The basic framework of NF-κB signaling was established within the first decade of the groundbreaking discovery of NF-κB.1 According to this framework, NF-κB is a transcription factor sequestered in the cytoplasm of unstimulated cells through its association with an inhibitory protein of the IκB family. Stimulation of cells with a variety of agents, including proinflammatory cytokines such as TNFα and IL-1β, leads to the activation of a protein kinase that phosphorylates IκB at specific sites. This signal-induced site-specific phosphorylation targets IκB for polyubiquitination and subsequent degradation by the proteasome, thereby allowing NF-κB to enter the nucleus to turn on target genes. Building on this framework, subsequent research in the second decade was centered on understanding the regulation and function of the IκB kinase, which apparently integrates signals from diverse pathways. In 1996, a large (∼700 kDa) IκB kinase complex was partially purified; surprisingly, this kinase could be activated by polyubiquitination through a mechanism independent of proteasomal degradation.2 This was a rather provocative finding as there was no precedence of proteasome-independent function of ubiquitin and the results were obtained entirely from in vitro biochemical experiments. The idea that ubiquitin can activate IκB kinase, which was later called IKK when the subunits of the kinase complex were molecularly cloned, was revived when some components of the NF-κB pathway were found to be connected to ubiquitination.3, 4 In particular, TRAF6, an essential signaling protein of the NF-κB pathway, was found to be a ubiquitin ligase (E3) that catalyzes the synthesis of a unique polyubiquitin chain required for IKK activation.5 Subsequent studies showed that TRAF6-catalyzed polyubiquitination leads to activation of the TAK1 kinase complex, which in turn phosphorylates and activates IKK.6 However, these studies were also carried out largely in vitro. Although the role of TAK1 in the NF-κB pathway was later supported by multiple lines of evidence, including those derived from RNA interference and Drosophila genetic experiments, the final in vivo proof in higher organisms was still lacking – until now. Through genetic ablation of TAK1 in mice, Sato et al.7 have now provided convincing in vivo evidence that TAK1 is essential for IKK activation in multiple signaling pathways. In this commentary, we will review the current knowledge of the connection between ubiquitin, TAK1 and IKK, and discuss some important questions that remain to be resolved.

The role of ubiquitin and TAK1 in IKK activation was discovered in the course of studying the interleukin-1 (IL-1) signaling pathway. In this pathway, the binding of IL-1β to its receptor (IL-1R) leads to the recruitment of several proteins including MyD88, IRAK1, IRAK4 and TRAF6 to the receptor complex. IRAK4 is a protein kinase that phosphorylates IRAK1, which is also a kinase but its catalytic activity is not required for signaling. Following phosphorylation, IRAK1 and TRAF6 are released to the cytoplasm to activate downstream kinases including IKK, JNK and p38. The IKK complex consists of two catalytic subunits IKKα and IKKβ, and an essential regulatory subunit NEMO/IKKγ.8 Recent genetic and biochemical studies have shown that IKKβ and NEMO regulate the phosphorylation and subsequent degradation of IκB proteins in response to stimulation by a large variety of NF-κB stimuli, including TNFα and IL-1β. IKKα, on the other hand, is responsible for the phosphorylation of the NF-κB precursor p100 in response to stimulation of a subset of TNF receptor (TNFR) superfamily in certain cells such as B cells. Phosphorylation of p100 leads to its ubiquitination and subsequent processing to the mature subunit p52 by the proteasome.

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