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The NF-kB Family of Transcription Factors andIts Regulation

Andrea Oeckinghaus1,2 and Sankar Ghosh1,2

1Department of Immunobiology and Department of Molecular Biophysics and Biochemistry, Yale UniversitySchool of Medicine, New Haven, Connecticut 06520

2Department of Microbiology and Immunology, Columbia University, College of Physicians and Surgeons,New York 10032

Correspondence: [email protected]

Nuclear factor-kB (NF-kB) consists of a familyof transcription factors that play critical roles ininflammation, immunity, cell proliferation, differentiation, and survival. Inducible NF-kBactivation depends on phosphorylation-induced proteosomal degradation of the inhibitorof NF-kB proteins (IkBs), which retain inactive NF-kB dimers in the cytosol in unstimulatedcells. The majority of the diverse signaling pathways that lead to NF-kB activation convergeon the IkB kinase (IKK) complex, which is responsible for IkB phosphorylation and is essen-tial for signal transduction to NF-kB. Additional regulation of NF-kB activity is achievedthrough various post-translational modifications of the core components of the NF-kB signal-ing pathways. In addition to cytosolic modifications of IKK and IkB proteins, as well as otherpathway-specific mediators, the transcription factors are themselves extensively modified.Tremendous progress has been made over the last two decades in unraveling the elaborateregulatory networks that control the NF-kB response. This has made the NF-kB pathway aparadigm for understanding general principles of signal transduction and gene regulation.

Following the identification of NF-kB(nuclear factor-kB) as a regulator of kB

light chain expression in mature B and plasmacells by Sen and Baltimore, inducibility of itsactivity in response to exogenous stimuli wasdemonstrated in various cell types (Sen et al.1986b; Sen et al. 1986a). Years of intenseresearch that followed demonstrated thatNF-kB is expressed in almost all cell types andtissues, and specific NF-kB binding sites arepresent in the promoters/enhancers of a largenumber of genes. It is now well-establishedthat NF-kB plays a critical role in mediating

responses to a remarkable diversity of externalstimuli, and thus is a pivotal element in multiplephysiological and pathological processes. Theprogress made in the past two decades in under-standing how different stimuli culminate inNF-kB activation and how NF-kB activation istranslated into a cell-type- and situation-specificresponse has made the NF-kB pathway a para-digm for understanding signaling mechanismsand gene regulation. Coupled with the largenumber of diseases in which dysregulation ofNF-kB has been implicated, the continuinginterest into the regulatory mechanisms that

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govern the activity of this important transcrip-tion factor can be easily explained.

Because of its ability to influence expressionof numerous genes, the activity of NF-kB istightly regulated at multiple levels. The pri-mary mechanism for regulating NF-kB isthrough inhibitory IkB proteins (IkB, inhibitorof NF-kB), and the kinase that phosphorylatesIkBs, namely, the IkB kinase (IKK) complex.A number of post-translational modificationsalso modulate the activity of the IkB and IKKproteins as well as NF-kB molecules themselves.In this article, we introduce the major playersin the NF-kB signaling cascade and describethe key regulatory steps that control NF-kBactivity. We highlight the basic principles thatunderlie NF-kB regulation, and many of thesetopics are discussed in depth in other articleson the subject.

NF-kB STIMULI AND kB-DEPENDENTTARGET GENES

NF-kB transcription factors are crucial playersin an elaborate system that allows cells toadapt and respond to environmental changes,

a process pivotal for survival. A large numberof diverse external stimuli lead to activation ofNF-kB and the genes whose expression is regu-lated by NF-kB play important and conservedroles in immune and stress responses, andimpact processes such as apoptosis, prolifer-ation, differentiation, and development.

Bacterial and viral infections (e.g., throughrecognition of microbial products by receptorssuch as the Toll-like receptors), inflamma-tory cytokines, and antigen receptor engage-ment, can all lead to activation of NF-kB,confirming its crucial role in innate and adap-tive immune responses. In addition, NF-kBactivation can be induced upon physical(UV- or g-irradiation), physiological (ischemiaand hyperosmotic shock), or oxidative stresses(Fig. 1) (Baeuerle and Henkel 1994; Haydenet al. 2006).

