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Identification of Distinct Signaling Pathways Leading to the Phosphorylation of Interferon Regulatory Factor 3

2001; Elsevier BV; Volume: 276; Issue: 1 Linguagem: Inglês

10.1074/jbc.m007790200

1083-351X

Marc J. Servant, Benjamin ten Oever, Cécile Le Page, Lucia Conti, Sandra Gessani, Ilkka Julkunen, Rongtuan Lin, John Hiscott,

NF-κB Signaling Pathways

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses. Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses. interferon interferon regulatory factor positive regulatory domain IκB kinase double stranded RNA lipopolysaccharide PMA, phorbol 12-myristate 13-acetate virus-activated kinase human embryonic kidney calf intestine alkaline phosphatase tumor necrosis factor measle virus Newcastle disease virus vesicular stomatitis virus c-Jun N-terminal kinase polyacrylamide gel electrophoresis amino acid(s) phenylmethylsulfonyl fluoride hemagglutinating units whole cell extracts regulated on activation normal T cell expressed 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid Virus infection of mammalian cells triggers multiple signal transduction cascades involved in the activation of a diverse set of immunoregulatory genes and proteins that together create the antiviral state, an intracellular environment that antagonizes virus replication. The type I interferon (IFN)1family is essential to the development of the antiviral state and the IFN gene family represents one of the best characterized models of virus inducible gene activation (1Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3343) Google Scholar). Once produced, these secreted proteins induce gene expression in neighboring cells through cell surface cytokine receptors and the JAK-STAT signaling pathways. STAT1/2 heterodimers, in conjunction with interferon-stimulated gene factor 3γ bind to interferon-stimulated response elements found in numerous IFN-induced genes such as 2′-5′ oligoadenylate synthase and the double stranded RNA (dsRNA) activated kinase (PKR), resulting in the induction of proteins which impair viral gene expression and replication (1Stark G.R. Kerr I.M. Williams B.R.G. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3343) Google Scholar). Molecular regulation of IFN gene transcription is tightly regulated by extra- and intracellular signals induced at the site of infection. One of the best characterized models of such regulation is the virus-inducible promoter/enhancer of the IFN-β gene (2Kim T.K. Maniatis T. Mol. Cell. 1997; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 3Wathelet M.G. Lin C.H. Parakh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 4Falvo J.V. Parekh B.S. Lin C.H. Fraenkel E. Maniatis T. Mol. Cell. Biol. 2000; 20: 4814-4825Crossref PubMed Scopus (111) Google Scholar). This promoter includes an overlapping set of regulatory elements designated positive regulatory domains (PRDs) I to IV, which interact with several signal-responsive transcription factors including NF-κB (p50-p65), ATF-2/c-Jun heterodimers, and interferon regulatory factors (IRF) that bind to PRD II, PRD IV, and PRD I-III, respectively. Together with the chromatin-associated HMG I(Y) proteins, these transcription factors form a stereospecific transcriptional enhancer complex, termed the enhanceosome (2Kim T.K. Maniatis T. Mol. Cell. 1997; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 3Wathelet M.G. Lin C.H. Parakh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 4Falvo J.V. Parekh B.S. Lin C.H. Fraenkel E. Maniatis T. Mol. Cell. Biol. 2000; 20: 4814-4825Crossref PubMed Scopus (111) Google Scholar) that stimulates the high level, transient activation of IFN-β transcription. The pathways involved in NF-κB and ATF-2/c-Jun activation have been well characterized. Following viral infection, treatment with proinflamatory stimuli like tumor necrosis factor (TNF)-α, interleukin-1, (IL-1), or exposure to dsRNA, these transcription factors are activated through stimulation of distinct kinase cascades. In unstimulated cells, the NF-κB factors are retained in the cytoplasm in association with inhibitory subunits, IκBs; virus-induced phosphorylation at conserved N-terminal residues is accomplished by the IκB kinase (IKK) complex. Phosphorylation triggers a signal that induces ubiquitin-dependent degradation of IκB, and subsequent nuclear translocation of the NF-κB dimers (reviewed in Ref. 5DeLuca C. Kwon H.J. Pelletier N. Wainberg M.A. Hiscott J. Virology. 