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7/27/2019 The NF-kB Family of Transcription Factors and Its Regulation
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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 065202Department 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 rolesininflammation, immunity, cell proliferation, differentiation, and survival. Inducible NF-kB
activation 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 componentsof 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 was
demonstrated 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 and
tissues, 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, andthus is a pivotal element in multiple
physiological and pathological processes. Theprogress made in the past two decades in under-standing how different stimuli culminate in
NF-kB activation and how NF-kB activation is
translated into a cell-type- and situation-specificresponse has made the NF-kB pathway a para-digm for understanding signaling mechanisms
and gene regulation. Coupled with the largenumber of diseases in which dysregulation of
NF-kB has been implicated, the continuinginterest into the regulatory mechanisms that
Editors: Louis M. Staudt and Michael KarinAdditional Perspectives on NF-kB available at www.cshperspectives.org
Copyright# 2009 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a000034Cite this article as Cold Spring Harb Perspect Biol2009;1:a000034
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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 is
tightly regulated at multiple levels. The pri-mary mechanism for regulating NF-kB isthrough inhibitory IkB proteins (IkB, inhibitor
of NF-kB), and the kinase that phosphorylatesIkBs, namely, the IkB kinase (IKK) complex.A number of post-translational modifications
also modulate the activity of the IkB and IKKproteins as well as NF-kB moleculesthemselves.
In this article, we introduce the major playersin the NF-kB signaling cascade and describe
the key regulatory steps that control NF-kBactivity. We highlight the basic principles thatunderlie NF-kB regulation, and many of these
topics 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 to
adapt and respond to environmental changes,
a process pivotal for survival. A large number
of diverse external stimuli lead to activation of
NF-kB and the genes whose expression is regu-lated by NF-kB play important and conserved
roles 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-kB
activation 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 as
IkBa, p105, or A20 are themselves NF-kB-dependent, thereby generating auto-regulatoryfeedback loops in the NF-kB response. Other
Antigen receptors
Infection
NF-kB
TCR
BCR
LPSdsRNA
VCAM,ICAM
TCRa, bMHC I
TNFa, IL-1,IL-6, IL-12 IkBa, c-Rel,
p105G-CSF,
M-CSF
Bcl-XL,IAPs
Serum amyloid protein,C3 complement
Immunregulatory proteins
Cytokines
IMMUNE RESPONSE/INFLAMMATION NF-kBREGULATION
PROLIFERATION
APOTOSIS/CELL SURVIVAL
Rel/IkBproteins
Cyclins,growth
factors
Regulators ofapoptosis
TNFaIL-1
UV-, g-radiationreactive oxygen
intermediates (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
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NF-kB target genes are central components of
the immune response, e.g., immune receptor
subunits 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. The
list 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), thereby
substantiating 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 the
bone, skin, and central nervous system add tothe importance of this pleiotropic transcription
factor (Hayden and Ghosh 2004).Because so many key cellular processes such
as cell survival, proliferation, and immunity are
regulated 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 TRANSCRIPTION
FACTOR FAMILYThe 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 to
form distinct transcriptionally active homo-and heterodimeric complexes (Fig. 2). They
all 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 with
IkBs, as well as nuclear translocation. Crystalstructures of p50 homo- andp50/p65 heterodi-mers bound to DNA revealed that the amino-terminal part of the RHD mediates specificDNA binding to the NF-kB consensus sequence
present in regulatory elements of NF-kB targetgenes (50 GGGPuNNPyPyCC-30), whereas the
carboxy-terminal part of the RHD is mainly
responsible for dimerization and interaction
with 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 in
most cells, NF-kB can also be detected as a con-stitutively active, nuclear protein in certain cell
types, 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 15
different dimers. However, the physiologicalexistence and relevance for all possible dimericcomplexes has not yet been demonstrated. The
p50/65 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 be
unique 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 B
cells (Grumont et al. 1994b; Miyamoto et al.1994). The NF-kB family of proteins can be
further divided into two groups based on theirtransactivation potential because only p65,RelB, and c-Rel contain carboxy-terminal
transactivation domains (TAD) (Fig. 2). RelBis unique in that it requires an amino-terminal
leucine zipper region in addition to its TAD tobe fully active (Dobrzanski et al. 1993). p50
and p52 are generated by processing of the pre-cursor molecules p105 and p100, respectively.The amino-termini of these precursors
contain the RHDs of p50 or p52, followed bya glycine rich region (GRR) and multiple
The NF-kB Family of Transcription Factors and Its Regulation
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copies of ankyrin repeats that are characteristic
for the IkB protein family. Therefore, not all
combinations of Rel dimers are transcription-ally active: DNA-bound p50 and p52 homo-and heterodimers have been found to repress
kB-dependent transcription, most probablyby preventing trancriptionally active NF-kB
dimers from binding to kB sites, or through
recruitment of deacetylases to promoter
regions (Zhong et al. 2002). An intriguing
feature of p50 and p52 is their ability to associ-ate with atypical IkB proteins such as IkBz andBcl-3, which most likely provide transcriptional
activation properties. Because of their carboxy-terminal ankyrin repeats, the precursors p105
and p100 can also inhibit nuclear localization
p65
IkBa
IkBb
IkBe
IkBz
IKKa
IKKb
NEMOIKKg
Bcl-3
p100
p105
RelB
NF-kB/RelFa
mily
IkBFam
ily
IK
Kcomplex
c-Rel
p100/p52
p105/p50
RelA
RHD
RHD
RHD
RHD
RHD
RHD
ANK
ANKGRR
GRR
PEST
PEST
RHD
GRR
GRR ANK
TAD
TADLZ
DD
DD
DD
DD
NBD
NBDHLH
LZ
LZ
LZ ZCC2CC1
Kinase domain
Kinase domain
HLH
TAD
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.
A. Oeckinghaus and S. Ghosh
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and transcriptional activity of NF-kB dimers
that they are associated with, and hence can be
classified as IkB proteins.The specific physiological role of individual
NF-kB dimers remains to be fully understood.Crystallographic structural analysis has helpeddelineate some of the principles that govern
selection of particular kB-sites by NF-kBdimers, and biochemical experiments revealedpreferential affinities of individual dimers for
particular sites in vitro. However, knockoutstudies demonstrated that biochemical affinity
data do not fully explain the specificity of NF-kB dimers in vivo and it has become obvious
that 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 promoters
together with the potential of particular NF-
kB dimers to trigger specific proteinproteininteractions at the promoter is likely to be
more important as transcriptional control
involves the concerted action of the transcrip-tion factor, coactivators, and corepressors inthe context of nucleosomal chromatin. The kB
sequence may also affect the conformationof the bound NF-kB dimers and thus
influence their ability to recruit coactivators.Furthermore, the kinetics of NF-kB dimer
binding and release from DNA is based on thepresence of sufficient IkBs, as well as the nature
of post-translational modifications of NF-kBsubunits. The combinatorial diversity of theNF-kB dimers certainly adds to their ability to
regulate distinct but overlapping sets of genesby influencing all these aspects of kB site
selection.
