NF-κB, the first quarter-century: remarkable progress and outstanding questions

Abstract

The ability to sense and adjust to the environment is crucial to life. For multicellular organisms, the ability to respond to external changes is essential not only for survival but also for normal development and physiology. Although signaling events can directly modify cellular function, typically signaling acts to alter transcriptional responses to generate both transient and sustained changes. Rapid, but transient, changes in gene expression are mediated by inducible transcription factors such as NF-κB. For the past 25 years, NF-κB has served as a paradigm for inducible transcription factors and has provided numerous insights into how signaling events influence gene expression and physiology. Since its discovery as a regulator of expression of the κ light chain gene in B cells, research on NF-κB continues to yield new insights into fundamental cellular processes. Advances in understanding the mechanisms that regulate NF-κB have been accompanied by progress in elucidating the biological significance of this transcription factor in various physiological processes. NF-κB likely plays the most prominent role in the development and function of the immune system and, not surprisingly, when dysregulated, contributes to the pathophysiology of inflammatory disease. As our appreciation of the fundamental role of inflammation in disease pathogenesis has increased, so too has the importance of NF-κB as a key regulatory molecule gained progressively greater significance. However, despite the tremendous progress that has been made in understanding the regulation of NF-κB, there is much that remains to be understood. In this review, we highlight both the progress that has been made and the fundamental questions that remain unanswered after 25 years of study.

Figure 1.

Twenty-five years of NF-κB literature. Graph indicates the total number of publications identified in PubMed using the keywords NF-κB, Rel, or IKK for each year since 1986 (shown in the left axis). Also graphed are the total publications identified with the above keywords as a percentage of all PubMed indexed publications in the same calendar year (shown in the right axis).

Figure 2.

Components of the NF-κB pathway. The mammalian Rel (NF-κB) protein family consists of five members: p65 (RelA), RelB, c-Rel (Rel), and the precursor proteins p100 (NF-κB2) and p105 (NF-κB1), the latter giving rise to p52 and p50, respectively. The IκB family consists of eight bona fide members, IκBα, IκBβ, IκBɛ, IκBζ, BCL-3, IκBNS, p100, and p105, which are typified by the presence of multiple ankyrin repeat domains. Not pictured is the potential IκB family member IκBη, which is discussed in the text. The IKK complex consists of IKKα (IKK1 or CHUK), IKKβ (IKK2), and NEMO (IKKγ). Relevant domains typifying each protein family are indicated. (ANK) Ankyrin repeat domain; (DD) death domain; (RHD) REL homology domain; (TAD) transactivation domain; (LZ) leucine zipper domain; (GRR) glycine-rich region; (SDD) scaffolding and dimerization domain; (ULD) ubiquitin-like domain; (Z) zinc finger domain; (CC) coiled-coil domain; (NBD) NEMO-binding domain; (α) α-helical domain; (IBD/DimD) IKK-binding domain/dimerization domain; (MOD/UBD) minimal oligomerization domain/ubiquitin-binding domain; (PEST) proline-rich, glutamic acid-rich, serine-rich, and threonine-rich.

Figure 3.

