Two Routes to NFkB

Nuclear factor-kappaB (NF-kB) is an evolutionary conserved family of transcription factors with key functions in many physiological and pathological processes (Ghosh and Karin 2002; Hayden and Ghosh 2004). The mammalian NF-kB family comprises five members, namely NFKB1 (p50/p105), NFKB2 (p52/p100), p65/RelA, cRel and RelB (Fig. 1). All NF-kB proteins are characterized by an N-terminal Rel homology domain (RHD) that confers hetero- or homodimerization between Rel proteins and sequence-specific DNA binding to gene regulatory elements in the chromatin. Only p65, c-Rel and RelB contain C-terminal transactivation domains (TAD) that are required for gene induction. In resting cells, NF-kB proteins are sequestered in the cytoplasm by inhibitory molecules (IkB: inhibitor of kappaB). The family of cytosolic IkBs consists of IkBœ, IkBP, IkBë as well as the NF-kB precursors NF-KB1/p105 and NF-KB2/p100 (Fig. 1). By an intramolecular cleavage, the precursors p105 and p100 give rise to transcription factors p50 and p52, respectively. As a common feature, all IkBs share an ankyrin repeat domain (ARD) that mediates interaction with the RHD and causes cytosolic retention by shielding the nuclear localization signals (NLS) of NF-kBs.

NF-kB activation is induced by a variety of stimulatory agents that include inflammatory cytokines, microbial pathogens, antigenic peptides, mitogens, morphogens and cellular stress. All upstream specific pathways converge at the IkB kinase (IKK) complex composed of two catalytic subunits IKKa and IKKp and a regulatory component IKKy/NEMO (NF-kB essential modifier) (Fig. 1). The first described canonical or type-1 NF-kB pathway is activated by all known physiological agents and involves site-specific serine phosphorylation of small IkBs or p105 by IKKp/IKKy in the cytosol (Fig. 2) (Li and Verma 2002). Phosphorylation targets IkBs for recognition by the SCF/pTRCP ubiquitin ligase complex that catalyzes the attachment of lysine 48-linked ubiquitin chains to the IkBs. Polyubiquitinated

NLS

AA 551

p65

□ l= lTADP

RelB q LZ

H

M ¥

557

NF-KB/Rel

c-Rel

-

619

p50

-

443

p52

-

447

SRD

Precursor

p100

p52

898

p105

P5C*- "

969

RHD SRD

IkBa

M0000ÜCP

317

IkB

IkBß

^HXXMXX>

361

IkBs

^^XXKXX^

500

ARD

AL

IKKa or IKKß

KD [] |=| LZ t=|HLH|=|b

745 or 756

IKK

NBD

IKKg/NEMO

KBD tj MOD/UBD t|ZF|]

419

RHD: Rel homology domain; TAD: Transactivation domain; LZ: Leucine zipper; AA: Amino acids; SRD: Signal

response domainARD: Ankyrin repeat domain; KD: Kinase domain; AL: Activation loop; HLH: Helix loop helix motif

KBD: kinase binding domain; MOD: Minimal oligomerization domain; UBD: Ubiquitin binding domain; ZF: Zinc fi

nger

Fig. 1 Scheme of NF-kB, IkB and IKK proteins. NF-kB transcription factors process an N-terminal Rel homology domain (RHD) that mediates dimerization, nuclear localization and DNA binding as a common characteristic. The C-terminal transactivation domain (TAD) of p65, RelB and c-Rel is needed for the recruitment of transcriptional coactivators. p100 and p105 represent precursor molecules that are proteolytically processed to p52 and p50, respectively. Inhibitory IkB proteins interact via their akyrin repeat domains with the C-terminal part of the RHD and prevent nuclear translocation of NF-kB by masking the nuclear localization sequence (NLS). The signal response domain (SRD) contains phosphor acceptor sites that are of critical function for stimulus-dependent degradation of IkB proteins. IKKa and IKKP are highly homologous and contain an activation loop (AL) within their N-terminal kinase domain (KD). In addition, leucine zippers and helix-loop-helix motifs are found in the C-terminus. Through the C-terminal Nemo-binding domain (NBD), IKKa and IKKP interact with the structurally unrelated IKKy/NEMO protein that is characterized by the N-terminal kinase-binding domain (KBD), a central minimal oligomerization/ubiquitin-binding domain (MOD/UBD), and a C-terminal zinc finger (ZF)

IkBs are subsequently degraded by the 26 S proteasome, which leads to the release and nuclear translocation of predominately p50/RelA- and p50/c-Rel-containing dimers. NF-kB activation by the canonical pathway is a rapid post-translational process that peaks within minutes and triggers induction of many immediate early antigens LPS

BAFF

TNFa

TNFa

BAFF

type 2

SCFß-TrCP

SCFß-TrCP

inflammation immunity cell growth apoptosis response: rapid, transient development differentiation delayed, sustained

Fig. 2 Type-1 and type-2 signaling to NF-kB. Extracellular stimuli activate NF-kB either by type-1 (IKKp/IKKy-dependent) or type-2 (IKKa-dependent) signaling. To date, all known inducers initiate type-1 signal transduction, leading to proteosomal degradation of cytosolic IkB proteins and subsequent nuclear translocation of predominately p65/p50 dimers that control the expression of target genes involved in inflammation, immunity and apoptosis. Type-1 signaling promotes a rapid response that is temporally restricted. A subset of NF-kB inducers is a strong activator of type-2 signaling, which induces the cleavage of the p100 precursor bound to RelB. Processing of p100 results in nuclear translocation of RelB/p52 dimers that activate target genes involved in development and differentiation. In contrast to type-1 signaling, type-2 responses are delayed and provoke sustained NF-kB activation genes involved in inflammation, immune response, apoptosis and proliferation. As a negative feedback mechanism, NF-kB induces expression of inhibitory IkB a proteins to assure the temporal restriction of canonical NF-kB activation.

A subset of tumor necrosis factor (TNF) receptor family members, e.g., B cell activating factor receptor (BAFFR), lymphotoxin P (LTP), CD40 or receptor activator of NF-kB (RANK) are potent inducers of the novel or type-2 NF-kB

pathway (Bonizzi and Karin 2004). However, also pathogenic stimulations by lipopolysccharides (LPS), Heliobacter pylori or human T cell leukemia virus (HTLV) have been shown to induce type-2 signaling (Fig. 2) (Mordmuller et al. 2003; Ohmae et al. 2005; Saccani et al. 2003). Type-2 activation promotes the processing of the p100 precursor to p52 and the generation of transcriptionally active p52/RelB complexes. Processing of p100 is initiated by NIK (NF-kB inducing kinase) dependent IKKa phosphorylation of p100. Phosphorylated p100 is processed in a way similar to IkB degradation in the type-1 pathway. However, in contrast to the rapid and transient induction of type-1 NF-kB complexes, p100 processing and p52/RelB activation are delayed for several hours and provoke sustained activation. Processing of p100 requires ongoing protein synthesis, which is rather in support of a co-translational mechanism (Mordmuller et al. 2003). The sustained activity of p52/RelB induces distinct sets of target genes that are often involved in the regulation of development and differentiation. However, a considerable crosstalk between both signaling systems can be anticipated (Grech et al. 2004; Lo et al. 2006).

The great interest in mechanistic details that govern the NF-kB signaling pathways stems from the many pathological conditions associated with deregulations in the NF-kB network. Constitutive IKK/NF-kB activation is associated with a variety of different neoplasias, e.g., colon cancer, mammary tumors, leukemias and lymphomas. Due to its anti-apoptotic and pro-proliferative activity, enhanced NF-kB activity is considered to be a central factor for neoplastic transformation and an attractive target in tumor therapy (Gilmore and Herscovitch 2006; Lin and Karin 2003). Other diseases related to constitutive NF-kB activity are autoimmune diseases such as rheumatoid arthritis or inflammatory airway diseases such as asthma or COPD (chronic obstructive pulmonary disease) (Burke 2003; Caramori et al. 2004).

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