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.
Pubmed ID: 20406818 RIS Download
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