NF-kB pathway


The eukaryotic transcription factor NF-kB was identified as a protein that bound to a specific decameric DNA sequence (ggg ACT TTC C), within the intronic enhancer of the immunoglobulin kappa light chain in mature B- and plasma cells but not pre B-cells (Sen and Baltimore, 1986, Cell, 46: 705-716). Later, it was demonstrated that NF-kB's DNA binding activity is induced by a variety of exogeneously applied stimuli, and that this activation is independent from de-novo protein synthesis. NF-kB has been detected in most cell types, and specific NF-kB binding sites (with the general consensus sequence: ggg RNN YYC C, R=purine Y=pyrimidine) have been identified in promoters and enhancers of a big number of inducible genes.

Rel/NF-kB proteins

The transcription factor NF-kB consists out of homo- or heterodimers of different subunits. The subunits are members of a family of structurally related proteins (Rel/NF-kB proteins). Five different Rel proteins (also called Rel/NF-kB proteins) have been identified so far: p50, p52, p65, RelB, and c-Rel. All those Rel proteins contain a conserved N-terminal region, called the Rel Homology Domain (RHD). The RHD contains the DNA-binding and dimerization domains and the nuclear localization signal of the Rel proteins. p50 (also called NF-kB1) and p65 (RelA) were the first NF-kB proteins to be identified. Their N-terminal 300 aa revealed high similarity to the oncoprotein v-Rel, its cellular homologue c-Rel and the Drosophila protein Dorsal what resulted in the terms Rel proteins and RHD.

The Rel/NF-kB proteins can be divided into two groups:
Only RelA (p65), RelB and c-Rel (and Dorsal and Dif in Drosophila) contain potent transactivation domains (TDs) within sequences C-terminal to the RHD. The TDs consist out of abundant serine, acidic and hydrophobic aminoacids which are essential for transactivation activity.
In contrast, p50 and p52 do not possess TDs, and therefore can not act as transcriptional activators by themselves. Homo- or heterodimers of p50 and p52 were even reported to repress kB site-dependent transcription in vivo (Lernbecher et al., 1993, Nature, 365: 767-770), possibly by competing with other transcriptionally active dimers (e.g. p50/RelA) for DNA binding! Interestingly, kB-site-dependent transcriptional activation by p50/p50 has been demonstrated in vitro (Lin et al., 1995, J. Biol. Chem. 270: 3123-3131).

There are additional differences between these two groups of Rel/NF-kB proteins. RelA, RelB and c-Rel mRNA transcripts code for proteins that basically consist out of the RHD and the TD, but p50 and p52 are synthesized as a p105 or p100 precursor protein, respectively. p105 and p100 belong to the IkB family. Their N-terminal portion constitutes the RHD while the C-terminal part contains multiple copies of ankyrin repeat sequences which are typical for IkB proteins. Between the RHD and ankyrin repeats lies a glycine-rich region (GRR). The GRR might provide a signal for endoproteolytic cleavage of p105 (and possibly p100) by an ATP- and Mg2+-dependent protease, what results in the release of p50 and the C-terminus which is ubiquitinated and degraded by the proteasome.

NF-kB subunit Size Special Characteristics
p50 50 kDa Synthesized as the precursor IkB protein p105; p50 does not posses TD.
p52 52 kDa Synthesized as the precursor IkB protein p100; p52 does not posses TD.
p65 (RelA) 551 aa = 65 kDa
RelB 582 aa = 68 kDa Does not possess PKA phosphorylation site, but an additional N-terminal leucin-zipper-like region which affects its transcriptional activity.
c-Rel 587 aa = 69 kDa

Functional and structural aspects of NF-kB action

The interaction of Rel/NF-kB proteins with DNA requires dimerization of the NF-kB subunits. The dimerization domain is located in the C-terminal region of the RHD, whereas the N-terminal part of the RHD contains the DNA-binding domain. Close to the C-terminal end of the RHD lies the Nuclear Localization Signal (NLS) which is essential for the transport of active NF-kB complexes into the nucleus. The RHD of all Rel proteins (except RelB) contains a phosphorylation site for PKA about 25 aa N-terminal to the NLS. Phosphorylation of this site may be required for nuclear localization of the Rel proteins and/or may be involved in the transcriptional activity and DNA-binding.
X-ray cristallography of a p50/p50 dimer bound to DNA gave insight into the structural basis of NFkB-DNA interaction: the RHD is composed out of two domains that have a beta-sandwich structure similiar the immunoglobulin fold. Two beta-sheets in the C-terminal portion (domain) of the RHD form a hydrophobic surface which mediate the dimerization of two NF-kB subunits, while several loops between the beta-strands of mainly the N-terminal domain interact with the DNA. This kind of interaction between loops and DNA is unusual for transcription factors which generally use alpha-helices or beta-sheets to contact DNA.
Once bound to a kB motif, Rel/NF-kB proteins also interact with DNA-associated factors as well as the general transcriptional apparatus, e.g. with TBP, TFIIB or CBP/p300. Promoter studies revealed that NF-kB acts in synergy with other transcription factors such as c-Jun or Sp1 in order to mediate an effective transcriptional activatiton. This suggests that a distinct combination of binding sites for different transcription factors within individual gene promoters contributes to the selective regulation of gene expression.