Consistent with the large number of signalsthat activate NF-kB, the list of target genes con-trolled by NF-kB is also extensive (Pahl 1999).Importantly, regulators of NF-kB such asIkBa, p105, or A20 are themselves NF-kB-dependent, thereby generating auto-regulatoryfeedback loops in the NF-kB response. Other

Antigen receptors

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TCRBCR

LPSdsRNA

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TCRa, bMHC I

TNFa, IL-1,IL-6, IL-12 IkBa, c-Rel,

p105G-CSF,M-CSF

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Serum amyloid protein,C3 complement

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IMMUNE RESPONSE/INFLAMMATION NF-kBREGULATION

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Cyclins,growthfactors

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TNFaIL-1

UV-, g-radiationreactive oxygenintermediates (ROI)

Cytokines

Genotoxic stress

Figure 1. NF-kB stimuli and target genes. NF-kB acts as a central mediator of immune and inflammatoryresponses, and is involved in stress responses and regulation of cell proliferation and apoptosis. Therespective NF-kB target genes allow the organism to respond effectively to these environmental changes.Representative examples are given for NF-kB inducers and kB-dependent target genes.

A. Oeckinghaus and S. Ghosh

2 Cite this article as Cold Spring Harb Perspect Biol 2009;1:a000034

NF-kB target genes are central components ofthe immune response, e.g., immune receptorsubunits or MHC molecules. Inflammatoryprocesses are controlled through NF-kB-depen-dent transcription of cytokines, chemokines,cell adhesion molecules, factors of the comple-ment cascade, and acute phase proteins. Thelist of kB-dependent target genes can befurther extended to regulators of apoptosis(antiapoptotic Bcl family members and inhibi-tor of apoptosis proteins/IAPs) and prolifer-ation (cyclins and growth factors), therebysubstantiating its role in cell growth, prolifer-ation, and survival (Chen and Manning 1995;Kopp and Ghosh 1995; Wissink et al. 1997).Furthermore, crucial functions of NF-kB inembryonic development and physiology of thebone, skin, and central nervous system add tothe importance of this pleiotropic transcriptionfactor (Hayden and Ghosh 2004).

Because so many key cellular processes suchas cell survival, proliferation, and immunity areregulated through NF-kB-dependent transcrip-tion, it is not surprising that dysregulation ofNF-kB pathways results in severe diseases suchas arthritis, immunodeficiency, autoimmunity,and cancer (Courtois and Gilmore 2006).

THE NF-kB TRANSCRIPTIONFACTOR FAMILY

The NF-kB transcription factor family inmammals consists of five proteins, p65 (RelA),RelB, c-Rel, p105/p50 (NF-kB1), and p100/52(NF-kB2) that associate with each other toform distinct transcriptionally active homo-and heterodimeric complexes (Fig. 2). Theyall share a conserved 300 amino acid longamino-terminal Rel homology domain (RHD)(Baldwin 1996; Ghosh et al. 1998), and se-quences within the RHD are required for di-merization, DNA binding, interaction withIkBs, as well as nuclear translocation. Crystalstructures of p50 homo- and p50/p65 heterodi-mers bound to DNA revealed that the amino-terminal part of the RHD mediates specificDNA binding to the NF-kB consensus sequencepresent in regulatory elements of NF-kB targetgenes (50 GGGPuNNPyPyCC-30), whereas the

carboxy-terminal part of the RHD is mainlyresponsible for dimerization and interactionwith IkBs (Ghosh et al. 1995; Muller et al.1995; Chen et al. 1998).

In most unstimulated cells, NF-kB dimersare retained in an inactive form in the cytosolthrough their interaction with IkB proteins.Degradation of these inhibitors upon theirphosphorylation by the IkB kinase (IKK)complex leads to nuclear translocation of NF-kB and induction of transcription of targetgenes. Although NF-kB activity is inducible inmost cells, NF-kB can also be detected as a con-stitutively active, nuclear protein in certain celltypes, such as mature B cells, macrophages,neurons, and vascular smooth muscle cells, aswell as a large number of tumor cells.

