in response to poly(I-C) treatment has been demonstrated. In this case, IRF-3 activation has been observed through nuclear accumulation, DNA binding activity, coactivator association, and gene induction (9Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Google Scholar, 12Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Google Scholar, 15Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Google Scholar, 24Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Mori W. Shiota K. Okabe Y. Namiki H. Fujita T. Genes Cells. 2001; 6: 375-388Google Scholar). On the other hand, phosphorylation of IRF-3 in response to LPS was demonstrated (26Kawai T. Takeuchi O. Fujita T. Inoue J. Muhlradt P.F. Sato S. Hoshino K. Akira S. J. Immunol. 2001; 167: 5887-5894Google Scholar) but not sufficiently to detail the precise phosphoacceptor sites. The in vivo signaling pathways leading to IRF-3 phosphorylation and activation as well as the precise phosphoacceptor sites remain to be elucidated. Previous studies have demonstrated inducible N-terminal phosphorylation of IRF-3 following the activation of mitogen-activated protein kinase kinase kinase pathways (13Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Google Scholar). More recently, the activation of DNA-PK following virus infection was shown to induce Thr135 phosphorylation (29Karpova A.Y. Trost M. Murray J.M. Cantley L.C. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2818-2823Google Scholar). However, no convincing physiological roles have been ascribed to these covalent modifications. In the present study, we characterized the minimal phosphoacceptor site(s) involved in the in vivo activation of IRF-3 following treatment with known inducers. Of the seven potential phosphoacceptor sites present in the C-terminal cluster, a single point mutation of Ser396 to Asp (S396D) was sufficient to generate a strong constitutively active form of IRF-3. Moreover, by using a novel phosphospecific antibody, we show for the first time that Ser396 is phosphorylated in vivofollowing virus infection, nucleocapsid (N) expression or dsRNA treatment. LPS (L-2654) was purchased from Sigma and dissolved in distilled water. Poly(I-C) was purchased from AmershamBiosciences and resuspended in phosphate-buffered saline. Sendai virus (SeV) was a generous gift of Dr. Ilkka Julkunen (Public Health Research Institute, Helsinki, Finland). wtIRF-3, wtIRF-3 5A, wtIRF-3 5D, wtIRF-3 3D, wtIRF-3 J2A, and wtIRF-3 J2D pFLAG constructs and the luciferase reporter plasmids IFNα promoter (IFNA1 pGL-3), IFNβ promoter (IFNB pGL-3), and the RANTES promoter (RANTES pGL-3) were described previously (13Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Google Scholar, 18Lin R. Heylbroeck C. Genin P. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1999; 19: 959-966Google Scholar, 21Lin R. Genin P. Mamane Y. Hiscott J. Mol. Cell. Biol. 2000; 20: 6342-6353Google Scholar). The expression constructs encoding different IRF-3 C-terminal point mutants pFLAG-IRF-3 3A, 2D, 2A, S396A, S396D, S398A, S398D, S398A/S402A, and S398D/S402D were generated by overlap PCR mutagenesis using Vent DNA polymerase. The pCMV-2 measles N expression construct has been described previously (30tenOever B.R. Servant M.J. Grandvaux N. Lin R. Hiscott J. J. Virol. 2002; 76: 3659-3669Google Scholar). SeV N cDNA was subcloned from pGEM SeV N plasmid (a gift from Dr. Illka Julkunen) using 5′-AATGGCTGGGTTGTTGAGCACCTTC-3′ and 5′-TTAGATTCCTCCCATCCCAGCTGCT-3′ as forward and reverse primers, respectively. The PCR-generated fragment was subcloned into pCMV-2. Human embryonic kidney (HEK) 293 cells were grown in α-minimum essential medium. HEC-1B cells and the astrocytoma cell line U373 overexpressing the Toll-like receptor (TLR)-4 coreceptor CD14 (U373/CD14), a gift from Dr. Michael David (University of California, San Diego, CA), were cultured in Dulbecco's modified Eagle's medium. The media were supplemented with 10% fetal bovine serum and antibiotics. U937 cells were grown in RPMI supplemented with 5% fetal clone (Hyclone) and antibiotics. All of the transfections were carried out on subconfluent HEK 293 cells (calcium phosphate coprecipitation method) or HEC-1B cells (FuGENE method) grown in 60-mm Petri dishes or 24-well plates (for the luciferase assay). 