Rel/NF-kB inducers and kB-dependent genes

NF-kB is involved in regulating many aspects of cellular activity, in stress, injury and especially in pathways of the immune response. Some examples are the response to and induction of IL-2, the induction of TAP1 and MHC molecules by NFkB, and many aspects of the inflammatory response, e.g. induction of IL-1 (alpha and beta), TNF-alpha and leukoyte adhesion molecules (E-selectin, VCAM-1 and ICAM-1). Moreover, NF-kB is involved in many aspects of cell growth, differentiation and proliferation via the induction of certain growth and transcription factors (e.g. c-myc, ras and p53).
NF-kB itself is induced by stimuli such as pro-inflammatory cytokines and bacterial toxins (e.g. LPS, exotoxin B) and a number of viruses/viral products (e.g. HIV-1, HTLV-I, HBV, EBV, Herpes simplex) as well as pro-apoptotic and necrotic stimuli (oxygen free radicals, UV light, gamma-irradiation).

Inhibitors of NF-kB

NF-kB dimers are sequestered in the cytosol of unstimulated cells via non-covalent interactions with a class of inhibitor proteins, called IkBs. To date seven IkBs have been identified: IkB-alpha, IkB-beta, IkB-gamma, IkB-epsilon, Bcl-3, p100 and p105. All known IkBs contain multiple copies of a 30-33 aa sequence, called ankyrin repeats which mediate the association between IkB and NF-kB dimers. The ankyrin repeats interact with a region in the RHD of the NF-kB proteins and by this mask their NLS and prevent nuclear translocation. Signals that induce NF-kB activity cause the phosphorylation of IkBs, their dissociation and subsequent degradation, allowing NF-kB proteins to enter the nucleus and induce gene expression.
Phosphorylation of IkBs results in their ubiquitination and subsequent degradation by the multicatalytic ATP-dependent 26S proteasome complex. Evidence for this was provided by inhibition experiments with peptide aldehyde inhibitors of the proteasome such as calpain inhibitor I (ALLN). The proteasome inibitors caused a block of the degradation of IkB-alpha after stimulation with e.g. TNF-alpha, and instead accumulation of phosphorylated IkB-alpha (at two N-terminal serine residues at positions 32 and 36). The phosphorylated IkBs then interacts with a protein called beta-TrCP, which triggersthe formation of a ubiquitin-ligase complex that adds multiple ubiquitin molecules to the IkB proteins at two N-terminal lysine residues (Maniatis, 1999, Genes Dev., 13: 505).

IkB kinases

After activation of cells by e.g. the binding of certain cytokines to their surface receptors, the IkB proteins are rapidly phosphorylated. Two kinases have been identified, that are responsible for this modification of the IkBs: IKK-alpha and IKK-beta. Both kinases were identified to be members of a high molecular complex which also contains IKK-gamma (also called NEMO, IKKAP) and IKAP. IKK-alpha and IKK-beta share significant sequence homology and contain identical structural domains. By their leucine-zipper domains they form heterodimers, in vivo.

IKK-alpha knockout mice die shortly after birth and exhibit developmental abnormalities: shortened and truncated limbs, ears, heads and snouts due to a defect of differentiation of skin epidermal cells (keratinocytes). In general, Ikk-alpha seems to be involved in skeletal development. Interestingly, IL-1 and TNF-alpha still can activate NF-kB in cells from Ikk-alpha -/- mice!
Ikk-beta knockout mice-embryos die from excessive loss of hepatocytes due to apoptosis. Apoptosis appears to be induced by TNF-alpha since Ikk-beta and TNFR1 double knockout mice are not affected by hepatocyte apoptosis and embryonic death. Additionally, fibroblasts from Ikk-beta -/- mice undergo apoptosis in response to TNF-alpha, presumably due to a missing "survival" signal usually provided by NF-kB activation (May and Gosh, 1999, Science, 284: 271-273).