IκB Kinase β Phosphorylates the K63 Deubiquitinase A20 To Cause Feedback Inhibition of the NF-κB Pathway

NF-κB signaling drives much of the initial acute inflammatory response to innate immune stimulation. Cells of the innate immune system express membrane-bound Toll-like receptors (TLRs) and intracellular pattern recognition receptors, such as the NOD proteins and RIG-I, all of which are responsible for recognizing specific components of bacterial, viral, or fungal organisms (21, 30, 31). Upon activation of these receptors, cells of the innate immune system activate divergent signal transduction pathways that ultimately converge to activate the IκB kinase (IKK) complex (11, 30). This complex consists, in its simplest form, of the kinases IKKα and IKKβ bound together by NEMO (IKKγ). Upon activation, this kinase complex phosphorylates IκBα, the inhibitor of NF-κB. This phosphorylation event leads to IκBα's ubiquitination and proteasomal degradation, allowing NF-κB transcription factors to stimulate the transcription of inflammatory genes (11). While these initial mechanisms of NF-κB activation have been well studied, the mechanisms by which NF-κB is downregulated are less well understood. The numerous signaling pathways causing NF-κB activation must be tightly regulated. Failure to mount a sufficient immune response results in susceptibility to infectious disease, and failure to properly downregulate NF-κB results in autoimmune or inflammatory disorders (11). Numerous studies have demonstrated that diverse inflammatory pathways are regulated in large part by coordinated phosphorylation and ubiquitination events (6, 16, 24, 28). The phosphorylation- and ubiquitin-mediated degradation of IκBα is the most well-known example of this coordination. In addition, TLR and RIP2/NOD2 activation results in the K63-linked ubiquitination of TRAF6 and NEMO (1, 2, 7, 30). Unlike K48-linked polyubiquitin chains, which target a protein for proteasomal degradation, the nondegradative K63-linked polyubiquitinated chains are thought to nucleate a signaling complex that activates the kinases TAK1 and IKKα/β (6, 16). In a similar way, K63-linked ubiquitination of TRAF2 and RIP1 is responsible for activating the IKK complex following activation of the tumor necrosis factor (TNF) receptor by TNF-α (6). Ubiquitination is a rapidly reversible modification, as deubiquitinating enzymes such as A20 (TNFAIP3) decrease the amplitude of ubiquitin-mediated NF-κB signaling (9, 29, 35). A20 contains a deubiquitinase domain at its N terminus and a series of seven zinc fingers at its C terminus (5, 9, 35). These C-terminal zinc fingers mediate K48-linked polyubiquitination. A20 deubiquitinates K63-polyubiquitinated proteins and then helps to conjugate K48-linked polyubiquitin chains such that the protein is targeted for degradation (5, 35). K63-linked polyubiquitination of many NF-κB regulators, such as RIP, TRAF6, TRAF2, and NEMO, positively regulates NF-κB activity (6, 16), and A20 has been shown to interact with and/or deubiquitinate the same proteins to ultimately inhibit the NF-κB signaling pathway (12, 29, 35, 37). The importance of A20 in the downregulation of inflammatory signaling pathways is further illustrated by A20−/− mice, which show uncontrolled whole-body inflammation and hypersensitivity to TNF-α and lipopolysaccharide (LPS) (5, 18). A20 is known to be transcriptionally upregulated following activation of NF-κB, but mechanisms regulating the intrinsic enzymatic activity of A20 have not been reported (17, 23). IKKβ is a serine/threonine kinase that is essential for the activation of the canonical NF-κB signaling pathway. Its activation of NF-κB is important for cell survival pathways, and a role in insulin signaling pathways was suggested following a report that IKKβ can phosphorylate IRS-1 (10, 25). IKKβ has also been shown to have a role in the development of a number of inflammation-related cancers (reviewed in references 3 and 15). While several IKKβ substrates have been recognized, both the importance of IKKβ and the breadth of signaling pathways it affects suggest that the complete repertoire of intracellular substrates has yet to be identified. Given this possibility, we developed and utilized an unbiased method to identify novel substrates of IKKβ. The optimal phosphorylation motif for IKKβ was identified using a positional scanning peptide library technique. A combination of several bioinformatic approaches was then used to identify the most likely candidate IKKβ substrates, and the deubiquitinating enzyme A20 was identified as a putative substrate of IKKβ. Serine 381 (S381) on A20 was identified as a target phosphorylation site for IKKβ and was verified as an in vitro and in vivo IKKβ phosphorylation site. We further found that phosphorylation of this site increases the ability of A20 to downregulate NF-κB in response to multiple stimuli. These findings show that phosphorylation of A20 by IKKβ modulates NF-κB signaling and represents a novel mechanism of regulation within innate immune signaling pathways.