Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation

J Biol Chem. 2004 Mar 12;279(11):9698-702. doi: 10.1074/jbc.M310616200. Epub 2003 Dec 31.

Abstract

Interferon regulatory factor (IRF)-3 is a critical transcription factor regulating innate immune responses against viral and bacterial infections. Signals activated by various pathogens are integrated by IRF-3 kinase, resulting in the specific phosphorylation of IRF-3 in the cytoplasm. This phosphorylation induces dimerization and association with the coactivators CREB-binding protein/p300, and the resultant complex activates the target genes in the nucleus. However, the phosphorylation sites that determine the active/inactive status of IRF-3 have been a source of controversy. In this study, we generated an antibody that specifically detects the phosphorylation of Ser-386 and used it as a probe. We found: 1) viral infection specifically induces phosphorylation of the Ser-386; 2) recently identified IRF-3 kinases (IKK-i/epsilon and TBK-1) phosphorylate Ser-386 and induce its dimerization; 3) phosphorylation of Ser-386 is exclusively observed with the dimer; 4) mutation at Ser-386 abolishes the dimerization potential; 5) a constitutively active 5D mutant designed to mimic phosphorylation of Ser/Thr residues other than Ser-385 and -386 is secondarily phosphorylated at Ser-386, presumably by an irrelevant kinase. These results strongly suggest that Ser-386 is the target of the IRF-3 kinase and critical determinant for the activation of IRF-3.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carrier Proteins / metabolism
  • Cell Line
  • Crystallography, X-Ray
  • Cytoplasm / metabolism
  • DNA-Binding Proteins / chemistry*
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Genetic Vectors
  • Humans
  • I-kappa B Kinase
  • Immunoblotting
  • Interferon Regulatory Factor-3
  • Mice
  • Molecular Sequence Data
  • Mutagenesis
  • Mutation
  • Phosphorylation
  • Plasmids / metabolism
  • Protein Binding
  • Protein Serine-Threonine Kinases / chemistry
  • Protein Serine-Threonine Kinases / metabolism
  • Sequence Homology, Amino Acid
  • Serine / chemistry*
  • Transcription Factors / chemistry*
  • Transfection

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • IRF3 protein, human
  • Interferon Regulatory Factor-3
  • Irf3 protein, mouse
  • Transcription Factors
  • citrate-binding transport protein
  • Serine
  • Tbk1 protein, mouse
  • Protein Serine-Threonine Kinases
  • TBK1 protein, human
  • I-kappa B Kinase