Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production

J Biol Chem. 2010 Jun 25;285(26):20252-61. doi: 10.1074/jbc.M109.089912. Epub 2010 Apr 20.

Abstract

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • Blotting, Western
  • Cell Line
  • Cell Line, Tumor
  • Chlorocebus aethiops
  • DEAD Box Protein 58
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Interferon Regulatory Factor-3 / genetics
  • Interferon Regulatory Factor-3 / metabolism
  • Interferon-beta / genetics
  • Interferon-beta / metabolism*
  • Interferon-beta / pharmacology
  • Microscopy, Confocal
  • Models, Molecular
  • Mutation
  • Phosphorylation / drug effects
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Protein Structure, Tertiary
  • Serine / genetics
  • Serine / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcriptional Activation
  • Transfection
  • Tripartite Motif Proteins
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Vero Cells

Substances

  • Adaptor Proteins, Signal Transducing
  • Interferon Regulatory Factor-3
  • Transcription Factors
  • Tripartite Motif Proteins
  • VISA protein, human
  • Green Fluorescent Proteins
  • Serine
  • Interferon-beta
  • TRIM25 protein, human
  • Ubiquitin-Protein Ligases
  • DDX58 protein, human
  • DEAD Box Protein 58
  • DEAD-box RNA Helicases