1998; 244: 27-38Crossref PubMed Scopus (32) Google Scholar). The rate-limiting step in this process is the activation of IKK which is composed of two catalytic subunits IKKα and β and one regulatory subunit IKKγ/NEMO. Numerous studies now suggest that the IKKβ catalytic subunit is required for IKK and NF-κB activation by TNF-α, interleukin-1, lipopolysaccharide (LPS), dsRNA, and viral infection (6Chu W.-M. Ostertag D. Li Z.-W. Chang L. Chen Y. Hu Y. Williams B. Perrault J. Karin M. Immunity. 1999; 11: 721-731Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 7Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (741) Google Scholar, 8Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Crossref PubMed Scopus (847) Google Scholar, 9Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (811) Google Scholar, 10Tanaka M. Fuentes M.E. Yamaguchi K. Durin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). Unlike NF-κB, the heterodimers ATF-2/c-Jun are expressed as nuclear proteins that are activated by phosphorylation of their activation domains by c-Jun amino-terminal kinases (JNKs) which are downstream of a well defined stress-activated kinase cascade (11Davis R.J. Biochem. Soc. Symp. 1999; 64: 1-12PubMed Google Scholar) The pathway(s) regulating IRF-3 phosphorylation and activation are also the focus of considerable investigation. IRF-3 belongs to the family of IRFs which include IRF-1 to IRF-7, interferon consensus sequence-binding protein (IRF-8), and interferon-stimulated gene factor 3γ (IRF-9) (12Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene (Amst .). 1999; 237: 1-14Crossref PubMed Scopus (458) Google Scholar). IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. IRF-3 demonstrates a unique response to viral infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to a conformational change, dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, stimulation of DNA binding and transcriptional activities (3Wathelet M.G. Lin C.H. Parakh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 13Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar, 14Navarro L. Mowen K. Rodems S. Weaver B. Reich N. Spector D. David M. Mol. Cell. Biol. 1998; 18: 3796-3802Crossref PubMed Scopus (133) Google Scholar, 15Sato M. Tanaka N. Hata N. Oda E. Taniguchi T. FEBS Lett. 1998; 425: 112-116Crossref PubMed Scopus (224) Google Scholar, 16Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (296) Google Scholar, 17Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar). Activated IRF-3 can in turn induce a specific subset of type 1 IFN genes in response to viral infection including IFN-β and human IFN -α1 (murine α4), as well as the CC-chemokine RANTES and the interleukin-15 (15Sato M. Tanaka N. Hata N. Oda E. Taniguchi T. FEBS Lett. 1998; 425: 112-116Crossref PubMed Scopus (224) Google Scholar, 18Juang 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: 9837-9842Crossref PubMed Scopus (236) Google Scholar, 19Lin R. Heylbroeck C. Genin P. Pitha P. Hiscott J. Mol. Cell. Biol. 1999; 19: 959-966Crossref PubMed Scopus (249) Google Scholar, 20Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar, 21Schafer S.L. Lin R. Moore P.A. Hiscott J. Pitha P.M. J. Biol. Chem. 1998; 273: 2714-2720Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 22Azimi N. Tagaya Y. Mariner J. Waldmann T.A. J. Virol. 