Through combinatorial associations, theRel protein family members can form up to 15different dimers. However, the physiologicalexistence and relevance for all possible dimericcomplexes has not yet been demonstrated. Thep50/65 heterodimer clearly represents themost abundant of Rel dimers, being found inalmost all cell types. In addition, dimeric com-plexes of p65/p65, p65/c-Rel, p65/p52, c-Rel/c-Rel, p52/c-Rel, p50/c-Rel, p50/p50, RelB/p50, and RelB/p52 have been described, someof them only in limited subsets of cells(Hayden and Ghosh 2004). RelB seems to beunique in this regard as it is only found inp50- or p52-containing complexes (Rysecket al. 1992; Dobrzanski et al. 1994). p50/c-Reldimers are the primary component of the con-stitutively active NF-kB observed in mature Bcells (Grumont et al. 1994b; Miyamoto et al.1994). The NF-kB family of proteins can befurther divided into two groups based on theirtransactivation potential because only p65,RelB, and c-Rel contain carboxy-terminaltransactivation domains (TAD) (Fig. 2). RelBis unique in that it requires an amino-terminalleucine zipper region in addition to its TAD tobe fully active (Dobrzanski et al. 1993). p50and p52 are generated by processing of the pre-cursor molecules p105 and p100, respectively.The amino-termini of these precursorscontain the RHDs of p50 or p52, followed bya glycine rich region (GRR) and multiple

The NF-kB Family of Transcription Factors and Its Regulation

Cite this article as Cold Spring Harb Perspect Biol 2009;1:a000034 3

copies of ankyrin repeats that are characteristicfor the IkB protein family. Therefore, not allcombinations of Rel dimers are transcription-ally active: DNA-bound p50 and p52 homo-and heterodimers have been found to represskB-dependent transcription, most probablyby preventing trancriptionally active NF-kBdimers from binding to kB sites, or through

recruitment of deacetylases to promoterregions (Zhong et al. 2002). An intriguingfeature of p50 and p52 is their ability to associ-ate with atypical IkB proteins such as IkBz andBcl-3, which most likely provide transcriptionalactivation properties. Because of their carboxy-terminal ankyrin repeats, the precursors p105and p100 can also inhibit nuclear localization

p65

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DD

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NF-kB2

NF-kB1

Figure 2. Members of the NF-kB, IkB, and IKK protein families. The Rel homology domain (RHD) ischaracteristic for the NF-kB proteins, whereas IkB proteins contain ankyrin repeats (ANK) typical for thisprotein family. The precursor proteins p100 and p105 can therefore be assigned to and fulfill the functions ofboth the NF-kB and IkB protein families. The domains that typify each protein are indicated schematically.CC, coiled-coil; DD, death domain; GRR, glycine-rich region; HLH, helix-loop-helix; IKK, IkB kinase; LZ,leucine-zipper; NBD, NEMO binding domain; PEST, proline-, glutamic acid-, serine-, and threonine-richregion; TAD, transactivation domain; ZF, zinc finger.



and transcriptional activity of NF-kB dimersthat they are associated with, and hence can beclassified as IkB proteins.

The specific physiological role of individualNF-kB dimers remains to be fully understood.Crystallographic structural analysis has helpeddelineate some of the principles that governselection of particular kB-sites by NF-kBdimers, and biochemical experiments revealedpreferential affinities of individual dimers forparticular sites in vitro. However, knockoutstudies demonstrated that biochemical affinitydata do not fully explain the specificity of NF-kB dimers in vivo and it has become obviousthat dimer selection cannot be reduced to thekB sequence alone (Udalova et al. 2002;Hoffmann et al. 2003; Hoffmann et al. 2006;Schreiber et al. 2006; Britanova et al. 2008).The complex structures of target promoterstogether with the potential of particular NF-kB dimers to trigger specific protein–proteininteractions at the promoter is likely to bemore important as transcriptional controlinvolves the concerted action of the transcrip-tion factor, coactivators, and corepressors inthe context of nucleosomal chromatin. The kBsequence may also affect the conformationof the bound NF-kB dimers and thusinfluence their ability to recruit coactivators.Furthermore, the kinetics of NF-kB dimerbinding and release from DNA is based on thepresence of sufficient IkBs, as well as the natureof post-translational modifications of NF-kBsubunits. The combinatorial diversity of theNF-kB dimers certainly adds to their ability toregulate distinct but overlapping sets of genesby influencing all these aspects of kB siteselection.

IkB PROTEINS

In most resting cells, the association of NF-kBdimers with one of the prototypical IkB pro-teins IkBa, IkBb, and IkB1, or the precursorRel proteins p100 and p105, determines theircytosolic localization, which makes the IkBproteins essential for signal responsiveness.In addition, expression of two atypical mem-bers of this protein family, namely Bcl-3 and

IkBz, can be induced upon stimulation andregulate the activity of NF-kB dimers in thenucleus. Finally, an alternative transcript ofthe p105 gene, apparently only expressed insome murine lymphoid cells, has been iden-tified and termed IkBg, but its physiologicalfunction remains unclear (Inoue et al. 1992;Gerondakis et al. 1993; Grumont and Geron-dakis 1994a).

All IkB proteins are characterized by thepresence of five to seven ankyrin repeat motifs,which mediate their interaction with the RHDof NF-kB proteins (Fig. 2). In general, individ-ual IkB proteins are thought to preferentiallyassociate with a particular subset of NF-kBdimers, however surprisingly little experimentalevidence concerning this important subjectexists. IkBa and IkBb are primarily believedto inhibit c-Rel and p65-containing complexes,with IkBa having a higher affinity for p65:p50than for p65:p65 complexes (Malek et al.2003). RelB exclusively binds p100, whereasBcl-3 and IkBz preferentially associate withhomodimers of p50 and p52 (Hoffmann et al.2006).

The Canonical IkBs

The amino-terminal signal responsive regions(SRR) of the typical IkB proteins IkBa, IkBb,and IkB1 contain conserved serine residues(e.g., Ser32 and Ser36 in IkBa) that are targetedby the IKKb subunit of the IKK complex in thecourse of canonical signaling to NF-kB (seealso paragraph: The IKK complex). Phosphor-ylated IkB proteins are then modified withK48-linked ubiquitin chains by ubiquitinligases of the SCF or SCRF (Skp1-Culin-Roc1/Rbx1/Hrt-1-F-box) family, upon recognitionof the DSPGXXSP motif of phosphorylatedIkB proteins through their receptor subunitb-TRCP (b-tranducin repeat containingprotein) (Yaron et al. 1998; Fuchs et al. 1999;Hatakeyama et al. 1999; Kroll et al. 1999; Wuand Ghosh 1999). Ubiquitination of IkBsultimately results in their degradation by theproteasome.

IkBa is the best-studied member of theIkB family of proteins and displays all defining



characteristics of an NF-kB inhibitor. The ca-nonical p65/p50 heterodimer is primarilyassociated with IkBa and rapid, stimulus-induced degradation of IkBa is critical fornuclear translocation and DNA binding ofNF-kB p65/p50. The traditional model of IkBfunction posits that IkB proteins sequesterNF-kB in the cytosol; however, the actual situ-ation is more complex. Crystallographic struc-tural analysis of an IkBa:p65:p50 complexrevealed that the IkBa molecule masks onlythe nuclear localization sequence (NLS) ofp65, whereas the NLS of p50 remains accessible(Huxford et al. 1998; Jacobs and Harrison1998). This exposed NLS together with thenuclear export sequence (NES) present inIkBa leads to constant shuttling of IkBa:NF-kB complexes between the nucleus andcytosol, despite a steady-state localization thatappears to be exclusively cytosolic. Degradationof IkBa shifts this dynamic equilibrium becauseit eliminates contribution of the NES inIkBa and exposes the previously masked NLSof p65. As active NF-kB promotes IkBa ex-pression, an important negative feedback regu-latory mechanism is generated that criticallyinfluences the duration of the NF-kB response.Consistent with such a role, termination of theNF-kB response upon TNFa stimulation isnotably delayed in the case of IkBa deficiency(Klement et al. 1996).

The roles of the other IkB family membersare less well established. Although IkBb showsNF-kB binding specificity similar to IkBa, italso possesses a number of unique properties.In contrast to IkBa, IkBb:NF-kB complexesdo not undergo nuclear-cytoplasmic shuttling(Tam and Sen 2001) and IkBb can associatewith NF-kB complexes bound to DNA, suggest-ing a possible regulatory function in the nucleus(Thompson et al. 1995; Suyang et al. 1996). Eventhough IkBb is degraded and resynthesized in astimulus-dependent manner—although withconsiderably delayed kinetics compared toIkBa—IkBb deletion does not affect thekinetics of NF-kB responses significantly(Hoffmann et al. 2002). Surprisingly, knockingin of IkBb to replace IkBa, thereby placingIkBb expression under the regulation of the

IkBa promoter, leads to normal kinetics ofNF-kB activation and termination despite thefact that IkBb normally does not function inthe early dampening of the NF-kB response(Cheng et al. 1998). This suggests that the func-tional characteristics of the different canonicalIkBs are at least in part caused by temporal dis-tinctions in their resynthesis based on thespecific transcriptional regulation of their pro-moters (Hoffmann et al. 2002).

Similar to IkBb, IkB1 is also degraded andresynthesized in response to NF-kB signalingwith significantly delayed kinetics (Kearns et al.2006). Besides, IkB1 expression and functionseem to be limited to cells of the hematopoeticlineage, reinforcing the hypothesis that distinctIkBs exert unique roles in NF-kB responses indifferent cellular scenarios.

The Precursor IkBs

The precursor proteins p100 and p105 areexceptional in their ability to function as Relprotein inhibitors, because they can dimerizewith other NF-kB molecules via their RHDs,whereas their carboxy-terminal ankyrin repeatsserve the function of IkB proteins (Rice et al.1992; Naumann et al. 1993; Solan et al. 2002).Although their overall sequence and organi-zation is similar, generation of p50 andp52 from their respective precursors occursthrough distinct processing mechanisms. p105is constitutively processed, resulting in a presetratio of p105 and p50 containing Rel dimers ineach cell. This constitutive processing has beensuggested to occur co- or post-translationally(Lin et al. 1998; Moorthy et al. 2006) anddepends on a GRR located carboxy-terminal tothe RHD that serves as termination signal forthe proteosomal proteolysis (Lin and Ghosh1996). However, p105 can, like IkBa, alsoundergo complete degradation upon phos-phorylation of carboxy-terminal serine residuesby IKKb, a process that is favored when p105 isbound to other NF-kB subunits (Harhaj et al.1996; Heissmeyer et al. 1999; Cohen et al. 2001;Cohen et al. 2004; Perkins 2006). Interestingly,this event has been suggested to be independentof ubiquitination (Moorthy et al. 2006).



p100 processing represents a predominantlystimulus-dependent event that defines the non-canonical NF-kB activation pathway (see alsoparagraph: The IKK complex). It is regulatedthrough phosphorylation of p100 by the IKKasubunit of the IKK complex, which results inubiquitination and subsequent partial degra-dation of p100 by the proteasome, releasingtranscriptionally active p50/RelB complexes(Senftleben et al. 2001). However, p100 canalso regulate p65-containing complexes down-stream of IKKa and limit p65-dependentresponses in T cells in a negative feedback loopsimilar to IkBa, thereby acting as an inhibitorin canonical signaling pathways (Ishimaruet al. 2006; Basak et al. 2007). Constitutiveprocessing of p100 has only been described tooccur at a low level in certain cell types(Heusch et al. 1999). The ability of p100 to regu-late RelB is of particular importance, as RelB-containing dimers exclusively associate withp100 and require p100 binding for stabilization(Solan et al. 2002). Therefore, p100 is able toaffect NF-kB transcriptional responses at a levelthat extends beyond its role as an IkB protein.

The Atypical IkBs

The role of the atypical IkB protein Bcl3 inNF-kB signaling is not fully understood andits mechanism of action is still unclear. Bcl3contains a TAD and is found primarily in thenucleus, where it associates with p50 and p52homo- or heterodimers. Bcl3 has been sug-gested to provide transcriptional activationfunction on otherwise repressive p50 or p52homodimers (Bours et al. 1993). Furthermore,Bcl3 may remove repressive dimers from pro-moters, thereby allowing transcriptionallyactive p65/50 to access and facilitate transcrip-tion in an indirect manner through release ofrepression (Wulczyn et al. 1992; Hayden andGhosh 2004; Perkins 2006). Conversely, induc-tion of Bcl3 expression has been shown toinhibit subsequent NF-kB activation. In sucha model, Bcl3 may stabilize repressing dimersat kB sites, thereby preventing access of TADcontaining NF-kB dimers and consequenttranscription (Wessells et al. 2004; Carmody

et al. 2007). The potential of Bcl3 to eitherpromote or impede transcription has beenshown to depend to some extent on differentpost-translational modifications (Nolan et al.1993; Bundy and McKeithan 1997; Massoumiet al. 2006).

Within the IkB family, IkBz is most similarto Bcl3. It is not constitutively expressed, butinduced upon engagement of IL-1 and TLR4receptors and is localized to the nucleus(Yamazaki et al. 2005). For the most part, IkBzassociates with p50 homodimers. Expressionof a subset of NF-kB target genes, includingIL-6, is not induced in IkBz-deficient cellsupon IL-1 or LPS stimulation. IkBz is hy-pothesized to act as a coactivator of p50 homo-dimers even though IkBz does not contain anapparent TAD (Yamamoto et al. 2004). IkBzhas also been suggested to negatively regulatep65-containing complexes and may thus alsobe able to selectively activate or impedespecific NF-kB activity (Yamamoto et al. 2004;Motoyama et al. 2005).

