Basic principles of NF-κB signalling
NF-κB proteins are sequestered in the cytoplasm as latent complexes by inhibitory proteins referred to as inhibitor of NF-κB (IκB) proteins, which prevent NF-κB nuclear translocation and DNA binding [19]. Whereas the majority of IκBs (IκBα, IκBβ, IκBε, p105 (also known as NF-κB1), p100 (also known as NF-κB2)) serve as inhibitors of NF-κB, IκBξ and Bcl-3 instead potentiate NF-κB transactivation in the nucleus. p100 and p105 are precursors of the p52 and p50 NF-κB subunits, respectively. There are two unique NF-κB signalling pathways, termed canonical (or classical) and noncanonical (or alternative) NF-κB pathways.
The canonical NF-κB pathway plays a major role in innate and adaptive immunity, and is triggered by many stimuli including proinflammatory cytokines (for example, TNF, IL-1), antigens, RANKL, and TLR ligands. NF-κB signalling initiated by different receptors requires the formation of proximal protein-protein interactions that are often receptor specific, but ultimately converge in the activation of the IκB kinase (IKK) complex, which mediates phosphorylation of the inhibitory IκB protein leading to its K48-polyubiquitination and degradation by the proteasome [20]. The IKK complex is comprised of the two catalytic subunits IKK1 and IKK2 (also known as IKKα and IKKβ) and the regulatory subunit NF-κB essential modulator (NEMO - also known as IKKγ) (Figure 1). Gene targeting experiments showed that IKK2 and NEMO, but not IKK1, are required for canonical NF-κB activation [21].
Figure 1  Canonical NF-κB signalling and negative regulators. Ligand engagement of specific membrane receptors such as TNFR1, CD40, RANK, and TLR4 trigger the recruitment of specific adaptor proteins (TNF receptor 1-associated death domain protein (TRADD), MyD88, MAL, TIR domain-containing adaptor-inducing IFNβ (TRIF)), kinases (RIP1, IRAK1, IRAK4), and ubiquitin ligases (TRAF2, TRAF6, cIAP1, cIAP2) to the receptor. K63-linked polyubiquitination of TRAFs, RIP1 and IRAK1, is recognised by NEMO and TAB proteins, resulting in the recruitment and activation of respectively IKK2 and TAK1. TAK1 then phosphorylates and activates IKK2, which in turn phosphorylates IκBα, triggering its K48-linked ubiquitination and proteasomal degradation. This allows NF-κB (here shown as a heterodimer of p65 and p50) to translocate to the nucleus and promote target gene expression. TRAF1, which has no ubiquitin ligase activity, can negatively regulate NF-κB activation, most probably by competing with other TRAFs. A20 and CYLD are deubiquitinating enzymes that control NF-κB activation by targeting specific signalling proteins including RIP1 and TRAF6, to which they are recruited using specific ubiquitin-binding adaptor proteins such as ABIN-1 and p62. miR-146 is thought to negatively regulate TLR signalling by inhibiting expression of IRAK1 and TRAF6. Finally, TLR signalling can also be inhibited by the transmembrane protein SIGIRR, which has been proposed to compete with TLR4 for binding to IRAK1 and TRAF6. The expression of many of these negative regulatory molecules is NF-κB dependent, implicating them in the negative feedback regulation of NF-κB activation. ABIN, A20-binding inhibitor of NF-κB; cIAP, cellular inhibitor of apoptosis; CYLD, cylindromatosis; IKK, IκB kinase; IκB, inhibitor of NF-κB; IRAK, IL-1R-associated kinase; MyD88, myeloid differentiation primary response gene 88; NEMO, NF-κB essential modulator; NF, nuclear factor; RANK, receptor activator of NF-κB; RIP1, receptor interacting protein 1; SIGIRR, single-immunoglobulinIL-1 receptor-related; TIR, Toll-like receptor/IL-1R; TRAF, TNF receptor-associated factor; TLR, Toll-like receptor; TNF, tumour necrosis factor. One of the best studied NF-κB signalling pathways is the TNF pathway. TNF stimulation results in the recruitment of TNF receptor-1-associated death domain (TRADD) protein and of receptor interacting protein 1 (RIP1), which function as adaptor proteins for the E3 ubiquitin ligases TNF receptor-associated factor (TRAF) 2 and TRAF5, which in turn bind the E3 ubiquitin ligases cellular inhibitor of apoptosis (cIAP) 1 and cIAP2 (Figure 1). On TNF stimulation, TNF-receptor bound RIP1 is rapidly modified by K63-linked polyubiquitin chains. TRAF2/5 and cIAP1/2 are good candidates for RIP1 ubiquitination, but the specific role of each is still unclear. The polyubiquitin chains on RIP1 are believed to create a scaffold to recruit the IKK and TAK1 complex via the ubiquitin-binding proteins NEMO and TAB1/2, respectively. The recent identification of a distinct E2/E3 enzyme complex that modifies NEMO with linear polyubiquitin chains and is essential for TNF-activated NF-κB signalling adds further complexity [22]. The exact role of protein-anchored polyubiquitin chains remains unclear, as it was recently suggested that unanchored polyubiquitin chains can directly activate the TAK1 complex [23].