2000; 74: 7338-7348Crossref PubMed Scopus (59) Google Scholar). As with NF-κB activation, the rate-limiting step in this process is C-terminal phosphorylation of IRF-3 by an uncharacterized virus activated kinase (VAK) activity. Previous studies have demonstrated that treatment with dsRNA was sufficient to trigger the nuclear accumulation of IRF-3 (17Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar) and the formation of an IRF-3 containing DNA binding complex (3Wathelet M.G. Lin C.H. Parakh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 16Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (296) Google Scholar). Recent studies also suggest that phosphorylation and activation of IRF-3 is not restricted to viral infection, since LPS, DNA-damaging and stress-inducing agents all stimulate nuclear accumulation of IRF-3, DNA binding activity, and transactivation (23Kim T. Kim T. Song Y.-H. Min I.M. Yim J. Kim T.K. J. Biol. Chem. 1999; 274: 30686-30689Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 24Navarro L. David M. J. Biol. Chem. 1999; 274: 35535-35538Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 25Kim T. Kim T.Y. Lee W.G. Yim J. Kim T.K. J. Biol. Chem. 2000; 275: 16910-16917Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Using a variety of pharmacological and molecular approaches, we now demonstrate that IRF-3 is uniquely activated via C-terminal virus-dependent phosphorylation. In addition to Sendai virus and Newcastle disease virus (NDV), measles virus (MeV) and vesicular stomatitis virus (VSV) are also identified as potent inducers of VAK activity. In contrast, exposure of cells to stress inducers, growth factors, DNA-damaging agents, and cytokines including doxorubicin and TNF-α, resulted in N-terminal phosphorylation but not C-terminal IRF-3 phosphorylation by a mitogen-activated protein kinase kinase kinase (MAPKKK)-related signaling pathway. N-terminal phosphorylation was not sufficient to promote nuclear translocation, transactivation, or degradation of IRF-3. The fact that numerous well characterized pharmacological inhibitors failed to block VAK activity suggests the involvement of a novel kinase in IRF-3 regulation by viruses. PDTC, sorbitol, LPS, and ribavirin were purchased from Sigma and dissolved in distilled water or phosphate-buffered saline. All other pharmacological inhibitors were from Calbiochem or Biomol and resuspended in dimethyl sulfoxide or ethanol. Recombinant macrophage inflammatory protein 1α, macrophage inflammatory protein 1β, and RANTES were from R&D Systems. Pertussis Toxin, epidermal growth factor, platelet-derived growth factor-BB, insulin, and thrombin were kind gifts from Dr. Sylvain Meloche. CMVBL-IRF-3wt, -IRF-3 5A, -IRF-3 5D; pFlag-IRF-3 1–240, the reporter plasmids containing two PRD II sites, pGL3-P2(2)tk-LUC and the IFNβ promoter, pGL3-IFN-β-LUC were described previously (13Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar, 19Lin R. Heylbroeck C. Genin P. Pitha P. Hiscott J. Mol. Cell. Biol. 1999; 19: 959-966Crossref PubMed Scopus (249) Google Scholar, 26Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (265) Google Scholar). The κB-mutated RANTES promoter, pGL3-κBm-RANTES-LUC, was prepared by cloning the BglII-SalI fragment (−397 to +5, filled in with the Klenow enzyme) from the κBm-RANTES-CAT reporter plasmid (19Lin R. Heylbroeck C. Genin P. Pitha P. Hiscott J. Mol. Cell. Biol. 1999; 19: 959-966Crossref PubMed Scopus (249) Google Scholar) into the NheI site (filled in with the Klenow enzyme) of the pGL3-basic vector. The expression constructs encoding different C-terminal IRF-3 truncations, pFlag-IRF-3-(1–198), -(1–186), -(1–174), and -(1–150) were generated by overlap polymerase chain reaction mutagenesis using Vent DNA polymerase. Constructs encoding for MAPKKKs, PCDNA3-MEKK1-HA, and pRK5-MYC-Cot were kind gifts from Drs. Richard Gaynor and Warner Greene, respectively. The rtTA-Jurkat, rtTA-Jurkat IRF-3wt, and rtTA-Jurkat IRF-3–5D were described previously (27Heylbroeck C. Balachandra S. Servant M.J. Deluca C. Barber G. Lin R. Hiscott J. J. Virol. 