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.
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Authors | Marc J Servant, Nathalie Grandvaux, Benjamin R tenOever, Delphine Duguay, Rongtuan Lin, John Hiscott |
Journal | The Journal of biological chemistry
(J Biol Chem)
Vol. 278
Issue 11
Pg. 9441-7
(Mar 14 2003)
ISSN: 0021-9258 [Print] United States |
PMID | 12524442
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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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
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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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