title: Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation.
abstract: The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-alpha/beta) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. 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. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.
authors.affiliation: Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec, Canada. mdli@musica.mcgill.ca
authors.forename: R
authors.initials: R
authors.lastname: Lin
authors.forename: C
authors.initials: C
authors.lastname: Heylbroeck
authors.forename: P M
authors.initials: PM
authors.lastname: Pitha
authors.forename: J
authors.initials: J
authors.lastname: Hiscott
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051548:Histone Acetyltransferases; D006801:Humans; D050838:Interferon Regulatory Factor-3; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D056921:Nuclear Receptor Coactivator 3; D010449:Peptide Mapping; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D018108:Respirovirus / Q000254:growth & development*; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; C494233:Irf3 protein, mouse; D009097:Multienzyme Complexes; D015534:Trans-Activators; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; C538867:Ncoa3 protein, mouse; D056921:Nuclear Receptor Coactivator 3; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex