The Mechanism Of Phosphatase Cdc25A Inhibiting RIG-â…  Mediated Immune Response

The RIG-Ⅰ-like receptor (RLR) family is composed of RIG-Ⅰ, MDA5 and LGP2. RLRs are composed of two N-terminal caspase recruitment domains (CARDs), a central DEAD box helicase/ATPase domain, and a C-terminal regulatory domain. They are localized in the cytoplasm and recognize the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses. RIG-Ⅰ and MDA5 recognize different RNA viruses by detecting short dsRNAs with 5’-triphosphate ends and long dsRNAs, respectively. LGP2 functions as a regulator in RIG-Ⅰ-mediated and MDA5-mediated virus recognition.RIG-Ⅰ mediated innate immune response is a critical component of host anti-virus defense system. It recognizes relatively short dsRNA (up to 1 kb), and the presence of a 5’-triphosphate end greatly enhances its type I IFN-inducing activity. RIG-Ⅰ is comprised of two N-terminal caspase-recruitment domains (CARDs) followed by a DExD/H box RNA domain. The C-terminal portion of RIG-Ⅰ, designated the repressor domain (RD), was shown to inhibit the triggering of RIG-Ⅰ signaling in the steady state and the N-terminal CARDs are responsible for activating downstream signaling pathways that mediate dsRNA induced type IIFN production. After recognizing viral RNA, RIG-Ⅰ conformation changes and then the CARDs motif is exposed. Activated RIG-Ⅰ then bind to and activate VISA, which localized on the mitochondrial outer membrane. The activated VISA recruits downstream signaling proteins such as TRAF3/6, TRADD, TANK and so on, and a signalsome is assembled on the membrane of mitochondrial. This complex phosphorylates TBK1 and IKK, and then finally activates NF-κB and IRF3/7. Activated NF-κB and IRF3/7 shuttle into the nucleus and promote the transcription of IFN-p.Because of playing a crucial role in the innate immunity, the host cells have to strongly regulate RIG-Ⅰ signaling pathway against the occurrence of auto-immune injury. Recently, the RIG-Ⅰ regulation study mainly focus on two fields:(1) the regulation comes from host cell which consists of positive and negative regulation. The RIG-Ⅰ conformation is known to be modulated by ubiquitination. TRIM25 and Riplet (also known as RNF135) act as E3 ubiquitin ligases that mediate the K63-linked polyubiquitination of RIG-Ⅰ. This modification is required for the activation of RLR signaling. On the other hand, K48-type polyubiquitination of RIG-Ⅰ by RNF125 leads to RIG-Ⅰ degradation by the proteasome and inhibition of RIG-Ⅰ signaling. Furthermore, there are other RIG-Ⅰ signaling negative regulators such as DUB A, CYLD, SIKE, SHP1 and so on. (2) Virus proteins regulate RIG-Ⅰ signaling. Such as NS1 of influenza A virus which binds with RIG-Ⅰ specifically, NS3/4A of HCV can cut VISA at Cys508 and release VISA into cytoplasm from mitochondria.It has been reported that phosphorylation plays an important role in the RIG-Ⅰ regulation system. For example, CKlyl can phosphorylate p65 at Ser536 and induce p65 ubiquitination and degradation and then suppress RIG-Ⅰ signaling; IKK complex phosphorylates IkB and induces the ubiquitination and degradation of IκB which causes the activation of NF-κB and promotes the IFNβ transcription; TBK1 can phosphorylate IRF3/7 which induces the formation of IRF3/7 heterodimer or homodimer and upregulates IFNP transcription.However, the function of dephosphorylation on RIG-Ⅰ pathway is still not well understand. In this study, by taking the approach of luciferase reporter assay, we found that phosphatase Cdc25 A can repress the activation of IFNβ promoter induced by SeV or IAV infection. In addition, we found that Cdc25A can reduce IFNβ promoter activation induced by overexpression of RIG-Ⅰ, VISA and TBK1. However, Cdc25A did not affect the activation of ISRE promoter induced by IRF3 or NF-κB promoter induced by p65. These data implied that Cdc25A may negatively regulate RIG-Ⅰ pathway at the level of TBK1. By using the co-immunoprecipitation analysis, we found that Cdc25A can bind with RIG-Ⅰ, VISA and TRAF6. More importantly, Cdc25A interfered with the interaction between VISA and TBK1/MITA. Meanwhile, we found that the interaction between Cdc25A and RIG-Ⅰ-N is much stronger than that between Cdc25A and full length RIG-Ⅰ. These results implied that Cad25A may bind with CARD domain of RIG-Ⅰ and VISA and block their interaction and eventually suppress VISA signalsome formation. Furthermore, we found that Cdc25A can inhibit the phosphorylation of TBK1 and IRF3 induced by Sendai virus. This data suggested that the phosphatase activity of Cdc25A is related to the RIG-Ⅰ pathway regulation.In conclusion, we propose the possible mechanism of Cdc25A negatively regulating RIG-Ⅰ signaling:Cdc25A binds with CARD domain of RIG-Ⅰ and VISA and then disturbs VISA signaling complex formation which eventually repress TBK1 phosphorylation and negatively regulate RIG-Ⅰ pathway. Meanwhile, as a phosphatase, Cdc25A can inhibit TBK1 phosphorylation directly and down-regulate RIG-Ⅰ pathway.