The most basic components of NF-κB signaling pathways include receptors, signal adapter proteins, IκB kinase (IKK) complex, IκB protein and NF-κB dimer. When cells are subjected to various intracellular and extracellular stimuli, IKK is activated, resulting in phosphorylation and ubiquitination of IκB protein, and then IκB protein is degraded and NF-κB dimer is released. The NF-κB dimer is further activated by various post-translational modifications and is transferred to the nucleus. In the nucleus, it binds to the target gene and promotes the transcription of the target gene. The NF-κB signaling pathway includes both canonical and non-canonical signaling pathways. In the canonical NF-κB signaling pathway, the degradation of IκB protein releases the NF-κB dimer. The non-canonical NF-κB signaling pathway is activated through the processing of p100 to p52.
The NF-κB protein family consists of five members: p50, p52, p65 (RelA), c-Rel, and RelB. They are encoded by the NFKB1, NFKB2, RELA, REL, and RELB genes, respectively. Structurally, they all have an N-terminal Rel homology domain (RHD) responsible for their binding to DNA and dimerization. In addition, p65, c-Rel and RelB have a transcriptional activation region (TAD) that positively regulates gene expression. p50 and p52 do not have transcriptional activation regions, and their homodimers can inhibit transcription.
Figure 1. Schematic representation of NF-κB family of proteins.
In general, NF-κB exists in the form of a dimer, and its dimer has two modes of existence, including the binding of NF-κB dimer to IκB protein, and the binding of NF-κB dimer to DNA. The amino terminus of the NF-κB dimer is an immunoglobulin-like region that has selectivity for a certain form of κB site. The C-terminal hydrophobic region provides a link between the various subunits of NF-κB.
Figure 2. Structures of NF-κB. (A) Space-filling model of the crystal structure of the p50/p65 heterocomplex bound to DNA. (B) The same structure shown in ribbon diagrams in two orientations. (Napetschnig J. 2013)
Many extracellular stimuli can cause activation of NF-κB signaling pathways, such as pro-inflammatory cytokines TNFα, interleukin IL-1, bacteria lipopolysaccharide (LPS), T-cell and B-cell mitogens, viral double-stranded RNA and various physical and chemical pressures. Although the intracellular early signal pathways produced by these extracellular stimuli vary, it is generally believed that the signaling response initiated by most of these extracellular stimuli will eventually activate the IKK complex. In this transmission process, adaptor proteins play an important role. In NF-κB signaling pathways, many signaling intermediates are common, especially upstream signals of the IKK complex. Different signaling pathways can utilize some common signaling elements to activate and inhibit pathways. Upstream signal adaptor proteins of the IKK complex include TNF receptor-associated factors (TRAFs) and receptor interacting proteins (RIPs). The kinases of the IKK complex include TGFβ-activating kinase 1 (TAK1) and NF-κB-inducing kinase (NIK). In the non-canonical signal pathway of NF-κB, TRAF and NIK can fully activate IKKα without NEMO. In the NF-κB canonical signaling pathway, TRAF, RIP and TAK1 are all required.
IκB kinase (IKK)
The IKK complex comprises three subunits, IKKα (IKK1), IKKβ (IKK2), and the regulatory subunit IKKγ (NEMO). Among specific NF-κB signaling pathways, IKKα and IKKβ are selectively required. IKKα and IKKβ have high sequence homology and similar structures. At the N-terminus, there is a protein kinase region, a leucine zipper region (LZ) and a helix-loop-helix (HLH) near the middle region. NEMO includes a large section of coiled-coli and a leucine zipper region near the C-terminus.
Figure 3. Structures of the IKK complex. (A) Domain organizations. (B) Crystal structure of IKKβ dimer. (C) Crystal structure of IKKβ NBD in complex with the N-terminal kinase-binding domain (HLX1) of NEMO. (Napetschnig J. 2013)
IKKα is not required in the canonical NF-κB signaling pathway. It is required in the NF-κB activation transduction pathway induced by receptor activator of NF-κB (RANK) and the NF-κB activation alteration pathway. Deletion of IKKα can lead to many developmental defects. IKKβ-deficient cells do not induce NF-κB activation in response to TNFα and IL-1 stimulation. The activation of IKKβ is necessary to avoid loss of multi-tissue function due to severe inflammatory reactions caused by ischemia or congestion. NEMO is required in the canonical NF-κB signaling pathway. NEMO may mediate the assembly of IKK complexes through direct interactions with IKKβ and IKKα and also facilitate the interaction of IκB proteins with IKK kinase complexes.
Activation of the IKK complex need a phosphorylation of T-loop serine of IKK subunits. However, the mechanism of phosphorylation is not clear yet. The phosphorylation sites of IKKβ are serine 177 and 181, and the phosphorylation sites of IKKα are serine 176 and 180. A common element in the activation of IKK complexes is the need for TRAF family members and the induction of oligomerization of TRAF to signal down. The C-terminal region of NEMO mediates IKK activation. Inducible oligomerization of NEMO mediated by RIP1 is thought to activate IKK, which has been demonstrated.
Through a series of studies, IKK activation models have been established. In the resting state, IKKs in the IKK complex are inhibited by interacting with NEMO. In the presence of stimuli, NEMO binds to a RIP protein, exposes the IKK protein kinase domain, induces trans-autophosphorylation of the T-loop serine residue, or phosphorylates T-ring serine residues by IKK-K. Activated IKK phosphorylates downstream enzyme substrates (e.g., IκB), thereby activating the NF-κB signaling pathway. Activated IKK can also phosphorylate serine 740 of IKKβ and serine 68 of NEMO, allowing the separation of NEMO dimers from IKK, preventing the repeated activation of kinases. Cdc37/HSP-90 (chaperone) and PP2A/PP2Cβ (phosphorylase) can mediate the recombination of the IKK complex.
