NF-κB refers to a group of structurally related transcription factors that play a central role in inflammation, lymphocyte activation, cell survival, and the formation of secondary lym phoid organs. NF-κB family members are important players in lymphocyte development and in the pathogenesis of many cancers, including malignant neoplasms derived from activated lymphocytes. NF-κB is prominently activated by cytokines of the IL-1, TNF, and IL-17 families, and is also induced downstream of TLR stimulation and antigen recognition. It is discussed here as the prototype of a transcription factor with fundamental roles in innate and adaptive immunity.
There are five NF-κB proteins. The domain that is common to all NF-κB proteins is a DNA-binding domain called a REL homology domain. For a transcription factor to be active, it must both bind DNA and contain an activation domain that can facilitate transcriptional initiation. Three NF-κB proteins have both REL homology domains and activation domains. These are p65/RELA, RELB, and c-REL. Two proteins, NF-κB1/p50 and NF-κB2/p52, contain a DNA-binding REL homology domain but lack activation domains. NF-κB1 typically forms active heterodimers with p65/RELA or with c-REL, and NF-κB2 forms heterodimers with RELB.
There are two pathways of NF-κB activation, called the canonical and noncanonical pathways (Fig. 1). Most stimuli that activate NF-κB do so by inducing the canonical pathway (hence its name). This pathway is activated by a number of receptors that drive inflammation, such as TLRs, the IL-1R, and some members of the TNFR family such as TNFRI. It is also activated in the context of lymphocyte activation by the BCR and the TCR. The canonical pathway results in the nuclear localization of transcriptionally active heterodimers of NF-κB1/p50 with p65/RELA or with c-REL. p50-containing heterodimers normally reside in the cytosol bound to an inhibitor of NF-κB called IκBα, and they cannot access the nucleus in nonactivated cells (see Fig. 1). The canonical NF-κB pathway induces the tagging and degradation of IκBα, allowing the unfettered heterodimeric NF-κB1 to migrate into the nucleus. Two very different types of polyubiquitination events are required for canonical NF-κB activation. There are a few common steps in the canonical pathway that apply to all upstream signal inputs.
• Upstream signaling leads to the activation of a unique type of ubiquitin E3 ligase that can add a lysine-63 type of ubiquitin chain to a protein called NEMO or IKKγ that is a noncatalytic subunit of a trimeric enzyme complex called the IκB kinase (IKK) complex. This complex contains two other subunits called IKKα and IKKβ, both of which have the potential to be catalytically active serine/threonine kinases. Ubiquitination of NEMO allows IKKβ to be activated by an upstream kinase.
• Active IKKβ phosphorylates the inhibitory protein bound to NF-κB, IκBα, on two specific serine residues, and thus tags this protein for lysine-48 ubiquitination.
Fig1. The canonical and noncanonical nuclear factor-κB (NF-κB) pathways. The canonical pathway is depicted on the left. Tumor necrosis factor (TNF) family receptors, Toll-like receptors (TLRs), and antigen receptors activate or induce an E3 ligase that can polyubiquitinate NEMO/IκB kinase γ (IKKγ), a component of the IKK complex, forming lysine-63–linked ubiquitin chains. This leads to the phosphorylation and activation of IKKβ by an upstream kinase. IKKβ phosphorylates the inhibitor of NF-κB (IκBα) and targets it for lysine-48 polyubiquitination and proteasomal degradation. Degradation of IκBα leads to the entry of active NF-κB into the nucleus. Antigen receptors activate specific protein kinase C, which in turn activate the CARMA-1/BCL10/ MALT-1 complex (not shown) in order to activate IKK. TNF receptor–associated factors (TRAFs) are E3 ligases that are activated downstream of TNF family receptors and TLRs. The noncanonical pathway is depicted on the right. In this pathway, most prominently activated downstream of the lymphotoxin β receptor and the BAFF receptor, TRAFs are activated downstream of these receptors, also in a lysine-63 ubiquitination-dependent manner (not shown). The activated TRAFs contribute to the activation of the NIK kinase, which phosphorylates and activates an IKKα containing complex. The activated IKKα in turn phosphorylates p100 that is bound to RELB, marking it for ubiquitination and partial degradation, yielding p52 or NF-κB2. p52/RELB heterodimers then migrate into the nucleus.
