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Upregulation of NF-κB proceeds through the inducible phosphorylation and resulting activation of the IκB kinase (IKK) complex. However 16 years after the IKK complex was first characterized, and in spite of the large volume of publications, it is still not completely clear how IKK is activated. Over the past decade, an increasing body of mostly in vitro data suggests a role for regulatory (K63-linked) polyubiquitination in IKK activation. The first part of this chapter critically examines the literature for the evidence of regulatory ubiquitination in different NF-κB pathways. Activation of NF-κB by regulatory ubiquitination has been proposed to be balanced by deubiquitinases which cleave the regulatory ubiquitin chains, thereby downregulating NF-κB activation. The second part of the chapter therefore takes a look at these deubiquitinases, focusing on a specific deubiquitinase called A20, which has been shown to be critical in downregulating NF-κB-mediated inflammation.

While in vitro biochemical data has suggested multiple targets for regulatory ubiquitination, a bona fide ubiquitination target has to be ultimately substantiated by genetic complementation experiments (such as generation of knock-in mouse, where the knock-in mutation selectively impairs the process of regulatory ubiquitination). The knock-in mouse that we have generated selectively abrogates the deubiquitinase function of A20 in vivo. These mice display normal NF-κB activation without showing any inflammatory phenotype. Our results clearly demonstrate that the well-characterized role of A20 in limiting inflammatory responses is due to effects other than deubiquitination of K63-ubiquitin chains. This is the concluding chapter which reiterates our finding that the “the deubiquitinase activity of A20 is dispensable for its role in NF-κB signaling” and provides a perspective on regulatory ubiquitination. As A20 clearly has an important role in many diseases, understanding the true molecular function of A20 remains an important and worthwhile goal for the future.

This chapter broadly introduces the innate and adaptive immune system, focusing on the critical role of the nuclear factor κB (NF-κB) family of transcription factors. While NF-κB was originally discovered 25 years ago as a regulator of κ light chain in B cells, research in the topic has diversified enormously and NF-κB has since been the focus of extensive research in multiple areas of biomedical science. Today, a search in PubMed using “NF-κB” alone results in more than 60,000 hits. The transcription factor controls the expression of multiple genes that are involved in regulating inflammation and shaping the immune response. Therefore in order to avoid autoimmunity, it is essential that the activation of NF-κB be tightly regulated so as to limit both the duration and magnitude of the immune response. This chapter explores broader themes that have emerged for NF-κB activation, and the possible role of regulatory ubiquitination in NF-κB activation.

Ubiquitination of multiple signaling adaptor molecules by K63-linked ubiquitin chains have been proposed to be a key regulatory mechanism in NF-κB activation. Deubiquitinase enzymes such as A20 have been suggested to limit the persistence of NF-κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 has garnered significant interest as mice lacking A20 die prematurely from multiorgan inflammation and cachexia, as a result of increased NF-κB signaling. Thus, it is evident that A20 is nonredundant in its ability to limit the persistence of NF-κB signaling. To understand the physiological relevance of A20-mediated deubiquitination, we generated knock-in mice that lack the deubiquitinating function of A20 (A20-OTU mice). We hypothesized that these mice would have an inflammatory phenotype because of increased, persistent NF-κB signaling. However our results show that A20 OUT mice display normal NF-κB activation and no inflammatory phenotype, thereby demonstrating that the deubiquitinase activity of A20 is dispensable for normal NF-κB signaling.