Nuclear factor κB (NF-κB) transcription factors are retained in the cytosol by the inhibitor of NF-κB (IκB) protein. In response to proinflammatory cytokines, such as tumor necrosis factor–α (TNF-α), IκB is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated, and degraded by the proteasome, which enables NF-κB to translocate to the nucleus to regulate gene expression. Inhibition of NF-κB signaling is required to prevent inappropriate inflammatory responses. A20 is an anti-inflammatory protein, which has an N-terminal ovarian tumor (OTU) domain, containing deubiquitinase (DUB) activity, and C-terminal zinc-finger domains, which are implicated in the ubiquitination of target proteins. Inhibition of NF-κB signaling by A20 was thought to result from its ubiquitin-editing functions (see commentary by Srinivasula and Ashwell); however, Skaug et al. have shown a noncatalytic mechanism by which A20 inhibits IKK function. Experiments in transfected human embryonic kidney–293 cells showed that increased amounts of A20 blocked IKK activation in response to TNF-α but did not prevent formation of the TNF-α receptor (TNFR) complex, which includes polyubiquitin chains to which are bound NEMO, a regulatory subunit of the IKK complex, and TAK1, which phosphorylates IKK. In a cell-free system, both wild-type A20 and a mutant A20 lacking DUB activity (C103A) blocked IKK activation downstream of the scaffold protein and E3 ubiquitin ligase TRAF6. A mutant A20 lacking the seventh zinc-finger domain failed to block IKK activation and, unlike wild-type A20, was unable to bind to polyubiquitin chains. Coimmunoprecipitation studies showed that the binding of A20 to polyubiquitin chains was required for its physical interaction with NEMO. Binding of A20 to polyubiquitin chains prevented the autophosphorylation of the kinase TAK1, which was required for its phosphorylation of IKK and IKK activation. Although the authors could not rule out the ligase activity of A20 in NF-κB inhibition, they did not observe an A20-dependent effect on target protein stability in response to TNF-α. Together, these data suggest that A20 inhibits NF-κB signaling through a noncatalytic mechanism and that polyubiquitin chains affect signaling outcomes by determining protein-protein interactions.
B. Skaug, J. Chen, F. Du, J. He, A. Ma, Z. J. Chen, Direct, noncatalytic mechanism of IKK inhibition by A20. Mol. Cell 44, 559–571 (2011). [Online Journal]
S. M. Srinivasula, J. D. Ashwell, A20: More than one way to skin a cat. Mol. Cell 44, 511–512 (2011). [Online Journal]