5 μg of DNA constructs (per 60-mm dish) or 10 ng of pRLTK reporter (Renilla luciferase for internal control), 100 ng of pGL3 reporter (firefly luciferase, experimental reporter), and 250 ng of pFLAG expression plasmids (24-well plate) were introduced into HEK 293 cells. At 36 h, the cells were collected, washed in ice-cold phosphate-buffered saline, and assayed for reporter gene activities (Promega). Infections with SeV as well as treatments with poly(I-C) and LPS were accomplished in serum-free medium for the first 2 h, after which 10% fetal bovine serum was added for the rest of the incubation period. Whole cell extracts (WCE) were prepared in Nonidet P-40 lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 5 mm EDTA, 10% glycerol, 30 mm NaF, 1.0 mm Na3VO4, 40 mmβ-glycerophosphate, 0.1 mm phenylmethylsulfonyl fluoride, and 5 μg/ml of each leupeptin, pepstatin, and aprotinin) and stored at −80 °C. Total RNA was harvested using Trizol reagent (Invitrogen) as recommended by the manufacturer. RNA was reverse transcribed with Superscript (Invitrogen), and the resulting cDNA were used in a PCR with the primers described for the original cloning of virus N cDNAs (see above) at an annealing temperature of 60 °C using Taq DNA polymerase (AmershamBiosciences). To generate the polyclonal antibody specific for IRF-3 phosphorylated at Ser396, termed HIS5033, a phosphopeptide corresponding to residues 388–402 of human IRF-3, KENTVDLHIS(PO 4−)NSHPLS, was synthetized (W. M. Keck Biotechnology Resource Center), coupled to keyhole limpet hemocyanin, and used to immunize rabbits (Pocono Rabbit Farms & Laboratory, Inc, Canadensis, PA). Briefly, 2 mg of peptide in phosphate-buffered saline was coupled to 5 mg of keyhole limpet hemocyanin (Sigma) in 0.2% glutaraldehyde. After neutralization with glycine, the coupled peptide was dialyzed overnight against cold phosphate-buffered saline and resuspended at a concentration of 2 mg/ml for immunization. To analyze the state of IRF-3 phosphorylation and to confirm the expression of the transgenes, WCE (30–60 μg) were subjected to electrophoresis on 7.5% or 10% acrylamide gels. The proteins were electrophoretically transferred to Hybond-C nitrocellulose membranes (Nycomed Amersham, Inc.) in 25 mm Tris, 192 mm glycine, and 20% methanol. The membranes were blocked in Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20 for 1 h at 25 °C before incubation for 2 h at 25 °C with anti-IRF-3 (Santa Cruz; SC-9082), anti-FLAG M2 (Sigma), anti-IκBα (MAD 10) (1 μg/ml), anti-MYC (9E10) (1 μg/ml), or anti-ISG56 (a gift from Dr. G. Sen (Cleveland, OH) (1:1000) in blocking solution. For the phosphospecific antibodies anti-IκBα Ser32 phosphospecific antibody (1:2000) (New England Biolab) and HIS5033 (1:10000), the membranes were incubated in blocking solution for 1 h at 25 °C followed by incubation in Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 for overnight at 4 °C. After washing four times in Tris-buffered saline, 0.1% Tween 20, the membranes were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:10000) in blocking solution. Immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences). For coimmunoprecipitation studies, WCE (500 μg) were incubated with 1 μg of anti-CBP antibody A-22 (Santa Cruz) or 1 μg of anti-FLAG antibody M2 linked to 30 μl of protein A- or protein G-Sepharose beads, respectively, for 3 h at 4 °C (AmershamBiosciences). The beads were washed five times with Nonidet P-40 lysis buffer and resuspended in denaturating sample buffer, and the eluted IRF-3 proteins associated with CBP were analyzed by immunoblotting. To delineate the minimal residues required for IRF-3 activation, the effects of various phosphomimetic point mutations (Fig.1 A) on the transactivating potential of IRF-3 were analyzed using reporter gene assays with the IRF-3-responsive promoters IFNα1, IFNβ, and RANTES (Fig.1 B). Overexpression of wtIRF-3 alone minimally induced IFNα1, IFNβ, and RANTES promoter activities, as demonstrated previously (10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar, 21Lin R. Genin P. Mamane Y. Hiscott J. Mol. Cell. Biol. 2000; 20: 6342-6353Google Scholar, 31Juang Y.T. Lowther W. Kellum M. Au W.C. Lin R. Hiscott J. Pitha P.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9837-9842Google Scholar), whereas introduction of the C-terminal point mutation, S396D, enhanced IRF-3 transactivating potential over wtIRF-3 by 13-, 14-, and 11-fold for the IFNα1, IFNβ, and RANTES promoters, respectively (Fig. 1 B). The IFNα1, IFNβ, and RANTES promoters were also activated by the double point mutant 2D (13-, 12-, and 12-fold over wtIRF-3, respectively) and, as previously reported (10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar, 16Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Google Scholar, 18Lin R. Heylbroeck C. Genin P. