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Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA.

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, (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.
AuthorsMarc J Servant, Nathalie Grandvaux, Benjamin R tenOever, Delphine Duguay, Rongtuan Lin, John Hiscott
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 278 Issue 11 Pg. 9441-7 (Mar 14 2003) ISSN: 0021-9258 [Print] United States
PMID12524442 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • CCAAT-Enhancer-Binding Proteins
  • Chemokine CCL5
  • DNA-Binding Proteins
  • Drosophila Proteins
  • IRF3 protein, human
  • Interferon Regulatory Factor-3
  • Interferon-alpha
  • Lipopolysaccharides
  • Membrane Glycoproteins
  • RNA, Double-Stranded
  • Receptors, Cell Surface
  • TLR4 protein, human
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Transcription Factors
  • Threonine
  • Aspartic Acid
  • Serine
  • Interferon-beta
  • Luciferases
Topics
  • Amino Acid Sequence
  • Aspartic Acid (chemistry)
  • Binding Sites
  • CCAAT-Enhancer-Binding Proteins (metabolism)
  • Cell Line
  • Chemokine CCL5 (metabolism)
  • DNA-Binding Proteins (chemistry, metabolism)
  • Drosophila Proteins
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Immunoblotting
  • Interferon Regulatory Factor-3
  • Interferon-alpha (metabolism)
  • Interferon-beta (metabolism)
  • Lipopolysaccharides (metabolism)
  • Luciferases (metabolism)
  • Membrane Glycoproteins (metabolism)
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Phosphorylation
  • Plasmids (metabolism)
  • Point Mutation
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Double-Stranded (chemistry, metabolism)
  • Receptors, Cell Surface (metabolism)
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sendai virus (genetics)
  • Serine (chemistry)
  • Threonine (chemistry)
  • Time Factors
  • Toll-Like Receptor 4
  • Toll-Like Receptors
  • Transcription Factors (chemistry, metabolism)
  • Transcriptional Activation
  • Transfection
  • Tumor Cells, Cultured
  • U937 Cells

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