Similar signalling principles apply to other receptors. For example, TLR4 stimulation by lipopolysaccharide induces the recruitment of Toll/IL-1 receptor adaptor protein (also referred to as Mal) and TRIF-related adaptor molecule (TRAM), which most probably serve as bridging factors to recruit myeloid differentiation primary response gene 88 (MyD88) and TIR domain-containing adaptor-inducing IFNβ (TRIF), respectively. MyD88 in turn recruits members of the IL-1R-associated kinase (IRAK) family and TRAF6, leading to oligomerisation and selfubiquitination of TRAF6 [24]. TRIF also recruits TRAF6 [25] and RIP1 [26] via a direct interaction. Both pathways then activate TAK1 and IKK in a ubiquitination-dependent manner similar to the TNF pathway (Figure 1).
The noncanonical NF-κB pathway can be activated by the lymphotoxin β receptor, BAFF receptor, CD40, and RANK (Figure 2). In this pathway, p100 is processed by the proteasome to p52, which together with the RelB NF-κB subunit regulates a distinct set of target genes that control B-cell development, secondary lymphoid organ development, and osteoclastogenesis [27] The noncanonical NF-κB pathway is strictly dependent on IKK1, which is activated upon phosphorylation by NF-κB inducing kinase (NIK). NIK is predominantly regulated at the post-translational level and is present at extremely low levels in most cell types. In unstimulated cells, NIK occurs in a cytoplasmic complex with TRAF2, TRAF3, and cIAP1/2, which K48-polyubiquitinates NIK, leading to its continuous degradation by the proteasome. Receptor ligation has been shown not only to remove TRAF3 from this complex by recruiting it to the receptor, but also to attract TRAF2 and cIAP1/2, which are essential for subsequent TRAF3 degradation. All this contributes to releasing NIK from its constitutive degradation, resulting in NIK accumulation and IKK1 phosphorylation [28,29] (Figure 2).
Figure 2  Noncanonical NF-κB signalling. CD40 and RANK can activate the noncanonical NF-κB pathway that is dependent on NF-κB inducing kinase (NIK) expression levels. In unstimulated cells NIK forms a cytosolic complex with the ubiquitin ligases TRAF2, TRAF3 and cIAP1/2, which facilitates the K48-linked polyubiquitination and proteasomal degradation of NIK, keeping NIK levels low. Upon ligand binding, TRAF3 is recruited to the receptor, where TRAF2 directs nondegradative K63-linked polyubiquitination of cIAP1/2, resulting in their activation. Subsequently cIAP1/2 directs its K48-linked polyubiquitination to TRAF3, rather than NIK. As a result, TRAF3 is degraded and NIK is stabilised, resulting in increased NIK levels in the cell. NIK then phosphorylates and activates IKK1, which mediates NF-κB p100 phosphorylation. This is followed by K48-linked polyubiquitination and partial proteasomal degradation of p100 to p52, which forms a heterodimer with RelB to activate transcription. Next to TRAF3, TRAF1 has also been identified as a negative regulator of this pathway, most probably by competing with other TNF receptor-associated factors. cIAP, cellular inhibitor of apoptosis; IKK, IκB kinase; NF, nuclear factor; RANK, receptor activator of NF-κB; TRAF, TNF receptor-associated factor; TNF, tumour necrosis factor. It should be mentioned that CD40, lymphotoxin β receptor and RANK mediate the activation of both canonical and noncanonical NF-κB signalling pathways. Upon binding of their ligand, CD40 and RANK interact with several TRAF members, including TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6, and this leads to the proteolysis of both TRAF2 and TRAF3, which represents an important step in the activation of the noncanonical pathway as described above. Specific TRAF molecules are associated with overlapping and distinct CD40-mediated functions. For example, in B cells TRAF6 is required for CD40-mediated JNK activation and IL-6 production, while TRAF2 is required for activation of NF-κB, and TRAF3 serves as a negative regulator of CD40 signalling [30,31].