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1999; 19: 959-966Google Scholar, 21Lin R. Genin P. Mamane Y. Hiscott J. Mol. Cell. Biol. 2000; 20: 6342-6353Google Scholar), the multiple point mutant 5D (9-, 5.5-, and 8-fold induction, respectively). However, the point mutants S398D, S398D/S402D, and S402D/S404D/S405D exhibited only intermediate effects, and the S385D/S386D mutant did not induce these promoters. Our initial result thus demonstrates that substitution of Ser396 with the phosphomimetic Asp is sufficient to generate a constitutively active form of IRF-3 that functions as a strong activator of promoters containing PRD I–III or ISRE regulatory elements. Formation of the IRF-3 holocomplex, which consists of an IRF-3 dimer and CBP or p300 coactivators, is a critical step in the activation of the transcription factor (32Suhara W. Yoneyama M. Kitabayashi I. Fujita T. J. Biol. Chem. 2002; 277: 22304-22313Google Scholar). Association with CBP/p300 is strictly localized to the nucleus and tethers IRF-3 in the nucleus after induction with viruses (16Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Google Scholar, 17Kumar K.P. McBride K.M. Weaver B.K. Dingwall C. Reich N.C. Mol. Cell. Biol. 2000; 20: 4159-4168Google Scholar, 32Suhara W. Yoneyama M. Kitabayashi I. Fujita T. J. Biol. Chem. 2002; 277: 22304-22313Google Scholar). Therefore, the relationship between the different IRF-3 point mutants and CBP association was evaluated using coimmunoprecipitation assays. As shown in Fig.2, infection with SeV stimulated wtIRF-3 and CBP association (Fig. 2, A and B, lanes 1 and 2). Mutation of Ser396 to Ala completely abrogated virus-induced CBP association (Fig. 2 A,lanes 3 and 4), whereas the phosphomimetic point mutant S396D constitutively associated with CBP (Fig. 2 B, compare lanes 1 and 3), an association that was enhanced following virus infection (Fig. 2 B, lane 4). Similar results were obtained with the other mutants IRF-3 2A (Fig. 2 A, lanes 7 and 8) and IRF-3 5A (Fig. 2 A, lanes 13 and 14), where no inducible association with CBP was observed. Conversely, strong constitutive binding to CBP was present with IRF-3 2D (Fig.2 B, lane 7) and IRF-3 5D (Fig. 2 B,lane 13). As a control for these results, the S398A form of IRF-3 was still able to associate with CBP following virus infection (Fig. 2 A, lanes 5 and 6), but only a basal association of the phosphomimetic counterpart IRF-3 S398D with CBP was observed in the absence of infection (Fig. 2 B, compare lanes 1 and 5). As previously reported (16Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Google Scholar), coactivator association was not observed with either the IRF-3 J2A or J2D mutants (Fig. 2, A and B, lanes 15 and 16). As discussed previously (30tenOever B.R. Servant M.J. Grandvaux N. Lin R. Hiscott J. J. Virol. 2002; 76: 3659-3669Google Scholar), Ser385/386 phosphorylation may be required for the sequential phosphorylation of the Ser/Thr cluster at amino acids 396–405. Together these data indicate that Ser396 is a critical phosphoacceptor site for coactivator CBP/p300 association and that a clear correlation exists between the capacity of the point mutants to induce promoter activity and to associate with the CBP coactivator. Recent studies have demonstrated that the561 gene encoding the ISG56 protein is strongly induced in response to virus infection, type I IFN, dsRNA (33Guo J. Peters K.L. Sen G.C. Virology. 2000; 267: 209-219Google Scholar) or direct expression of IRF-3 5D (20Grandvaux N. Servant M.J. tenOever B.R. Sen G.C. Balachandran S. Barber G.N. Lin R. Hiscott J. J. Virol. 2002; 76: 5532-5539Google Scholar). The effect of IRF-3 on 561 gene induction is direct, because the promoter contains two tandem ISRE sites, mutation of which results in the complete loss of promoter activation by IRF-3 5D (20Grandvaux N. Servant M.J. tenOever B.R. Sen G.C. Balachandran S. Barber G.N. Lin R. Hiscott J. J. Virol. 