IkB PROTEINS
In most resting cells, the association of NF-kB
dimers with one of the prototypical IkB pro-teins IkBa, IkBb, and IkB1, or the precursor
Rel 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 and
regulate the activity of NF-kB dimers in the
nucleus. Finally, an alternative transcript ofthe p105 gene, apparently only expressed in
some 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 the
presence of five to seven ankyrin repeat motifs,which mediate their interaction with the RHD
of NF-kB proteins (Fig. 2). In general, individ-ual IkB proteins are thought to preferentially
associate with a particular subset of NF-kBdimers, however surprisingly little experimentalevidence concerning this important subject
exists. 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, whereas
Bcl-3 and IkBz preferentially associate with
homodimers 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 the
course of canonical signaling to NF-kB (seealso paragraph: The IKK complex). Phosphor-
ylated IkB proteins are then modified withK48-linked ubiquitin chains by ubiquitin
ligases of the SCF or SCRF (Skp1-Culin-Roc1/Rbx1/Hrt-1-F-box) family, upon recognitionof the DSPGXXSP motif of phosphorylated
IkB proteins through their receptor subunit
b-TRCP (b-tranducin repeat containing
protein) (Yaron et al. 1998; Fuchs et al. 1999;Hatakeyama et al. 1999; Kroll et al. 1999; Wu
and 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
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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 for
nuclear translocation and DNA binding ofNF-kB p65/p50. The traditional model of IkBfunction posits that IkB proteins sequester
NF-kB in the cytosol; however, the actual situ-ation is more complex. Crystallographic struc-tural analysis of an IkBa:p65:p50 complex
revealed that the IkBa molecule masks onlythe nuclear localization sequence (NLS) of
p65, whereas the NLS of p50 remains accessible(Huxford et al. 1998; Jacobs and Harrison
1998). 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 that
appears to be exclusively cytosolic. Degradationof IkBa shifts this dynamic equilibrium becauseit eliminates contribution of the NES in
IkBa and exposes the previously masked NLS
of p65. As active NF-kB promotes IkBa ex-pression, an important negative feedback regu-latory mechanism is generated that critically
influences the duration of the NF-kB response.Consistent with such a role, termination of the
NF-kB response upon TNFa stimulation isnotably delayed in the case of IkBa deficiency
(Klement et al. 1996).The roles of the other IkB family members
are 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 associatewithNF-kB complexes bound to DNA, suggest-ing a possible regulatory function in the nucleus
(Thompsonet al.1995; Suyang et al.1996). Eventhough IkBb is degraded and resynthesized in a
stimulus-dependent manneralthough withconsiderably delayed kinetics compared to
IkBaIkBb deletion does not affect thekinetics of NF-kB responses significantly(Hoffmann et al. 2002). Surprisingly, knocking
in of IkBb to replace IkBa, thereby placingIkBb expression under the regulation of the
IkBa promoter, leads to normal kinetics of
NF-kB activation and termination despite the
fact 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 signaling
with significantly delayed kinetics (Kearns et al.2006). Besides, IkB1 expression and function
seem to be limited to cells of the hematopoeticlineage, reinforcing the hypothesis that distinctIkBs exert unique roles in NF-kB responses in
different cellular scenarios.
The Precursor IkBs
The precursor proteins p100 and p105 are
exceptional in their ability to function as Rel
protein inhibitors, because they can dimerizewith other NF-kB molecules via their RHDs,whereas their carboxy-terminal ankyrin repeats
serve 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 and
p52 from their respective precursors occursthrough distinct processing mechanisms. p105is constitutively processed, resulting in a preset
ratio of p105 and p50 containing Rel dimers ineach cell. This constitutive processing has been
suggested to occur co- or post-translationally(Lin et al. 1998; Moorthy et al. 2006) and
depends on a GRR located carboxy-terminal tothe RHD that serves as termination signal forthe proteosomal proteolysis (Lin and Ghosh
1996). However, p105 can, like IkBa, alsoundergo complete degradation upon phos-
phorylation of carboxy-terminal serine residuesby IKKb, a process that is favored when p105 is
bound 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).