TNFR1 signaling to NF-κB. TNFR1 activates multiple signaling pathways, including NF-κB, AP-1, and the apoptosis and necroptosis cell death pathways. TNF-induced activation of NF-κB is mediated by a series of intermediary adapters. The cytoplasmic tail of TNFR1 exhibits several protein-binding domains, most notably a death domain (DD) that mediates signaling events following TNF binding. Signaling events are partially organized by subcellular compartmentalization of receptor complexes, and the TNFR cytoplasmic tail contains adapter protein-binding motifs that direct trafficking following TNF binding (Schutze and Schneider-Brachert 2009). Upon ligand binding, the DD of TNFR1 binds TRADD (TNFR-associated protein with a DD) and the DD-containing kinase RIP1 (Box 3). Mechanisms coordinating binding between the DDs of TRADD, RIP1, and TNFR1 are not fully established. Nevertheless, it is clear that each of these DD-containing proteins are capable of binding to other DD-containing proteins (Wajant and Scheurich 2011). TRADD also provides an assembly platform for recruitment of another DD adapter protein, FADD (Fas receptor-associated DD). TNFR1 lacks a TRAF interaction motif, and TRAF recruitment is thus also dependent on TRADD, which has a TRAF-binding domain. Although RIP1 also has a TRAF-binding domain and may contribute to TRAF2 recruitment under some circumstances (Pobezinskaya et al. 2008), it is generally thought that TRAF2 recruitment is primarily dependent on TRADD (Chen et al. 2008; Ermolaeva et al. 2008; Pobezinskaya et al. 2008). TRAF2 recruits cIAP1 and cIAP2 (Box 2), which are essential for IKK activation (Mahoney et al. 2008; Varfolomeev et al. 2007; Vince et al. 2007). The cIAPs can function as E3 ubiquitin ligases and are also responsible for the recruitment of the linear ubiquitin chain assembly complex, which is required for efficient activation of IKK and JNK pathways (LUBAC [linear ubiquitin assembly complex]) (Box 4; Haas et al. 2009; Rahighi et al. 2009; Tokunaga et al. 2009, 2011; Gerlach et al. 2011; Ikeda et al. 2011). RIP1 and TRAF2 cooperate in the recruitment of the TAK1 and IKK kinase complexes, leading to IKK activation and activation of NF-κB.

Figure 4.

TLR4 signaling to NF-κB. Both TLR and IL-1R receptor families are defined by the presence of cytoplasmic TIR (Toll IL-1R) domains. Upon ligand binding, TIR domains mediate the recruitment of TIR-containing adapter proteins such as MyD88, TRIF, Mal, or TRAM (Yamamoto et al. 2004a). TLR4, which responds to bacterial lipopolysaccharide, has a complex bifurcating signaling scheme. MyD88 is the prototypical TIR adapter and is used in all characterized TLR signaling pathways, with the exception of TLR3. TLR4 recognizes LPS bound to either LPS-binding protein or MD2, and signaling is also dependent on the glycoprotein CD14. Formation of a complex between LPS and TLR4:MD2:CD14 results in the homodimerization of TLR4 and recruitment of the TIR-containing adapters Mal and TRAM. Mal serves as an adapter to recruit MyD88 to TLR4, while TRAM is an adapter between TLR4 and TRIF. Following recruitment to the receptor complex, dimerized MyD88 recruits IL-1R-associated kinases-4 (IRAK-4) through the DD of MyD88 and IRAK-4. IRAK-4 recruits IRAK-1, and the IRAK-1:IRAK-4 complex is responsible for binding to TRAF6. TRAF6, in turn, recruits the TAK1 and IKK complexes, leading to activation of NF-κB. TRIF, recruited by TRAM, predominantly activates the interferon pathway through an N-terminal TRAF3-binding motif. TRAF3 recruits the IKK family members IKKɛ and TBK1, which phosphorylate IRF3, leading to the induction of type I interferons. TRIF may also induce NF-κB activation through a C-terminal RHIM (RIP homology interaction motif) domain capable of recruiting RIP1 and also the IKK complex (Cusson-Hermance et al. 2005).

Figure 5.