It is obvious that our perception of IkBproteins has changed significantly over theyears from relatively simple inhibitory mol-ecules to complex regulators of NF-kB-drivengene expression. As described previously,IkBs can influence the makeup of cellularNF-kB through specific stabilization of un-stable dimers, stabilize DNA-bound dimers,thereby repressing or prolonging transcrip-tional responses, or even act as transcriptionalcoactivators. Therefore, it is probably moreappropriate to consider IkBs as multifacetedNF-kB regulators.

THE IKK COMPLEX: CANONICAL ANDNON-CANONICAL SIGNALING TO NF-kB

The first biochemical experiments assigned thecytosolic IkB kinase activity to a large proteincomplex of 700–900 kDa capable of specificallyphosphorylating IkBa on serines 32 and 36.Purification of this complex revealed thepresence of two catalytically active kinases,IKKa and IKKb, and the regulatory subunitIKKg (NEMO) (Chen et al. 1996; Mercurioet al. 1997; Woronicz et al. 1997; Zandi et al.



1997). IKKa and IKKb are ubiquitously ex-pressed and contain an amino-terminal kinasedomain, a leucine zipper, and a carboxy-terminal helix-loop-helix domain (HLH)(Fig. 2). The leucine zipper is responsible fordimerization of the kinases and mutations inthis region render the kinases inactive, whereasthe HLH is dispensable for dimerization, butessential for optimal kinase activity (Zandiet al. 1998). The carboxy-terminal portionsof IKKa and -b are critical for their interac-tion with the regulatory subunit IKKg, whichis mediated by a hexapeptide sequence(LDWSWL) on IKKs termed the NEMO bind-ing domain (NBD) (May et al. 2000; Mayet al. 2002). Activation of the IKK complex isdependent on the phosphorylation of twoserines in the sequence motif SLCTS of theT-loop regions in at least one of the IkB kinases(Mercurio et al. 1997; Ling et al. 1998).Whether these phosphorylation events occurthrough trans-autophosphorylation or throughphosphorylation by an upstream kinase con-tinues to be debated (Hayden and Ghosh 2004;Scheidereit 2006).

The physiological significance of IKKa andIKKb has been analyzed extensively in knock-out mice. These studies have revealed twogeneral types of signal propagation pathwaysto NF-kB activation, which are distinct inrespect to the inducing stimuli, the IKK sub-units involved, and the NF-kB/IkB substratestargeted (Fig. 3). In the canonical NF-kBpathway, which is induced by inflammatorycytokines, pathogen-associated molecules, andantigen receptors, IKKb is both necessary andsufficient to phosphorylate IkBa or IkBb inan IKKg-dependent manner. Thus, IKKb-deficient mice exhibit embryonic lethalitycaused by severe liver apoptosis because ofdefective TNF signaling to NF-kB in the devel-oping liver (Beg et al. 1995; Li et al. 1999a;Li et al. 1999b). Congruently, IKKb-deficientcells were shown to be unable to activateNF-kB upon stimulation with proinflamma-tory cytokines such as TNFa or interleukin-1(IL-1) (Li et al. 1999a; Li et al. 1999b). Therole of IKKa in canonical NF-kB signalingremains unclear; however, more recent studies

have established a role for IKKa in regulatinggene expression by modifying the phosphory-lation status of histones and p65 (Anest et al.2003; Yamamoto et al. 2003; Chen and Greene2004).

In contrast to the canonical pathway, thenon-canonical pathway that is induced by speci-fic members of the TNF cytokine family, such asBAFF, lymphotoxin-b, or CD40 ligand relieson IKKa, but not IKKb or IKKg. IKKa isbelieved to selectively phosphorylate p100associated with RelB (Senftleben et al. 2001;Scheidereit 2006). Although the upstreamsignal propagation events are poorly under-stood, IKKa clearly requires activation of NIK(NF-kB-inducing kinase), which seems to func-tion as both an IKKa-activating kinase as well asa scaffold linking IKKa and p100 (Xiao et al.2004). Phosphorylation of p100 entails recruit-ment of SCFbTrCP and subsequent polyubiq-uitination of Lys855, which is situated in apeptide sequence homologous to that targetedin IkBa (Fong and Sun 2002; Amir et al.2004). Similar to p105 processing, the GRR ofp100 is required for partial processing to gener-ate p52. In addition to non-canonical signalingto NF-kB, IKKa regulates developmental pro-cesses that are both dependent, as well as inde-pendent, of NF-kB-induced gene transcription(Sil et al. 2004).