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p100 processing represents a predominantly
stimulus-dependent event that defines the non-
canonical NF-kB activation pathway (see alsoparagraph: The IKK complex). It is regulated
through 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 can
also regulate p65-containing complexes down-stream of IKKa and limit p65-dependent
responses in T cells in a negative feedback loopsimilar to IkBa, thereby acting as an inhibitor
in canonical signaling pathways (Ishimaruet al. 2006; Basak et al. 2007). Constitutiveprocessing of p100 has only been described to
occur 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 to
affect 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 and
its mechanism of action is still unclear. Bcl3contains a TAD and is found primarily in thenucleus, where it associates with p50 and p52
homo- or heterodimers. Bcl3 has been sug-gested to provide transcriptional activation
function 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 and
Ghosh 2004; Perkins 2006). Conversely, induc-tion of Bcl3 expression has been shown to
inhibit subsequent NF-kB activation. In sucha model, Bcl3 may stabilize repressing dimersat kB sites, thereby preventing access of TAD
containing NF-kB dimers and consequenttranscription (Wessells et al. 2004; Carmody
et al. 2007). The potential of Bcl3 to either
promote or impede transcription has been
shown 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 similar
to 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. Expression
of a subset of NF-kB target genes, includingIL-6, is not induced in IkBz-deficient cells
upon IL-1 or LPS stimulation. IkBz is hy-pothesized to act as a coactivator of p50 homo-dimers even though IkBz does not contain an
apparent TAD (Yamamoto et al. 2004). IkBzhas also been suggested to negatively regulate
p65-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 multifaceted
NF-kB regulators.
THE IKK COMPLEX: CANONICAL ANDNON-CANONICAL SIGNALING TO NF-kB
The first biochemical experiments assigned thecytosolic IkB kinase activity to a large protein
complex 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 subunit
IKKg (NEMO) (Chen et al. 1996; Mercurioet al. 1997; Woronicz et al. 1997; Zandi et al.
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1997). IKKa and IKKb are ubiquitously ex-
pressed and contain an amino-terminal kinase
domain, 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, whereas
the HLH is dispensable for dimerization, butessential for optimal kinase activity (Zandiet al. 1998). The carboxy-terminal portions
of IKKa and -b are critical for their interac-tion with the regulatory subunit IKKg, which
is 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 two
serines 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 through
phosphorylation by an upstream kinase con-
tinues to be debated (Hayden and Ghosh 2004;Scheidereit 2006).
The physiological significance of IKKa and
IKKb has been analyzed extensively in knock-out mice. These studies have revealed two
general types of signal propagation pathwaysto NF-kB activation, which are distinct in
respect 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, and
antigen receptors, IKKb is both necessary andsufficient to phosphorylate IkBa or IkBb in
an IKKg-dependent manner. Thus, IKKb-deficient mice exhibit embryonic lethalitycaused by severe liver apoptosis because of
defective 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 activate
NF-kB upon stimulation with proinflamma-tory cytokines such as TNFa or interleukin-1(IL-1) (Li et al. 1999a; Li et al. 1999b). The
role of IKKa in canonical NF-kB signalingremains unclear; however, more recent studies
have established a role for IKKa in regulating
gene expression by modifying the phosphory-
lation status of histones and p65 (Anest et al.2003; Yamamoto et al. 2003; Chen and Greene
2004).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 is
believed 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 asbothan IKKa-activating kinaseas well as
a 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 targeted
in 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 signaling
to 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 thewide range of biological processes thatare affected by NF-kB, andthe disastrous conse-quences of dysregulated NF-kB signaling, intri-
cate and highly regulated mechanisms exist forcontrolling NF-kB activity. These added layers
of 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. In
addition, the NF-kB subunits themselvesare subject to a wide range of regulatory
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protein modifications, including phosphory-
lation, ubiquitination, or acetylation (Perkins2006). Some of these modifications also rep-resent important means for cross talk between
different signaling pathways (Perkins 2007).
Importantly, degradation of IkBs andnuclear translocation of the NF-kB dimersalone are not sufficient in eliciting a maximal
NF-kB response, which has been shown toalso critically depend on protein modifications
of NF-kB subunits. Post-translational modifi-cations of p65 have been most thoroughlyinvestigated in this context. Phosphorylation
by protein kinase A (PKA) was the first
modification of p65 to be recognized and has
since 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 part
of the protein and the RHD, thereby facilitating
NEMOP
P P
IKKaIKKa
NEMOP
IKKbIKKa
RelBRelB
p100
p52
RelBp52
P
p50 p65
IkR
UbUb
Proteasome
Ub P
p50p65
NIK
Canonical pathway(TNFa, IL-1, LPS)
Non-canonical pathway(CD40, BAFF, lymphotoxin-b)
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 translocate
to 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.
