Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation

Mol Cell Biol. 1998 May;18(5):2986-96. doi: 10.1128/mcb.18.5.2986.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biological Transport
  • Cell Compartmentation
  • Cell Nucleus / metabolism
  • Cysteine Endopeptidases / metabolism
  • Cytoplasm / metabolism
  • DNA Mutational Analysis
  • DNA-Binding Proteins / metabolism*
  • Histone Acetyltransferases
  • Humans
  • Interferon Regulatory Factor-3
  • Mice
  • Molecular Sequence Data
  • Multienzyme Complexes / metabolism
  • Nuclear Receptor Coactivator 3
  • Peptide Mapping
  • Phosphorylation
  • Proteasome Endopeptidase Complex
  • Protein Binding
  • Protein Processing, Post-Translational*
  • Respirovirus / growth & development*
  • Trans-Activators / metabolism
  • Transcription Factors / metabolism*
  • Transcriptional Activation

Substances

  • DNA-Binding Proteins
  • IRF3 protein, human
  • Interferon Regulatory Factor-3
  • Irf3 protein, mouse
  • Multienzyme Complexes
  • Trans-Activators
  • Transcription Factors
  • Histone Acetyltransferases
  • NCOA3 protein, human
  • Ncoa3 protein, mouse
  • Nuclear Receptor Coactivator 3
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex