Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production.
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.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Blotting, Western, Cell Line, Cell Line, Tumor, Cercopithecus aethiops, DEAD-box RNA Helicases, Green Fluorescent Proteins, Humans, Interferon Regulatory Factor-3, Interferon-beta, Microscopy, Confocal, Models, Molecular, Mutation, Phosphorylation, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Serine, Transcription Factors, Transcriptional Activation, Transfection, Ubiquitin-Protein Ligases, Vero Cells
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Blotting, Western, Cell Line, Cell Line, Tumor, Cercopithecus aethiops, DEAD-box RNA Helicases, Green Fluorescent Proteins, Humans, Interferon Regulatory Factor-3, Interferon-beta, Microscopy, Confocal, Models, Molecular, Mutation, Phosphorylation, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Serine, Transcription Factors, Transcriptional Activation, Transfection, Ubiquitin-Protein Ligases, Vero Cells
J. Biol. Chem.
Date: Jun. 25, 2010
PubMed ID: 20406818
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