title: 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.affiliation: Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA.
authors.forename: Estanislao
authors.initials: E
authors.lastname: Nistal-Villán
authors.forename: Michaela U
authors.initials: MU
authors.lastname: Gack
authors.forename: Gustavo
authors.initials: G
authors.lastname: Martínez-Delgado
authors.forename: Natalya P
authors.initials: NP
authors.lastname: Maharaj
authors.forename: Kyung-Soo
authors.initials: KS
authors.lastname: Inn
authors.forename: Heyi
authors.initials: H
authors.lastname: Yang
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.forename: Aneel K
authors.initials: AK
authors.lastname: Aggarwal
authors.forename: Jae U
authors.initials: JU
authors.lastname: Jung
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism; D016899:Interferon-beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018613:Microscopy, Confocal; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D050838:Interferon Regulatory Factor-3; C505371:MAVS protein, human; D011971:Receptors, Immunologic; D014157:Transcription Factors; D000070584:Tripartite Motif Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; D016899:Interferon-beta; C084523:TRIM25 protein, human; D044767:Ubiquitin-Protein Ligases; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases
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