2000; 74: 3781-3792Crossref PubMed Scopus (149) Google Scholar). Human embryonic kidney (HEK) 293 cells and HeLa cells were grown in α-minimal essential medium and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum, glutamine, and antibiotics. The monocytic cell line U937 was cultured in RPMI supplemented with 5% fetal bovine serum. The human bronchial lung carcinoma cell line A549 was purchased from ATCC (CCL-185) and cultured in F12K supplemented with 10% fetal bovine serum. Extracts of primary monocytes uninfected or infected with NDV were a kind gift of Dr. Sandra Gessani, ISS, Rome. All transfections were carried out on subconfluent HEK 293 cells grown in 60-mm Petri dishes or 24-well plates (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 250–500 ng of expression plasmids (24-well plate) were introduced into target cells by calcium phosphate coprecipitation method. At 24 h post-transfection, cells were infected with Sendai virus for 12 h (80 hemagglutinating units (HAU)/ml) or treated with the different inducers for the indicated times. At 36 h, cells were collected, washed in ice-cold phosphate-buffered saline and assayed for reporter gene activities (Promega); whole cell extracts (WCE) were prepared in Nonidet P-40 lysis buffer (50 mmTris, pH 7.4, 150 mm NaCl, 30 mm NaF, 5 mm EDTA, 10% glycerol, 1.0 mmNa3VO4, 40 mm β-glycerophosphate, 10−4m phenylmethylsulfonyl fluoride (PMSF), 5 μg/ml of each leupeptin, pepstatin, and aprotinin, and 1% Nonidet P-40) and stored at −80 °C. To verify the state of phosphorylation of IRF-3 and to confirm the expression of the transgenes, WCE (30–60 μg) were subjected to electrophoresis on 7.5, 10, or 12% acrylamide gels. Proteins were electrophoretically transferred to Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech, Inc.) in 25 mm Tris, 192 mm glycine, and 20% methanol. The membranes were blocked in TBS containing 5% nonfat dry milk and 0.1% Tween 20 for 1 h at 25 °C before incubation for 1.5 h at 25 °C with anti-IRF-3 (a kind gift from Dr. Paula Pitha), anti-α-actin (Sigma), or anti-Flag M2 (Sigma) (1:500 to 1:1000) in blocking solution. After washing four times in TBS, 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 (Amersham Pharmacia Biotech, Inc). For co-precipitation studies, WCE (200–1000 μg) were incubated with 1 μg of anti-CBP antibody A-22 (Santa Cruz) cross-linked to 30 μl of protein A-Sepharose beads for 3 h at 4 °C (Amersham Pharmacia Biotech). The beads were washed five times with Nonidet P-40 lysis buffer, resuspended in denaturating sample buffer, and the eluted IRF-3 proteins associated with CBP were analyzed by immunoblotting. To examine subcellular localization of the IRF-3 protein, nuclear and cytoplasmic extracts were prepared from HeLa cells after treatment with different inducers for 8 h. The cells were washed in buffer A (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 0.5 mm PMSF) and were resuspended in buffer A containing 0.1% Nonidet P-40. The cells were then chilled on ice for 10 min before centrifugation at 10,000 × g. This procedure was performed twice to remove cytoplasmic contaminants in the nuclear extracts. After centrifugation, supernatants were kept as cytoplasmic extracts. The pellet were then resuspended in buffer B (20 mm HEPES, pH 7.9, 25% glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 0.5 mm PMSF, 5 μg/ml of each leupeptin, pepstatin, aprotinin, spermine, and spermidine). Samples were incubated on ice for 15 min before being centrifuged at 10,000 × g. Nuclear extract supernatants were diluted with buffer C (20 mm HEPES, pH 7.9, 20% glycerol, 0.2 mm EDTA, 50 mm KCl, 0.5 mmdithiothreitol, 0.5 mm PMSF). Equivalent amounts of nuclear and cytoplasmic extracts (20 μg) were subjected to SDS-PAGE in a 10% polyacrylamide gel. Proteins were electrophoretically transferred to Hybond-C nitrocellulose membranes which were probed with IRF-3 antiboby as described earlier. HEK 293 cells were left untransfected or transfected with expression plasmids encoding wild-type or mutated forms of IRF-3. At 36 h post-transfection, cells were stimulated and WCE were prepared. Endogenous IRF-3 (400 μg) or overexpressed IRF-3 (150 μg) proteins were immunoprecipitated with anti-IRF-3 antibody (Santa Cruz) or anti-Flag antibody (Sigma) cross-linked to 30 μl of protein G-Sepharose beads for 4 h at 4 °C. Precipitates were washed two times in Nonidet P-40 lysis buffer followed by two washes in phosphatase buffer (50 mm Tris, pH 9.0, 1 mm MgCl2, 0.1 mm ZnCl2, 1 mm spermidine, 0.5 mm PMSF, 5 μg/ml aprotinin, and 