T-cell receptor (TCR) signaling to NF-κB. TCR-induced NF-κB activation requires ligation of both the TCR and the associated coreceptor CD28. Signaling involves the formation of large supramolecular clusters at the interface of the T-cell and antigen-presenting cell (APC). In vivo TCR ligation occurs upon presentation of cognate antigen in MHC-I or MHC-II for CD8 and CD4 T cells, respectively, by activated APCs expressing costimulatory molecules such as B7.1 or B7.2. Antigen:MHC complexes are engaged by T cells expressing somatically encoded antigen-specific TCRs. TCR:MHC binding is augmented by CD8:MHC-I or CD4:MHC-II interactions. ITAM motifs on CD3ζ are phosphorylated, leading to recruitment and activation of the ZAP70 kinase, which in turn activates PLCγ. Active PLCγ leads to DAG production. Coreceptor ligation involves recognition of B7 molecules on the APC surface by the TCR coreceptor CD28. CD28 ligation results in activation of PI3K and phosphorylation of PIP₂ to PIP₃. PDK1 binds PIP₃ and undergoes autophosphorylation, revealing a PKC-binding site. DAG, in conjunction with PDK1-mediated recruitment and phosphorylation, leads to activation of the atypical PKC family member PKCθ. PKCθ (Sun et al. 2000) and perhaps also the related family members PKCɛ and PKCη (Quann et al. 2011) are selectively required for the activation of NF-κB downstream from TCR. In addition to activating PKCθ, PDK1 also facilitates the formation of a signaling complex (Lee et al. 2005; Park et al. 2009) in which PKCθ and potentially other kinases phosphorylate CARD11 (Matsumoto et al. 2005; Sommer et al. 2005; Shinohara et al. 2007). Phosphorylation of CARD11 results in a structural change that allows the formation of the CARD11, BCL10, and MALT1 (CBM) (Ruefli-Brasse et al. 2003; Ruland et al. 2003) signaling complex. Recruitment of the IKK complex to the CBM through PKCθ (Lee et al. 2005) or ubiquitinated MALT1 (Oeckinghaus et al. 2007), together with recruitment of the TAK1 complex, perhaps through TRAF6-mediated ubiquitination of BCL10 (Sun et al. 2004), results in the activation of IKK and the NF-κB signaling pathway.

Figure 6.

CD40 signaling to the alternative NF-κB pathway. CD40 possesses multiple TRAF interaction motifs, and binding of CD40L to CD40 triggers direct binding to multiple TRAF proteins. Noncanonical NF-κB activation requires the NF-κB-inducing kinase (NIK). NIK is constitutively active; thus, the noncanonical pathway is activated through the post-translational regulation of NIK protein levels. In the steady state, NIK is subject to constitutive ubiquitination by TRAF3 and consequent degradation. Upon CD40 ligation, TRAF2 is recruited to the receptor and, in conjunction with cIAP, targets TRAF3 for proteasomal degradation (Liao et al. 2004). CD40 ligation may also promote NIK stabilization through an “allosteric model” in which binding of NIK and binding of CD40 by TRAF proteins are mutually exclusive events (Sanjo et al. 2010). Thus, upon TRAF binding to CD40, NIK is displaced from the TRAF2:TRAF3 complex. As a result of displacement of NIK and the loss of TRAF3, active NIK accumulates. NIK binds and phosphorylates IKKα, leading to activation of IKKα kinase activity. Phosphorylation of p100 C-terminal serine residues by IKKα results in p100 ubiquitination by the SCF^βTRCP complex and proteasomal processing to p52. Activation of the noncanonical pathway has also been well characterized for LTβR, BAFFR, and RANK.

Figure 7.

IκB functions. (A) Typical IκB proteins function by promoting cytosolic sequestration of NF-κB dimers. Upon stimulation of the canonical signaling pathway, such IκB proteins are phosphorylated by IKK and targeted for proteasomal degradation. Activation of NF-κB results in resynthesis of IκB proteins. (B) The precursor IκB proteins p100 and p105 have multiple functions. Their constitutive processing results in the generation of p50 and p52 subunits. Unprocessed precursor proteins may, alternatively, form complexes with other NF-κB proteins. Proteasomal degradation or processing, in the case of p100:RelB complexes, is induced by activation of the noncanonical pathway. Proteasomal degradation of p105-containing complexes is mediated by the canonical pathway and can result in the activation of both NF-κB and ERK via release of p105-bound Tpl2. (C) Atypical IκB proteins are induced by various stimuli, including NF-κB activation, and exert both positive and negative effects on NF-κB-mediated transcription. Atypical IκB proteins function by binding to nuclear DNA-associated NF-κB dimers. In the case of IκBβ, newly synthesized hypophosphorylated IκBβ protein acts to augment transcription of p65:c-Rel dimers. BCL-3 may promote or inhibit transcription, depending on various PTMs. IκBζ promotes transcription, whereas IκBNS inhibits transcription, by binding p50 dimers.