POST-TRANSLATIONAL MODIFICATIONSOF NF-kB

Given the wide range of biological processes thatare affected by NF-kB, and the disastrous conse-quences of dysregulated NF-kB signaling, intri-cate and highly regulated mechanisms exist forcontrolling NF-kB activity. These added layersof regulation involve a variety of posttrans-lational modifications of various core com-ponents of this signaling pathway, therebyinfluencing NF-kB activity at multiple levels.The critical phosphorylation and ubiquitina-tion events of IkB proteins and the IKKcomplex have been described previously. Inaddition, the NF-kB subunits themselvesare subject to a wide range of regulatory



protein modifications, including phosphory-lation, ubiquitination, or acetylation (Perkins2006). Some of these modifications also rep-resent important means for cross talk betweendifferent signaling pathways (Perkins 2007).

Importantly, degradation of IkBs andnuclear translocation of the NF-kB dimersalone are not sufficient in eliciting a maximalNF-kB response, which has been shown toalso critically depend on protein modificationsof NF-kB subunits. Post-translational modifi-cations of p65 have been most thoroughlyinvestigated in this context. Phosphorylationby protein kinase A (PKA) was the first

modification of p65 to be recognized and hassince been demonstrated to be critical for p65-driven gene expression of many but not alltarget genes (Naumann and Scheidereit 1994;Neumann et al. 1995; Chen and Greene 2004).The catalytic subunit of PKA (PKAc) exists incomplex with cytosolic IkB:NF-kB complexesand is activated upon IkB degradation to phos-phorylate Ser276 of p65. p50 and c-Rel are alsomodified by PKAc at equivalent sites to RelA(Perkins 2006). In p65, Ser 276 phosphory-lation is thought to break an intramolecularinteraction between the carboxy-terminal partof the protein and the RHD, thereby facilitating

NEMOP

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Figure 3. Canonical and non-canonical signaling to NF-kB. The canonical pathway is, e.g., induced by TNFa,IL-1, or LPS and uses a large variety of signaling adaptors to engage IKK activity. Phosphorylation of serineresidues in the signal responsive region (SRR) of classical IkBs by IKKb leads to IkB ubiquitination andsubsequent proteosomal degradation. This results in release of the NF-kB dimer, which can then translocateto the nucleus and induce transcription of target genes. The non-canonical pathway depends on NIK (NF-kB-inducing kinase) induced activation of IKKa. IKKa phosphorylates the p100 NF-kB subunit, whichleads to proteosomal processing of p100 to p52. This results in the activation of p52-RelB dimers, whichtarget specific kB elements.



DNA binding and enabling interaction with thetranscriptional coactivators p300 and CBP(CREB-binding protein) (Zhong et al. 1998;Zhong et al. 2002). In addition to PKA, MSK1and MSK2 (mitogen- and stress-activatedprotein kinase) have been shown to targetSer276, and in doing so possibly mediatingcross talk with the ERK and p38 MAPK path-ways (Vermeulen et al. 2003; Perkins 2006).Several additional phosphorylation eventshave been described but the physiologicalconsequences in most cases remain less clear.Ser536 within the TAD of p65 seems to be aphosphorylation site that is targeted by severalkinases, including IKKb. Numerous effectshave been ascribed to this modification, includ-ing regulation of p65 nuclear localization andCBP/p300 interaction (Chen et al. 2005;Perkins 2006). CK2 has been shown to induciblymodify Ser529; however, it remains unclearwhether this phosphorylation affects p65 tran-scriptional activity (Wang et al. 2000). Ser311can be phosphorylated by the atypical proteinkinase PKCz in response to TNF stimulation,which has also been demonstrated to positivelyinfluence p65:CBP interaction (Duran et al.2003). Interestingly, of the four phosphory-lation sites mentioned above, two are found inthe RHD (Ser276 and Ser311), whereas theother two are located within the TAD (Ser529and Ser 536).