5 μg/ml leupeptin). The phosphatase treatment was started by resuspending the beads in a total volume of 40 μl of phosphatase assay buffer containing 5 units of calf intestine alkaline phosphatase (CIP; Amersham Pharmacia Biotech) in the absence or presence of a phosphatase inhibitor mixture containing (final concentration) 10 mm NaF, 1.5 mmNa2MoO4, 1 mm β-glycerophosphate, 0.4 mm Na3VO4, and 0.1 μg of okadaic acid per ml. The reactions were incubated at 37 °C for 2 h and stopped by washing the beads once with Nonidet P-40 lysis buffer and addition of 50 μl of 2 × denaturating sample buffer. The samples were resolved by SDS-gel electrophoresis and analyzed by immunoblotting using anti-IRF-3 and anti-Flag antibodies. C-terminal phosphorylation of IRF-3 following paramyxovirus infection is a prerequisite for its nuclear translocation, association with CBP/p300 co-activators, and transcriptional activation (13Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar, 16Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (296) Google Scholar, 17Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar, 28Kumar K.P. McBride K.M. Weaver B.K. Dingwall C. Reich N.C. Mol. Cell. Biol. 2000; 20: 4159-4168Crossref PubMed Scopus (172) Google Scholar). VAK activity is relatively easy to detect in extracts from virus-infected cells, since phosphorylated IRF-3 migrates slower in SDS-PAGE than nonphosphorylated IRF-3 (13Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar, 16Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (296) Google Scholar, 17Yoneyama M. Suhara W. Fukuhara Y. Fukada M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar), a phenomenon observed with many phosphoproteins. To characterize the different forms of phosphorylated IRF-3 in virus-infected cells, IRF-3 specific immunoblotting was used to reveal two forms of IRF-3 (designated forms I and II) in uninfected HEK 293, U937 and Jurkat cells (Fig.1 B, lanes 1, 3, and 7). These forms were also present in human epithelial HeLa cells, human bronchial epithelial A549 cells, primary human monocytes (see Figs. 3 and 6) and freshly isolated primary B cells (data not shown). Sendai virus infection resulted in the appearance of two slowly migrating forms of IRF-3 (forms III and IV) in HEK 293, U937, and IRF-3 expressing Jurkat cells (Fig. 1 B, lanes 2, 4, 5, 6, and 8). Forms III and IV represent IRF-3 phosphorylated at a cluster of serines near the C-terminal end of the protein (Ref. 13Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar and see Fig. 4 C). In addition, a net decrease in the amount of IRF-3 was observed between 4 and 12 h after virus infection of U937 cells, supporting the idea that C-terminal phosphorylated IRF-3 is subject to proteasome-mediated degradation (13Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar). Overexpression of the constitutively active form of IRF-3(5D) (13Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar, 19Lin R. Heylbroeck C. Genin P. Pitha P. Hiscott J. Mol. Cell. Biol. 1999; 19: 959-966Crossref PubMed Scopus (249) Google Scholar, 26Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (265) Google Scholar, 27Heylbroeck C. Balachandra S. Servant M.J. Deluca C. Barber G. Lin R. Hiscott J. J. Virol. 2000; 74: 3781-3792Crossref PubMed Scopus (149) Google Scholar) in Jurkat cells demonstrated that the phosphomimetic form migrated slower in SDS-PAGE than endogenous IRF-3 protein, at a position similar to form IV observed in cells infected with Sendai virus (Fig. 1 B, lane 9). This initial experiment, while largely confirming previous observations, nevertheless clearly demonstrates that multiple forms of IRF-3 phosphoprotein exist in unstimulated and virus-infected cells.Figure 3Activation of IRF-3 is restricted to virus infections. A, phosphorylation of IRF-3. Whole cell extracts (75 μg), prepared from HEK 293 cells, freshly isolated primary monocytes, and A549 cells uninfected (−) or infected with MeV (multiplicity of infection of 1.0), NDV (100 HAU/ml), and VSV (multiplicity of infection of 10) for different time points, were resolved by 7.5% SDS-PAGE and transferred