De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling

NF-kappaB transcription factors mediate the effects of pro-inflammatory cytokines such as tumour necrosis factor-alpha and interleukin-1beta(1). Failure to downregulate NF-kappaB transcriptional activity results in chronic inflammation and cell death, as observed in A20-deficient mice(2). A20 is a potent inhibitor of NF-kappaB signalling, but its mechanism of action is unknown(2). Here we show that A20 downregulates NF-kappaB signalling through the cooperative activity of its two ubiquitin-editing domains. The amino-terminal domain of A20, which is a de-ubiquitinating (DUB) enzyme of the OTU (ovarian tumour) family(3), removes lysine-63 (K63)-linked ubiquitin chains from receptor interacting protein ( RIP), an essential mediator of the proximal TNF receptor 1 (TNFR1) signalling complex(4,5). The carboxy-terminal domain of A20, composed of seven C-2/C-2 zinc fingers(6), then functions as a ubiquitin ligase by polyubiquitinating RIP with K48-linked ubiquitin chains, thereby targeting RIP for proteasomal degradation. Here we define a novel ubiquitin ligase domain and identify two sequential mechanisms by which A20 downregulates NF-kappaB signalling. We also provide an example of a protein containing separate ubiquitin ligase and DUB domains, both of which participate in mediating a distinct regulatory effect.