Positive regulatory role of c-Src-mediated TRIM25 tyrosine phosphorylation on RIG-I ubiquitination and RIG-I-mediated antiviral signaling pathway.

Retinoic acid-inducible gene I (RIG-I) detects viral RNAs and induces antiviral responses. During viral RNA recognition by RIG-I, tripartite motif protein 25 (TRIM25) plays a critical regulatory role by inducing K63-linked RIG-I polyubiquitination. Previous proteomics analysis revealed several phosphorylation sites on TRIM25, including tyrosine 278 (Y278), yet the roles of these modifications remain elusive. Here, we demonstrated that TRIM25 interacted with c-Src and underwent tyrosine phosphorylation by c-Src kinase upon viral infection and the phosphorylation is required for the complete activation of RIG-I signaling. Analysis using a c-Src inhibitor and TRIM25 mutant, in which tyrosine 278 is substituted by phenylalanine (Y278F), suggested that the phosphorylation positively regulates K63-linked polyubiquitination of RIG-I and subsequent antiviral signaling. The TRIM25 Y278F mutant displayed decreased E3-ubiquitin ligase activity in vitro, suggesting that this phosphorylation event affects the E3-ligase activity of TRIM25. Thus, we provide a molecular mechanism of c-Src-mediated positive regulation of RIG-I signaling.