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DNA binding and enabling interaction with the
transcriptional coactivators p300 and CBP
(CREB-binding protein) (Zhong et al. 1998;Zhong et al. 2002). In addition to PKA, MSK1
and MSK2 (mitogen- and stress-activatedprotein kinase) have been shown to targetSer276, and in doing so possibly mediating
cross talk with the ERK and p38 MAPK path-ways (Vermeulen et al. 2003; Perkins 2006).Several additional phosphorylation events
have 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 several
kinases, including IKKb. Numerous effectshave been ascribed to this modification, includ-ing regulation of p65 nuclear localization and
CBP/p300 interaction (Chen et al. 2005;Perkins 2006). CK2has been shown to inducibly
modify Ser529; however, it remains unclearwhether this phosphorylation affects p65 tran-scriptional activity (Wang et al. 2000). Ser311
can be phosphorylated by the atypical protein
kinase 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 in
the RHD (Ser276 and Ser311), whereas theother two are located within the TAD (Ser529
and Ser 536).The diversity of the p65 phosphorylation
sites 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 transcriptional
activity, 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 to
augment transcriptional activity without affect-
ing DNA or IkB binding, presumably throughgenerating a binding site for a coactivator
protein. In contrast, acetylated Lys221 impedesassociation with IkBa, thereby enhancingDNA binding (Chen et al. 2002; Chen and
Greene 2004). Different histone deacetylases(HDAC) such as HDAC-3 and SIRT1 have beenimplicated in removing p65 acetylations (Chen
and 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 of
the NF-kB response involves the resynthesis ofIkB proteins induced by activated NF-kB(Pahl 1999). Newly synthesized IkBa can
enter the nucleus, remove NF-kB from the
DNA, and relocalize it to the cytosol (Haydenand Ghosh 2004). Also, the precursors p105and p100, as well as c-Rel and RelB, are NF-kB
inducible 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). In
the recent years, additional inhibitory mecha-nisms have been identified that function later
in 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 cofactor
binding or mediating displacement and degra-dation of NF-kB dimers. Because acetylation
of p65 has been demonstrated to negativelyimpact its DNA-binding affinity (Kiernan
et al. 2003), regulation of p65 associationwith HATs and HDACs influences terminationof the NF-kB response (Ghosh and Hayden
2008). An alternative scenario involvesubiquitin-dependent proteosomal degradation
A. Oeckinghaus and S. Ghosh
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of promoter-bound p65, facilitated by the ubiq-
uitin ligase SOCS-1 (Ryo et al. 2003; Saccani
et al. 2004). In parallel, the nuclear ubiquitinligase PDLIM2 has been shown to target p65
for ubiquitination and proteasomal degra-dation, as well as to transport p65 to promyelo-cytic leukemia (PML) nuclear bodies, thereby
supporting transcriptional silencing (Tanakaet al. 2007). In addition, IKKa has been demon-strated to promote p65 and c-Rel turnover and
removal 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 begun
to understand the complex network of modi-fications regulating NF-kB transcriptional
activity in the nucleus. As a detailed knowledgeof these mechanisms is crucial for our under-standing of the function of NF-kB, this area of
investigation is likely to continue to attract sig-
nificant attention in the future.
CONCLUSIONS
The transcription factors of the NF-kB family
control the expression of a large number oftarget genes in response to changes in the
environment, thereby helping to orchestrateinflammatory and immune responses. As aber-rant NF-kB activation underlies various disease
states, 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-kB
regulation, although their versatile effects onNF-kB activity make them more complex thansimple inhibitory proteins. A diverse array of
post-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 of
the well-characterized links between NF-kBand diseases like cancer or arthritis, unraveling
the complexity of NF-kB regulation remains a
major goal to help act on specific steps of the
NF-kB pathway, thereby avoiding the risk ofharmful side effects that could result from
general inhibition of NF-kB.
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14 Cite this article as Cold Spring Harb Perspect Biol2009;1:a000034