Ubiquitin signalling in the NF-kappaB pathway

Abstract

The transcription factor NF-kappaB (nuclear factor kappa enhancer binding protein) controls many processes, including immunity, inflammation and apoptosis. Ubiquitination regulates at least three steps in the NF-kappaB pathway: degradation of IkappaB (inhibitor of NF-kappaB), processing of NF-kappaB precursors, and activation of the IkappaB kinase (IKK). Recent studies have revealed several enzymes involved in the ubiquitination and deubiquitination of signalling proteins that mediate IKK activation through a degradation-independent mechanism.

Figure 1. The NF-κB signaling pathways

In the canonical pathway (left), stimulation of TNF receptors (TNFR), IL-1 receptors (IL-1R) or Toll-like receptors (TLR) with their cognate ligands activates TRAF proteins and subsequently the kinase TAK1, which phosphorylates and activates IKKβ. IKKβ then phosphorylates IκB resulting in its ubiquitination by the SCF-βTrCP ubiquitin ligase complex and subsequent degradation by the proteasome. The NF-κB dimer consisting of p50 and Rel-A can then enter the nucleus to regulate the expression of targets genes involved in inflammation, immunity and cell survival. In the non-canonical pathway, a subset of receptors belonging to the TNFR superfamily, such as the B cell receptor for BAFF (BAFF-R), activates the kinase NIK through an unknown mechanism. NIK then phosphorylates IKKα, which in turn phosphorylates the NF-κB precursor p100. p100 is subsequently polyubiquitinated and then processed to the mature subunit p52 by the proteasome. p52 and its binding partner Rel-B translocate to the nucleus to turn on genes that are important for the maturation of B cells.

Figure 2. A model for the processing of p100 by the proteasome

Polyubiquitinated p100 is recruited to the proteasome, allowing the flexible glycine rich region (GRR) to insert into the proteasome as a loop. Degradation of the polypeptide proceeds in both the N- and C-terminal directions. The degradation of the C-terminus proceeds to the end, whereas the degradation along the N-terminus stops when the tightly folded Rel-homology domain (RHD) cannot be unfolded to allow insertion into the proteasome. The resulting p52 subunit and its dimeric partner Rel-B is then released from the proteasome.

Figure 3. A model for IKK activation by TRAF6 ubiquitination

Stimulation of IL-1R or TLR leads to the recruitment of MyD88, IRAK and TRAF6 to the receptor complex. This may facilitate TRAF6 oligomerization and activate its ubiquitin ligase activity, leading to K63-linked polyubiquitination of targets including Nemo (not shown) and TRAF6 itself. This polyubiquitination reaction requires E1 and Ubc13/Uev1A, and can be reversed by deubiquitination enzymes such as CYLD and A20. Ubiquitinated TRAF6 is recruited to the TAK1/TAB2 complex through binding of K63-linked polyubiquitin chains to the NZF domain of TAB2 as well as by the direct interaction between TRAF6 and TAB2. This binding may facilitate the dimerization or oligomerization of the TAK1/TAB2 complex, promoting its autophosphorylation and TAK1 activation. TAK1 then phosphorylates IKKβ, resulting in its activation.

Figure 4. A central role for ubiquitin in multiple signaling pathways

Polyubiquitination by TRAF2 and TRAF6 not only mediates signaling by TNFR, IL1R and TLR (see Fig. 3) in the innate immunity pathways, but also plays an important role in adaptive immune responses by activating IKK in response to stimulation of TCR. In T cells, BCL10 and MALT1 activate the ligase activity of TRAF6 (and possibly TRAF2), to catalyze the K63-linked polyubiquitination of NEMO and TRAF6 itself. These ubiquitination events mediate the activation of TAK1 and IKK. NOD2 and RIP2, which trigger innate immune responses against intracellular bacteria, also facilitate the K63-linked polyubiquitination of NEMO. DNA damage leads to the sumoylation and subsequent ubiquitination of NEMO in the nucleus. The ubiquitinated NEMO then exits the nucleus and associates with the other subunits of the cytosolic IKK complex, resulting in IKK activation. Some receptors of the TLR family, including TLR7, 8 and 9, are localized in the endosomal membrane. Stimulation of TLR7/8 and TLR9 by viral RNA and CpG DNA, respectively, leads to the activation of both NF-κB and IRF7, which coordinately regulate interferon production. IRF7 binds to MyD88, which is associated with the endosomal TLRs as well as IRAK and TRAF6. TRAF6 polyubiquitination is required for the activation of IRF7, presumably by promoting the phosphorylation and subsequent nuclear translocation of IRF7.

Table 1. Protein and enzymes involved in ubiquitin-mediated activation of IKK

Protein name abbreviations: c-IAP1: cellular inhibitor of apoptosis 1; RIP: receptor interacting protein; NEMO: NF-κB essential modulator; MALT1: mucosal associated lymphoid tissue 1; TIFA: TRAF-interacting protein with a forkhead-associated domain. CYLD: cylindromatosis suppressor; Protein domain abbreviations: Ubc: ubiquitin conjugating enzyme; RING: really interesting new gene; CUE: cue1 homologous; NZF: novel zinc finger; UBA: Ub association; DD: death domain; HLH: helix-loop-helix; CARD: caspase activation and recruitment domain; Ig-like: immunoglobulin like; FHA: forkhead-associated; UCH: ubiquitin C-terminal hydrolase; CAP-Gly: cytoskeleton-associated protein; SH3: Src homology-3; OTU: ovarian tumor type cysteine protease.