to nitrocellulose. IRF-3 was analyzed by immunoblotting for the presence of phosphorylated IRF-3 forms (II to IV) with anti-IRF-3 antibody. B,transactivation of PRD I-III- and interferon-stimulated response elements containing promoters. HEK 293 cells were transiently transfected with reporter constructs containing IFN-β enhancer (IFN-β-LUC) and the κB-mutated RANTES promoter (κBm-RANTES-LUC). At 24 h post-transfection, cells were treated as indicated in the figure and LUC activity was analyzed 12 h later. Relative LUC activity was measured as fold activation as described under "Materials and Methods." Each value represents the mean ± S.E. of triplicate determinations. The data are representative of at least two different experiments with similar results. The concentration of viruses used was: Sendai virus (Sv), 80 HAU/ml; measles virus (MeV), multiplicity of infection of 1.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6N-terminal phosphorylation does not alter IRF-3 subcellular localization or function. A, IRF-3 phosphorylation. Whole cell extracts prepared from HEK 293 cells untreated (−) or treated with different agents or infected with Sendai virus (Sv; 80 HAU/ml) for 8 h (except for TNF-α-treated cells where a 30-min stimulation is shown) were prepared. Protein extracts (75 μg) were analyzed by immunoblotting for the presence of phosphorylated IRF-3 (II to IV) with anti-IRF-3 antibody. The concentration of agents used were: sorbitol (S), 0.3 m; NaCl (N), 0.25m; anisomycin (A), 1 μm; doxorubicin (D), 1 μg/ml; PMA (P), 100 ng/ml; TNF-α (T), 25 ng/ml. B, interaction between IRF-3 and CBP coactivator. HeLa, HEK 293, and U937 cells were treated as described in A. Whole cell extracts (500 μg) were immunoprecipitated with anti-CBP antibody A22, covalently linked to protein A-Sepharose beads. The immunoprecipitated proteins were resolved by SDS-gel electrophoresis on 7.5% acrylamide gel and transferred to nitrocellulose membrane. The membrane was probed with anti-IRF-3 antibody. As indicated, only forms III and IV were found to bind CBP. Lane 10, WCE (30 μg) prepared from uninfected HEK 293 cells were used to show the position of forms I and II. The concentration of agents used are described in A. LPS (L), 10 μg/ml. C, cytoplasmic to nuclear translocation of IRF-3. Hela cells were treated as indicated in A and B. Cytoplasmic and nuclear fractions were prepared as described under "Materials and Methods." Each isolated fraction was subjected to 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-IRF-3 antibody. Lower panels, membranes were stripped and reblotted with an anti-α-actin antibody.D, transactivation of interferon-stimulated response elements and PRD II containing promoters. HEK 293 cells were transfected with the κB-mutated RANTES promoter (κBm-RANTES-LUC) or P2(2)tk-LUC reporter plasmids and the MEKK1 (M, 250 ng) or ΔNIRF-3 (ΔN, 500 ng) expression plasmids when indicated. At 24 h post-transfection, cells were treated as indicated below the bar graph and LUC activity was analyzed 12 h later. Relative LUC activity was measured as fold activation. Each value represents the mean ± S.E. of triplicate determinations. The data are representative of at least three different experiments with similar results. The concentration of agents used were: Sendai virus (Sv), 80 HAU/ml; PMA (P), 100 ng/ml; LPS (L), 10 or 100 μg/ml; TNF-α (T), 10 or 100 ng/ml; doxorubicin (D), 1 μg/ml; anisomycin (A), 1 μm; and sorbitol (S), 0.20m.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Stress inducers, DNA-damaging agent, and NF-κB inducers stimulate N-terminal IRF-3 phosphorylation. A and B, phosphatase treatment. HEK 293 cells were left untreated (−) or treated for 8 h with the indicated agents (A) or co-transfected with Flag-IRF3wt and two MAPKKKs, MEKK1, and Cot (B). At 36 h post-transfection or following different treatments, whole cell extracts were prepared and subjected to immunoprecipitation using IRF-3 antibody covalently linked to protein A-Sepharose beads or Flag antibody immobilized onto protein-G Sepharose beads. The immunoprecipitated IRF-3 proteins were