Ubiquitination of STING on Lysine 224 Facilitates Cytosolic DNA Activated Host Defense

Conserved structures among microbial species, known as pathogen associated molecular patterns (PAMPs), are recognized by host pattern recognition receptors (PRRs), which then initiate a serial of innate immune signaling transductions to protect the host from pathogen invading. A recently identified endoplasmic reticulum-associated protein STING (stimulator of interferon genes) has been shown to be an essential component of the innate immunity, especially with regard to the cytosolic DNA sensing pathway. Cytosolic DNA species derived from invading microbes or leaked from the nuclear or mitochondrial compartments of the cell can potently trigger the induction of host defense genes by activating STING-dependent pathway. Posttranslational modifications of STING, such as phosphorylation, palmitoylation and ubiquitination, have been shown to play critical roles in regulating STING activity. However, previous studies about STING ubiquitination frequently appears conflicting and thus need to be further clarified. By using a mass spectrometry and mutagenesis approach, we identified lysine 224 as the most critical ubiquitination site of STING. We demonstrated that after association with cyclic dinucleotides (CDN’s), STING-dependent trafficking which is required to deliver Tank binding kinase 1 (TBK1) to interferon regulatory transcription factors (IRF) such as IRF3, was reliant upon the K63-linked polyubiquitination on K224. The inhibition of K224 ubiquitination specifically prevented IRF3 but not NF-?B activation, additionally indicating that STING trafficking is not required to activate the latter signaling pathway. siRNA-based screening further identified mitochondrial E3 ubiquitin protein ligase 1 (MUL1) as an E3 ligase able to catalyze the K63-linked polyubiquitination of STING on K224. Suppression of MUL1 expression in murine cells inhibited DNA-induced STING trafficking, which lead to impaired IRF3 activation and type I IFN production. Collectively, these findings demonstrate the critical role of K224 ubiquitination in STING function and provide molecular insight into the mechanisms governing host defense responses