The diversity of the p65 phosphorylationsites detected so far suggests that possibly evenmore modification sites will be discovered inthe future, in particular in other NF-kB sub-units. It is likely that these modificationswould serve to fine-tune NF-kB transcriptionalactivity, e.g., in determining target-gene speci-ficity and timing of gene expression, ratherthan acting as simple on–off switches.

p65 can also be inducibly acetylated atdifferent sites (Lys122, 123, 218, 221, and 310)with variable effects on its activity, probablyby the action of CBP/p300 and associatedhistone acetyltransferases (HAT). While acety-lation of lysines 218, 221, and 310 has beenshown to promote p65 transactivation, acety-lated lysines 122 and 123 act in an inhibitorymanner. Acetylation at Lys310, promoted by

phosphorylation of Ser276, has been shown toaugment transcriptional activity without affect-ing DNA or IkB binding, presumably throughgenerating a binding site for a coactivatorprotein. In contrast, acetylated Lys221 impedesassociation with IkBa, thereby enhancingDNA binding (Chen et al. 2002; Chen andGreene 2004). Different histone deacetylases(HDAC) such as HDAC-3 and SIRT1 have beenimplicated in removing p65 acetylations (Chenand Greene 2004; Yeung et al. 2004).

TERMINATION OF THE NF-kB RESPONSE

Our knowledge about the mechanisms leadingto NF-kB activation far exceeds what we knowabout the regulatory mechanisms that deter-mine its inactivation. The best studied andwell accepted mechanism for termination ofthe NF-kB response involves the resynthesis ofIkB proteins induced by activated NF-kB(Pahl 1999). Newly synthesized IkBa canenter the nucleus, remove NF-kB from theDNA, and relocalize it to the cytosol (Haydenand Ghosh 2004). Also, the precursors p105and p100, as well as c-Rel and RelB, are NF-kBinducible genes, and hence the composition ofNF-kB complexes in a cell can vary over time.Significant progress has also been made inunderstanding how negative regulators influ-ence signaling upstream of the IKK complex(Hayden and Ghosh 2004; Krappmann andScheidereit 2005; Hacker and Karin 2006). Inthe recent years, additional inhibitory mecha-nisms have been identified that function laterin the pathway and directly affect the active,DNA-bound NF-kB. These include post-translational modifications of NF-kB subunitsand have been shown to participate in shuttingoff the NF-kB response by altering cofactorbinding or mediating displacement and degra-dation of NF-kB dimers. Because acetylationof p65 has been demonstrated to negativelyimpact its DNA-binding affinity (Kiernanet al. 2003), regulation of p65 associationwith HATs and HDACs influences terminationof the NF-kB response (Ghosh and Hayden2008). An alternative scenario involvesubiquitin-dependent proteosomal degradation



of promoter-bound p65, facilitated by the ubiq-uitin ligase SOCS-1 (Ryo et al. 2003; Saccaniet al. 2004). In parallel, the nuclear ubiquitinligase PDLIM2 has been shown to target p65for ubiquitination and proteasomal degra-dation, as well as to transport p65 to promyelo-cytic leukemia (PML) nuclear bodies, therebysupporting transcriptional silencing (Tanakaet al. 2007). In addition, IKKa has been demon-strated to promote p65 and c-Rel turnover andremoval from proinflammatory gene promotersin macrophages (Lawrence et al. 2005). Finally,NF-kB subunits have been shown to be inhib-ited by oxidation and alkylation of a redox-sensitive cysteine located in the DNA-bindingloop (Perkins 2006). This list is far from beingcomplete and it seems that we have just begunto understand the complex network of modi-fications regulating NF-kB transcriptionalactivity in the nucleus. As a detailed knowledgeof these mechanisms is crucial for our under-standing of the function of NF-kB, this area ofinvestigation is likely to continue to attract sig-nificant attention in the future.

CONCLUSIONS

The transcription factors of the NF-kB familycontrol the expression of a large number oftarget genes in response to changes in theenvironment, thereby helping to orchestrateinflammatory and immune responses. As aber-rant NF-kB activation underlies various diseasestates, precise activation and termination ofNF-kB is ensured by multiple regulatory pro-cesses. It is fair to say that the IkB proteins rep-resent one of the primary means of NF-kBregulation, although their versatile effects onNF-kB activity make them more complex thansimple inhibitory proteins. A diverse array ofpost-translational modifications of IKK, IkB,and NF-kB proteins provides the means to fine-tune the NF-kB response at multiple levels.Tremendous progress has been made in under-standing the regulatory mechanisms shapingthe NF-kB response, yet it is striking howmuch still remains to be discovered. Because ofthe well-characterized links between NF-kBand diseases like cancer or arthritis, unraveling

the complexity of NF-kB regulation remains amajor goal to help act on specific steps of theNF-kB pathway, thereby avoiding the risk ofharmful side effects that could result fromgeneral inhibition of NF-kB.

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Documents

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identification of nf

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additional regulation

inducible nfkbactivation

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