Negative Role of RIG-I Serine 8 Phosphorylation in the Regulation of Interferon-β 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-β 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-β 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-β 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.

Conventional Protein Kinase C-α (PKC-α) and PKC-β Negatively Regulate RIG-I Antiviral Signal Transduction

“…The mutation of either S 8 or T 170 to phosphomimetic D or E (aspartic/glutamic acid), but not to A (alanine), strongly suppressed TRIM25-mediated RIG-I ubiquitination and antiviral signaling, indicating that RIG-I phosphorylation and TRIM25-mediated RIG-I ubiquitination functionally antagonize each other (7,22). To define the distinct roles of S 8 and T 170 phosphorylation in RIG-I regulation, we generated additional point mutants of the RIG-I N-terminal CARDs fused to mammalian glutathione S-transferase (GST-RIG-I 2CARD), as well as of full-length RIG-I: the S 8 E/T 170 E and S 8 A/T 170 A mutants mimicking constitutive phosphorylation or nonphosphorylation at both residues, and the S 8 A/T 170 E and S 8 E/T 170 A mutants at which one site is nonphosphorylated while the other mimics constitutive phosphorylation.…”

“…1B). Given that S 8 E or T 170 E mutation strongly suppressed RIG-I binding to TRIM25 (7,22), which apparently abolished RIG-I ubiquitination, we tested Flag-tagged RIG-I S 8 A/T 170 E, S 8 E/T 170 A, S 8 A/T 170 A, and S 8 E/T 170 E mutants for their TRIM25 binding abilities (Fig. 1C).…”

“…phorylation, suggesting that, upon viral RNA binding, conformational changes in RIG-I not only cause the dissociation of the RIG-I/PKC complex but also allow the recruitment of a yet-to-beidentified phosphatase for RIG-I S 8 and T 170 dephosphorylation. In fact, our previous studies demonstrated that RIG-I S 8 /T 170 phosphorylation is robustly enhanced by phosphatase inhibitor treatment (7,22), suggesting that S 8 and T 170 phosphorylations are tightly regulated by phosphatase-dependent dephosphorylation. The data presented here further indicate that the dephosphorylation of both S 8 and T 170 ultimately allows efficient TRIM25 binding, RIG-I CARD ubiquitination, and MAVSmediated downstream signaling.…”

“…Furthermore, a recent study showed that TRIM25 can induce RIG-I signaling in an in vitroreconstituted cell-free system (32). In addition to ubiquitination, we have recently shown that serine/threonine phosphorylation of the RIG-I CARDs represents an important mechanism to regulate RIG-I antiviral activity (7,22). The RIG-I CARDs undergo phosphorylation at S 8 and T 170 under normal conditions, which suppresses RIG-I downstream signaling by inhibiting RIG-I-TRIM25 binding and thereby TRIM25-mediated RIG-I ubiquitination.…”

“…For immunoblotting, the following primary antibodies were used: anti-Myc (1:2,000) (Covance), anti-V5 (1:5,000) (Invitrogen), anti-Flag (M2; 1:2,000) (Sigma), anti-hemagglutinin (HA) (1:2,000) (clone HA-7; Sigma), anti-glutathione S-transferase (anti-GST) (1:2,000) (Sigma), anti-ubiquitin (P4D1; 1:500) (Santa Cruz), antipolyubiquitin (Lys 63 linkage specific) (1:200) (Biomol), monoclonal anti-TRIM25 (1:2,000) (BD Biosciences), monoclonal anti-RIG-I (Alme-1; 1:1,000) (Alexis), anti-IRF3 (1:1,000) (Santa Cruz), anti-PKC-␣ (clone M4; 1:1,000) (Millipore), anti-PKC-␤ I (1:1,000) (Santa Cruz), anti-PKC-␤ II (1:1,000) (Santa Cruz), anti-PKC-␤ II (1:200) (Abcam), anti-phospho-S 657 -PKC-␣ (1:500) (Millipore), anti-phospho-T 500 PKC-␤ I&II (1:500) (Upstate), anti-PKC-(1:1,000) (Millipore), anti-PKC-(1:1,000) (Millipore), anti-␤-actin (Abcam), and anti-phosphothreonine (pThr; 1:500) (Cell Signaling Technology). The phospho-specific pS 8 -RIG-I and pT 170 -RIG-I antibodies were generated by immunizing rabbits with phosphopeptides and have been previously described (7,22). Protein kinase C inhibitors bisindolylmaleimide I (BIM I), Gö6976, and Ro-32-0432 were purchased from Calbiochem.…”