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.