The E3 ligase β-TRCP1 earmarks OTUD3 for destruction to fine-tune cGAS activation.

Activation of cytosolic DNA sensing through cyclic GMP-AMP synthase (cGAS) induces the production of type I interferons and proinflammatory cytokines, which are essential for antiviral and antibacterial responses, inflammation, and immune modulation. While hyperactivation of cGAS leads to autoimmune diseases, its inactivation contributes to immune evasion and resistance to immunotherapies. Therefore, cGAS activity must be tightly regulated. One mechanism involves the deubiquitination and stabilization of cGAS by the deubiquitinase OTUD3; however, the upstream signals and pathophysiological cues governing OTUD3 regulation remain poorly understood. Here, we report that the E3 ubiquitin ligase β-TRCP1 targets OTUD3 for ubiquitination and proteasomal degradation. This recognition is dependent on RSK3-mediated phosphorylation of a conserved "ESG" motif in OTUD3, which serves as a phospho-degron for β-TRCP1 binding. Intriguingly, cytosolic DNA challenge inactivates the β-TRCP1/RSK3 pathway, resulting in OTUD3 stabilization and enhanced cGAS activation, representing a fine-tuning mechanism of innate immune signaling. Notably, this DNA-induced inactivation of RSK3 is independent of canonical Ras/MEK/extracellular signal-regulated kinase signaling and DNA damage-responsive kinases, but dependent on mTORC2 signaling. Collectively, our studies identify β-TRCP1/RSK3 as a previously unrecognized upstream signaling axis that regulates OTUD3 protein stability in response to DNA stress, thereby modulating cGAS-driven innate immune responses. This pathway presents a potential therapeutic target for modulating innate immunity in autoimmune diseases and cancer.