Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA.
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, (382)GGASSLENTVDLHISNSHPLSLTSDQY(408) at 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 beta, IFNalpha1, 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 Ser(396) following Sendai virus infection, expression of viral nucleocapsid, and double-stranded RNA treatment. These results demonstrate that Ser(396) 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 Ser(396) phosphorylation in lipopolysaccharide-treated cells suggests that other major pathways may be involved in IRF-3 activation following Toll-like receptor 4 stimulation.
Servant
Marc J
MJ
Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research and the Departments of Microbiology & Immunology and Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada.
Grandvaux
Nathalie
N
tenOever
Benjamin R
BR
Duguay
Delphine
D
Lin
Rongtuan
R
Hiscott
John
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D002460:Cell Line; D018946:Chemokine CCL5 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D050838:Interferon Regulatory Factor-3; D016898:Interferon-alpha / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029082:Sendai virus / Q000235:genetics; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D020298:U937 Cells
D022762:CCAAT-Enhancer-Binding Proteins; D018946:Chemokine CCL5; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D016898:Interferon-alpha; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D012330:RNA, Double-Stranded; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014157:Transcription Factors; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D016899:Interferon-beta; D008156:Luciferases