TRAF6-mediated degradation of DOK3 is required for production of IL-6 and TNFα in TLR9 signaling

•TRAF6 is able to catalyse Lys 48 polyubiquitylation of target protein.•CpG induces degradation of DOK3 via Lys 48 polyubiquitylation of lysine 27 of DOK3.•DOK3 degradation critically influences the TNFα and Il-6 production in TLR9 signaling.

Our previous study showed that the downstream of kinase 3 (DOK3) is degraded during macrophage stimulation with CpG. However, the underlying mechanism and role in Toll-like receptor 9 (TLR9) signaling remains elusive. In this study, we demonstrate that CpG treatment leads to ubiquitin-mediated degradation of DOK3 via interaction with an E3 ligase TNFR-associated factor 6 (TRAF6). We also identified the 27th amino acid (lysine) of DOK3 is responsible for Ly48 polyubiquitination of DOK3. Furthermore, reintroduction of DOK3 (K27R) into DOK3-deficient macrophages abolishes DOK3 degradation induced by CpG and suppresses the production of IL-6 and TNFα. More importantly, our study uncovers a novel role of an E3 ligase TRAF6, namely, TRAF6 is also able to catalyse Lys 48 polyubiquitylation of target protein except for Lys 63 polyubiquitylation.