The IκB protein family includes 7 members: IκBα, IκBβ, IκBζ, IκBε, Bcl-3, p100, and p105. IκB binds to the NF-κB dimer in the cytoplasm and plays an important role in signal responses. The IκB protein structure has an ankyrin repeat region, i.e., a plurality of closely linked hook repeats each containing 33 amino acids. The main role of IκB protein is to mask the nuclear localization signal of NF-κB, prevent its entry into the nucleus and its binding to DNA, and make NF-κB exist in the cytoplasm of cells in an inactive form. Therefore, for the study of NF-κB signaling pathway, the study of IκB protein is very important.
Figure 4. Schematic representation of IκB family of proteins.
Typical IκB proteins include IκBα, IκBβ, and IκBε. Among them, IκBα is the strongest negative feedback factor in the activation of NF-κB, which ensures the rapid occurrence and closure of NF-κB activation. However, IκBβ and IκBε can buffer the fluctuation of system activation so that NF-κB can maintain a relatively long response time. IκBα and IκBβ specifically inhibit the dimer containing p65 and c-Rel.
Precursor IκB proteins include p100 and p105. p100 occurs mainly in non-canonical pathways and is eventually degraded to p52. The process of p100 processing into p52 is a regulated process that includes phosphorylation and ubiquitination. p100 is the only regulatory factor for RelB because the NF-κB dimer containing RelB binds only p100. p105 can be induced to degrade by the activated IKK complex. p105 is processed through the proteasome to form p50, which occurs during the translation stage.
Atypical IκB proteins include IκBζ and Bcl-3. Bcl-3 is found in the nucleus along with isoforms and heterodimers composed of p50 and p52. The homodimer of p50 can bind to the κB site of the target DNA, thereby inhibiting the NF-κB signaling pathway. Bcl-3 has two modes of action. The first can mediate the release of transcriptional repressors by exposing p50 homodimers from the κB site, and the κB sites are exposed, thereby activating the NF-κB signaling pathway. Second, it can also stabilizes p50 homodimers and blocks p65, p50, or other dimers containing TAD from entering the κB site, inhibiting signaling pathways. IκBζ is similar to Bcl-3 and has weaker homology with other IκB proteins. IκBζ is up-regulated in IL-1 and TLR4 induced NF-κB responses, and its expression is mainly concentrated in the nucleus.
Figure 5. Canonical and non-canonical NF-κB signaling pathways.
NF-κB/p65: p50 dimer activation through the canonical pathway, such as those transduced through TNFR1, involves signal responsive activation of IKK. In general, the catalytic activity of IKKβ is required for canonical signaling. In response to a variety of inflammatory stimuli, the IKK complex phosphorylates IκBs at specific N-terminal serine residues (serine 32 and serine 36 for IκBα). Subsequent β-TrCP-mediated ubiquitination of canonical IκBs promotes complete proteasomal degradation of the inhibitors to liberate bound NF-κB dimers. Interestingly, neither phosphorylation nor ubiquitination is sufficient to dissociate IκBs from the p65: p50 dimer and proteasomal degradation of IκBs is absolutely required for p65: p50 nuclear translocation. This mode of NF-κB activation in the canonical pathway is protein synthesis-independent. NF-κB transcriptional activity is thought to be further modulated through phosphorylation and other posttranscriptional modifications of p65, but a clear understanding that relates p65 modification with its function is yet to emerge.
LTβR stimulation was shown to induce nuclear accumulation of RelB: p52 via the NIK/IKK1 pathway and the phosphorylation and processing of de novo synthesized, rather than preexisting p100 protein. Indeed, a first phase of p65 activity was proposed to induce p100 synthesis to amplify RelB: p52 dimer activation, but recent evidence indicates that p65-responsive expression of RelB is more important in this regard. Signaling through multiple other TNF receptor superfamily members, such as BAFFR, CD40R, BCMA, TAC1 or RANK, was also reported to activate the RelB: p52 dimer via the non-canonical pathway.
Regulation of NF-κB Transcriptional Activity
Figure 6. Regulation of NF-κB Transcriptional Activity. (Hayden, et al. 2008)
In non-stimulated cells, the κB site of the gene of interest is occupied by homodimers of p50 or p52. At this stage, Bcl-3 and IκB can positively and negatively regulate the NF-κB signaling pathway by removing and stabilizing p50 or p52 homodimers. When the cells are stimulated, along with the signal transduction, after degrading the IκBα protein, p65 can be phosphorylated in the cytoplasm by IKKα or PKA, and can also be phosphorylated in the nucleus by MSK1/2 or RSK-1. Phosphorylated p65 is more likely to bind to CBP/P300, thereby acetylating histones and p65, and promoting the expression of the target gene. Under the action of IKKα, the co-activator CBP/P300 was phosphorylated, and thus exchanged with the co-rejector HDACs, and the phosphorylated coactivator CBP/P300 was bound to the NF-κB dimer. IKKα and MSK1 can also directly phosphorylate histones to promote transcription. When NF-κB binds to the target gene, IκBα and IκBε can reverse the regulation of NF-κB signaling pathway by separating NF-κB from the target gene.
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