NF-κB signaling: Flipping the Switch with Polyubiquitin Chains

Cells respond rapidly to changes in their internal and external environments, often by using post-translational protein modifications to transmit signals from the cell surface or internal sites to the nucleus. These signaling pathways allow cells to respond to growth signals, tolerate stresses or trigger programmed cell death. In general, signaling pathway activation is transient, with feedback mechanisms that reset the regulatory network. Protein phosphorylation, for example, is a well-characterized post-translational mechanism for transmitting transient cellular signals [1]. Phosphate groups, covalently attached to target proteins by substrate-specific protein kinases, can be removed by specific phosphatases, thereby terminating the signaling cascade. Recent studies on the NF-κB signaling network indicate that, in addition to transient protein phosphorylation, polyubiquitin chain attachment and removal work as on/off switches at several points along the signal transduction pathways that lead to activation of NF-κB family transcription factors. The carboxyl terminus of the ubiquitin polypeptide is enzymatically appended to lysines on target proteins as well as on other ubiquitin molecules to form polyubiquitin chains. Protein ubiquitination involves the sequential action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3) [2]. E3s bind both E2 and substrate, and facilitate the transfer of ubiquitin molecules from the E2 to the substrate. Ubiquitin is best known as a signal that targets proteins for degradation by the proteasome. In the ubiquitin–proteasome pathway, proteins destined for degradation are usually attached to a polyubiquitin chain in which ubiquitin molecules are linked to one another by amide bonds involving a specific ubiquitin lysine, Lys48 [2]. Proteasomes recognize the Lys48-linked chain, leading to degradation of the substrate and recycling of the ubiquitin molecules. Ubiquitin removal from proteins is catalyzed by a diverse group of enzymes collectively termed deubiquitinating enzymes. Interplay of Ubiquitination and Phosphorylation in NF-κB Regulation As noted, protein phosphorylation and ubiquitination are now both known to participate in the NF-κB signaling pathway (Figure 1) [3]. NF-κB molecules can concentrate in the nucleus and activate specific genes only when inhibitory controls on NF-κB are relieved. This occurs in response to DNA damage, during stimulation by proinflammatory cytokines such as tumor necrosis factor α (TNFα) or with an innate immunity response initiated when a ligand interacts with a Tolllike receptor (TLR) [4]. Mature NF-κB is normally kept inactive by tight binding to an inhibitory protein called IκB. Activation of NF-κB is triggered by site-specific phosphorylation of IκB, which allows IκB to be recognized by a ubiquitin-ligation complex. Polyubiquitinated IκB is degraded by the proteasome, releasing active NF-κB [3]. Regulation of NF-κB is thus directed toward controlling cellular levels of IκB. Remarkably, multiple studies now indicate that polyubiquitin chain formation promotes protein–protein associations that activate several steps in the signaling cascades leading to activation of the IκB kinase (IKK) complex [4]. These ubiquitin polymers are Lys63-linked chains, rather than the Lys48-linked chains that target proteins for proteasomal degradation. In the case of TNFα stimulation (Figure 1), activated TNF receptor recruits several adaptor proteins, including receptorinteracting protein (RIP) and TNF receptor-associated factor 2 (TRAF2). TNFα also induces polyubiquitination of RIP (and TRAF2), allowing binding of TAB2, a regulatory subunit in the TAK1 kinase complex, and this activates the TAK1 catalytic subunit [4] [5]. Active TAK1 then phosphorylates, and thereby activates, IKK (Figure 1).