Polyubiquitinated IκBα is targeted for degradation in the proteasome, and the canonical NF-κB heterodimer is then free to enter the nucleus (see Fig. 1). We discussed earlier how TCR and BCR signaling contributes to the activation of PKCθ and PKCβ, respectively. These PKCs can phosphorylate a protein called CARMA1 that forms a complex with two proteins called BCL10 and MALT1. The CARMA1/MALT1/BCL10 complex can contribute to the activation of a lysine-63 type of ubiquitin E3 ligase called TRAF6. Active TRAF6 can activate TAK1 and also add a lysine-63 ubiquitin chain to NEMO, thus facilitating the activation of IKKβ. TLRs, IL-17R, and the IL-1R also acti vate TRAF6 to initiate IKK activation. Many members of the TNF receptor family, including the TNF receptor and CD40, can activate canonical NF-κB signaling through the activation of other TRAF proteins, such as TRAF2, TRAF3, and TRAF5.
A separate NF-κB signaling pathway, called the noncanonical pathway, processes a precursor protein called p100 into p52, thus allowing heterodimers of NF-κB2/p52 and its partner RELB to enter the nucleus. In nonactivated cells, the p100 precursor is bound to RELB, and the p100/RELB complex is unable to enter the nucleus until p100 is converted to p52. This pathway is activated downstream of a few TNFR family signaling receptors, most notably the lymphotoxin-β receptor (LTβR), which drives lymphoid organogenesis, and the BAFF receptor (BAFFR), which facilitates B-cell survival. Receptors such as LTβR and BAFFR that induce the noncanonical NF-κB pathway also use TRAFs to activate a kinase called NIK that in turn activates an IKK-like complex that contains IKKα homodimers. This leads to the phosphorylation of p100, marking it for ubiquitination and degradation in the cytosol, and leads to the generation of noncanonical p52/RelB NF-κB complexes that can migrate into the nucleus (Fig. 2).
Fig2. The transforming growth factor–β (TGF-β) signaling pathway. Processed TGF-β dimers bind to the TGβRII receptor, which is a dimeric receptor serine/threonine kinase. TGFβRII is activated and then associates with and phosphorylates the TGFβRI dimer, also a receptor serine/threonine kinase. Catalytically active TGFβRI then phosphorylates a receptor-regulated SMAD transcription factor (an R-SMAD, such as SMAD2 or SMAD3) that resides in the cytosol and which then forms a heterodimer with a Co-SMAD (such as SMAD4). This SMAD heterodimer enters the nucleus and induces the transcription of target genes.
Transforming Growth Factor–β Signaling
Transforming growth factor–β1 (TGF-β1) is a growth fac tor of importance in T- and B-cell biology. It is one member of the widely studied TGF-β superfamily of structurally related growth factors that play crucial roles in numerous aspects of invertebrate and vertebrate development as well as in cellular transformation in cancer. During T-cell differentiation, TGF-β participates in regulatory T-cell development and function and the development of Th17 cells. In humoral immunity, TGF-β is important for class switching to IgA.
Newly synthesized TGF-β is cleaved in the Golgi into two fragments, a C-terminal peptide that dimerizes to form the inacive cytokine and an N-terminal pro-domain, called latency associated peptide, that also dimerizes and remains bound to the inactive TGF-β dimer. After secretion, the latency-associated peptide dimer, still bound to the inactive TGF-β dimer, associates covalently (forming disulfide bonds) with a specific protein on the extracellular matrix, forming what is known as the latent complex. αV integrins on another cell bind to the latency-associated peptide and the activated integrin then exerts physical force, disrupting the latent complex and leading to the release of active TGF-β dimers. TGF-β dimers then bind to the dimeric TGFβRII receptor protein on an adjacent cell surface.
Mammalian cells contain seven different TGF-β family type I receptors and five different type II receptors. Both the TGFβRI and the TGFβRII proteins are transmembrane protein dimers whose extracellular domains bind TGF-β and whose cytosolic tails contain serine/threonine kinase domains. After TGFβRII has been ligated it is catalytically activated and phosphorylates and activates TGFβRI. Catalytically active TGFβRI then phosphorylates a cytosolic receptor-regulated SMAD family transcription factor or R-SMAD (such as SMAD2 or SMAD3), which then acquire a high affinity for a co-SMAD (such as SMAD4). The R-SMAD-co-SMAD complex then goes to the nucleus and induces transcription of TGF-β target genes (see Fig. 2). Depending on the cell type, these target genes differ. In the induction of peripheral or induced T regulatory cells, for instance, TGF-β signaling induces the expression of the FoxP3 transcription factor that is essential for the inhibitory function of these regulatory T cells.