2002; 76: 5532-5539Google Scholar). To determine the effect of transient expression of the different phosphomimetic IRF-3 forms on the induction of the endogenous ISG56 in the IFN-unresponsive HEC-1B cells, induction of ISG56 was analyzed by immunoblot (Fig.3). Sendai virus infection resulted in 25-fold induction of ISG56 (Fig. 3, lane 10), whereas wtIRF-3 expression alone resulted in a weak induction of ISG56 protein of 2.6-fold (Fig. 3, lane 2). Transfection of S396D as well as 2D and 5D resulted in a stronger induction of the protein of 5.2-, 6.4-, and 10-fold, respectively (Fig. 3, lanes 3,5, and 8). However, only weak inductions between 2.2- and 3.5-fold were observed with the other point mutants (Fig. 3,lanes 4, 6, 7, and 9). Thus, mutation at position 396 is sufficient to create a phosphomimetic form of IRF-3 that allows stable binding to CBP/p300 coactivator, transactivation of reporter genes controlled by PRD and ISRE response elements, and, importantly, enhancement of endogenous expression of ISG56. To verify whether in vivophosphorylation of IRF-3 occurred at Ser396, an antibody directed against a phosphopeptide spanning Ser396, named HIS5033, was raised (see “Materials and Methods”). Fig.4 A shows that the antibody reacted only toward the transfected FLAG-wtIRF-3 transgene when cells were infected with SeV; indeed, no signal was observed in IRF-3 S396A-overexpressing cells infected with SeV. In contrast, Fig.4 B shows a specific endogenous signal when extracts derived from SeV-infected HEK 293 cells were analyzed by immunoblot using the phosphospecific 396-P antibody HIS5033 (Fig. 4 B, lanes 2 and 3). Reblotting of the stripped membrane with an anti-IRF-3 antibody (SC-9082) showed that the specific signal observed with HIS5033 antibody corresponded to the slowly migrating form of IRF-3, previously shown to represent the activated form of IRF-3 (Fig.4 B, compare lanes 4–6) (10Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Google Scholar, 13Servant M.J. ten Oever B. LePage C. Conti L. Gessani S. Julkunen I. Lin R. Hiscott J. J. Biol. Chem. 2001; 276: 355-363Google Scholar). Although IRF-3 degradation had already reduced the amount of IRF-3 in these cells at 6 h post-infection (Fig. 4 B, lane 5) and the IRF-3 signal was difficult to detect with anti-IRF-3 antibody (SC-9082), the phosphospecific antibody HIS5033 reacted strongly under these conditions (Fig. 4 B, compare lanes 2and 5 and lanes 3 and 6). To investigate whether the phosphorylation of Ser396 could be mimicked by expression of viral N protein (30tenOever B.R. Servant M.J. Grandvaux N. Lin R. Hiscott J. J. Virol. 2002; 76: 3659-3669Google Scholar), transfection experiments were performed with constructs expressing measles virus and SeV N for 36 h post-transfection. As shown in Fig. 4 C, expression of either SeV or measles virus N induced higher migrating forms of IRF-3 (middle panel). Use of HIS5033 antibody revealed that Ser396 was phosphorylated under these conditions (Fig. 4 C, top panel). Taken together, these results demonstrate that virus infection or expression of viral nucleocapsid protein is sufficient to induce Ser396phosphorylation. To study the kinetics of Ser396phosphorylation in response to virus, dsRNA, or LPS, the LPS-sensitive human U373/CD14 astrocytoma cell line was used as previously described (34Orr S.L. Tobias P. J. Endotoxin Res. 2000; 6: 215-222Google Scholar). Detailed kinetics of SeV, LPS, and poly(I-C) induction was performed, and the phosphorylation state of Ser396 was analyzed by immunoblot using the phosphospecific antibody HIS5033. As shown in Fig. 5 (A andB), both SeV and poly(I-C) induced Ser396phosphorylation of IRF-3 with maximum signals detected at 4 and 2 h, respectively (Fig. 5, A, lanes 4–7, andB, lanes 3 and 4). Specific phosphoserine 396 signal was detected when IRF-3 first displayed a retarded mobility in SDS-PAGE, as observed by the use of the SC-9082 antibody (Fig. 5, A, lanes 12–15, andB, lanes 11–13). However, no signal was detected when cells were treated with LPS (Fig. 5 C, lanes 1–8). Furthermore, qualitative and temporal differences in the kinetics of IRF-3 Ser396 phosphorylation by SeV and poly(I-C) were identified: 1) Although phosphorylation of IRF-3 in response to SeV infection, first detected at 4 h post-infection, was followed by degradation (Fig. 5 A, lanes 12–16), phosphorylation in response to poly(I-C) was detected at 2 h and did not lead to degradation. Rather, IRF-3 appeared to be dephosphorylated over time and returned to basal forms (Fig.5 B, lanes 12–16). 2) Although Ser396phosphorylation was not detected after LPS stimulation, a transient shift from form I to form II (Fig. 5 C, lanes 13–15) was observed. This" @default.
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• W2072916053 title "Identification of the Minimal Phosphoacceptor Site Required for in Vivo Activation of Interferon Regulatory Factor 3 in Response to Virus and Double-stranded RNA" @default.
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