The Rel/NF-kappaB Signal Transduction Pathway

Rel/NF-kB transcription factors include a collection of proteins, conserved from the fruit fly Drosophila melanogaster to humans. Among the commonly-used model organisms, these transcription factors are notably absent in yeast and the nematode Caenorhabditis elegans; in part, this may be because one of the primary roles of these factors is to control a variety of physiological aspects of immune and inflammatory responses. A pathway similar to the Rel/NF-kB signaling pathway may also control certain defense responses in plants. Rel/NF-kB proteins are related through a highly conserved DNA-binding/dimerization domain called the Rel homology (RH) domain. However, Rel/NF-kB proteins can be divided into two classes based on sequences C-terminal to the RH domain

. Members of one class (p105, p100, and Drosophila Relish) have long C-terminal domains that contain multiple copies of ankyrin repeats, which act to inhibit these molecules. Members of this class become active, shorter DNA-binding proteins (p105 to p50, p100 to p52) by either limited proteolysis or arrested translation. As such, members of this first class are generally not activators of transcription, except when they form dimers with members of the second class of Rel/NF-kB transcription factors. The second class includes c-Rel (and its retroviral homologue v-Rel), RelB, RelA (p65), and the Drosophila Dorsal and Dif proteins. This second class of Rel proteins contains C-terminal transcription activation domains, which are often not conserved at the sequence level across species, even though they can activate transcription in a variety of species. The cDNA and predicted protein sequences of Rel/NF-kB transcription factors can be rapidly accessed via this site.

Rel/NF-kB transcription factors bind to 9-10 base pair DNA sites (called kB sites) as dimers. All vertebrate Rel proteins can form homodimers or heterodimers, except for RelB, which can only form heterodimers. This combinatorial diversity contributes to the regulation of distinct, but overlapping, sets of genes, in that the individual dimers have distinct DNA-binding site specificities for a collection of related kB sites. The term NF-kappaB commonly refers specifically to a p50-RelA heterodimer, which is one of the most avidly forming dimers and is the major Rel/NF-kB complex in most cells. The x-ray crystallographic structures of several Rel/NF-kB dimers on DNA (including p50-p50, p65-p65, p50-p65, c-Rel-c-Rel, p50-p65-IkB) have now been solved, and these structures can be accessed from this site.

The activity of NF-kB is tightly regulated by interaction with inhibitory IkB proteins. As with the Rel/NF-kB proteins, there are several IkB proteins, which have different affinities for individual Rel/NF-kB complexes, are regulated slightly differently, and are expressed in a tissue-specific manner. The IkB proteins include, at least, p105, p100, IkBa, IkBb, IkBg, IkBe, IkBz, Bcl-3, and the Drosophila Cactus protein. The cDNA and predicted protein sequences of these IkBs can be obtained through this site.

The best-studied NF-kB-IkB interaction is that of IkBa with the NF-kB p50-RelA dimer. This interaction blocks the ability of NF-kB to bind to DNA and results in the NF-kB complex being primarily in the cytoplasm due to a strong nuclear export signal in IkBa. That is, the NF-kB-IkBa complex is continuously shuttling between the nucleus and the cytoplasm, but its rate of nuclear export exceeds its rate of import and thus the complex is generally cytoplasmic. From biochemical studies and direct structural determinations , it is clear that IkBa makes multiple contacts with NF-kB. These interactions cover sequences of NF-kB that are important for DNA binding. In contrast, when IkBb interacts with the NF-kB complex, the complex is retained in the cytoplasm (i.e., does not undergo nucleo-cytoplasmic shuttling). Thus, not all NF-kB-IkB interactions are the same.

In most cells, NF-kB is present as a latent, inactive, IkB-bound complex in the cytoplasm. When a cell receives any of a multitude of extracellular signals , NF-kB rapidly enters the nucleus and activates gene expression. Therefore, a key step for controlling NF-kB activity is the regulation of the IkB-NF-kB interaction. Many of the molecular details of this control are now understood . Almost all signals that lead to activation of NF-kB converge on the activation of a high molecular weight complex that contains a serine-specific IkB kinase (IKK). IKK is an unusual kinase in that in most cells IKK contains (at least) three distinct subunits: IKKalpha, IKKbeta and IKKgamma. IKKa and IKKb are related catalytic kinase subunits, and IKKg is a regulatory subunit that serves as a sensing scaffold for the catalytic subunits. In the classical or canonical pathway, activation of IKK complex leads to the phosphorylation by IKKb of two specific serines near the N terminus of IkBa, which targets IkBa for ubiquitination (generally by a complex called beta-TrCP) and degradation by the 26S proteasome. In the non-canonical pathway, the p100-RelB complex is activated by an IKKa homodimer-mediated phosphorylation of the C-terminal region of p100, which leads to ubiquitination followed by degradation of the p100 IkB-like C-terminal sequences to generate p52-RelB. In either pathway, the unmasked NF-kB complex can then enter the nucleus to activate target gene expression. In the canonical pathway, one of the target genes activated by NF-kB is that which encodes IkBa. Newly-synthesized IkBa can enter the nucleus, remove NF-kB from DNA, and export the complex back to the cytoplasm to restore the original latent state. Thus, the activation of the NF-kB pathway is generally a transient process, lasting from 30-60 minutes in most cells.

A variety of recent evidence, however, suggests that the control of the NF-kB pathway is more complex than simply IKK-mediated regulation of the IkB-NF-kB interaction. For example, it appears that RelA and p50 are regulated by acetylation and prolyl isomerization, and that the transactivation activity of RelA can be affected by phosphorylation. Moreover, as a consequence of induction of NF-kB activity (at least by tumor necrosis factor) IKKa is also induced to enter the nucleus where it becomes associated with kB site promoters/enhancers to phosphorylate histone H3 which enhances the transcription of kB site-dependent genes.

In some normal cells, such as B cells, some T cells, Sertoli cells and some neurons, NF-kB is constitutively located in the nucleus. In addition, in many cancer cells (including breast cancer, colon cancer, prostate cancer, lymphoid cancers, and probably many others; NF-kB is constitutively active and located in the nucleus. In some cancers, this is due to chronic stimulation of the IKK pathway, while in other cases (such as some Hodgkin’s and diffuse large B-cell lymphoma cells) the gene encoding IkBa is sometimes mutated and defective. Moreover, several human lymphoid cancer cells have mutations or amplifications of genes encoding Rel/NF-kB transcription factors, which may enable these factors to accumulate in or cycle through the nucleus. It is thought that continuous nuclear Rel/NF-kB activity protects cancer cells from apoptosis and in some cases stimulates their growth. Therefore, many current anti-tumor therapies seek to block NF-kB activity as a means for inhibiting tumor growth or sensitizing the tumor cells to more conventional therapies, such as chemotherapy.

The Rel/NF-kB family is arguably the most-studied collection of eukaryotic transcription factors. For a collection of reviews on these transcription factors, the reader is directed to the November 22, 1999 issue of Oncogene, which contains a series of reviews on Rel/NF-kB, or the 2004 collection of reviews in the book edited by R Beyaert (both cited below).

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