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

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.

• Authors: Estanislao Nistal-Villán, Michaela U Gack, Gustavo Martínez-Delgado, Natalya P Maharaj, Kyung-Soo Inn, Heyi Yang, Rong Wang, Aneel K Aggarwal, Jae U Jung, Adolfo García-Sastre
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 285 Issue 26 Pg. 20252-61 (Jun 25 2010) ISSN: 1083-351X [Electronic] United States
• PMID: 20406818 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

• Adaptor Proteins, Signal Transducing (genetics, 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, genetics, metabolism)
• Green Fluorescent Proteins (genetics, metabolism)
• Humans
• Interferon Regulatory Factor-3 (genetics, metabolism)
• Interferon-beta (genetics, metabolism, pharmacology)
• Microscopy, Confocal
• Models, Molecular
• Mutation
• Phosphorylation (drug effects)
• Promoter Regions, Genetic (genetics)
• Protein Binding
• Protein Structure, Tertiary
• Receptors, Immunologic
• Serine (genetics, metabolism)
• Transcription Factors (genetics, metabolism)
• Transcriptional Activation
• Transfection
• Tripartite Motif Proteins
• Ubiquitin-Protein Ligases (genetics, metabolism)
• Vero Cells