Introduction to NF-jB Players, Pathways, Perspectives

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REVIEW

Introduction to NF- j B: players, pathways, perspectives

TD Gilmore

Biology Department, Boston University, Boston, MA, USA

This article serves as an introduction to the collection of reviews on nuclear factor-kappaB (NF- j B). It provides anoverview of the discovery and current status of NF- j B as aresearch topic. Described are the structures, activities andregulation of the proteins in the NF- j B family of transcription factors. NF- j B signaling is primarilyregulated by inhibitor j B (I j B) proteins and the I j B

kinase complex through two major pathways: thecanonical and non-canonical NF- j B pathways. Theorganization and focus of articles included in the followingreviews are described, as well as likely future areas of research interest on NF- j B.Oncogene (2006) 25, 66806684. doi:10.1038/sj.onc.1209954

Keywords: NF-kappaB; IkappaB; IKK; Rel; signaltransduction; transcription factor

Introduction

With great pleasure, I have edited this second compila-tion of reviews on nuclear factor-kappa B (NF- kB)transcription factors and NF- kB signaling. It is approxi-mately 20 years since the coincident discovery of threeproteins classical NF- kB, v-Rel and Dorsal thatshow variable nucleo-cytoplasmic subcellular localiza-tion (Gilmore and Temin, 1986; Sen and Baltimore,1986; Baeuerle and Baltimore, 1988; Steward et al .,1988), which were soon demonstrated to be members of the same family of proteins (Stephens et al ., 1983;Wilhelmsen et al ., 1984; Steward, 1987; Ghosh et al .,1990; reviewed by Gilmore, 1990; Kieran et al ., 1990).

Notably, the biological processes immunity (NF- kB),oncogenesis (v-Rel) and development (Dorsal) inves-tigated in those early studies continue to be areas thatprovoke much of the interest in NF- kB.

Today, the study of NF- kB signaling is essentially anindustry, complete with website (www.nf-kb.org), patent(Baltimore et al ., 2002) and approximately 25 000publications. For those few unfamiliar with the NF- kBtranscription factor family, it includes a collectionof proteins conserved from (at least) the phylumCnidaria to humans. Among model organisms, these

transcription factors are notably absent in yeast andCaenorhabditis elegans ; in the latter, it is likely that NF-kB-like genes/proteins have been lost (given that theyare present in the more primitive organism, the seaanenome Nematostella vectensis (Sullivan et al ., 2006)).As described below, the term NF- kB is somewhatconfusing, as it can be used to refer to the superfamily

(of Rel and NF- kB proteins across species), thesubfamily (e.g., p100, p105 and Relish) or the specicp50-RelA heterodimer, which is the major NF- kB dimerin many cells.

The larger NF- kB family of proteins is composed of two subfamilies: the NF- kB proteins and the Relproteins. All of these proteins share a highly conservedDNA-binding/dimerization domain called the Relhomology domain (RHD) (Gilmore, 1990) (Figure 1).The Rel subfamily includes c-Rel, RelB, RelA (aka p65)and Drosophila Dorsal and Dif. The Rel proteinscontain C-terminal transactivation domains, which areoften not conserved at the sequence level across species,even though they can activate transcription in a varietyof species including yeast. Members of the NF- kBsubfamily (p105, p100 and Drosophila Relish) aredistinguished by their long C-terminal domains thatcontain multiple copies of ankyrin repeats, which act toinhibit these proteins. The NF- kB proteins becomeshorter, active DNA-binding proteins (p105 to p50 andp100 to p52) by either limited proteolysis or, possiblysometimes, by arrested translation. As such, members of the NF- kB subfamily are generally not activators of transcription, except when they form dimers withmembers of the Rel subfamily. Of note, the nuclearfactor of activated T-cell (NFAT) transcription factorsalso contain the RHD and bind to similar DNAsequences as the Rel/NF- kB dimers, but NFAT proteinsgenerally have not been found to form dimers with Reland NF- kB proteins.

Collectively, NF- kB transcription factor dimers bindto 910 base pair DNA sites ( kB sites), which have agreat amount of variability (5 0-GGGRNWYYCC-3 0; R,A or G; N, any nucleotide; W, A or T; Y, C or T). Allvertebrate NF- kB family proteins can form homodimersor heterodimers in vivo, except for RelB, which onlyforms heterodimers in vivo. This combinatorial diversitycontributes to the regulation of distinct, but over-lapping, sets of genes for at least three reasons: becausethe individual dimers have distinct DNA-bindingsite specicities for a collection of related kB sites,

because of the different proteinprotein interactions the

Correspondence: Dr TD Gilmore, Biology Department, BostonUniversity, 5 Cummington Street, Boston, MA 02215, USA.

E-mail: [email protected]

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individual dimers make at target promoters, andbecause of the gene activation prole of different dimersunder specic physiological conditions.

The activity of NF- kB is tightly regulated byinteraction with inhibitory I kB proteins. As with theNF- kB transcription factors, there are several I kBproteins (e.g., I kBa , IkBb , IkBg, IkBe and DrosophilaCactus) that have different afnities for individualNF- kB dimers. Individual I kBs are also regulatedslightly differently by phosphorylation and proteolysis,and show differences in their tissue-specic expressionpatterns. From biochemical studies and direct structuraldeterminations (reviewed by Chen and Ghosh, 1999), itis clear that I kBa makes multiple contacts with NF- kB.Generally these interactions cover the nuclear localiza-tion signal of the given NF- kB dimer and interfere withsequences involved in DNA binding.

Thus, in most cells, NF- kB is present as a latent,inactive, I kB-bound complex in the cytoplasm. Thereare two, and possibly three, pathways leading to theactivation of NF- kB (see Figure 2 for details). The twobest-described pathways are called either the cano-nical and non-canonical pathways or the classical

and alternative pathways, respectively. The common

upstream regulatory step in both of these pathways isactivation of an I kB kinase (IKK) complex, whichconsists of catalytic kinase subunits (IKK a and/orIKK b) and a scaffold, sensing protein called NF- kBessential modulator (NEMO). As such, activation of NF- kB dimers is the result of IKK-mediated, phospho-rylation-induced degradation of the I kB inhibitor, whichenables the NF- kB dimers to enter the nucleus andactivate specic target gene expression. In most cases,the activation of NF- kB is transient and cyclical in thepresence of continual inducer. For example, in mousebroblasts maintained in the presence of tumor necrosisfactor, nuclear NF- kB DNA-binding activity appearsand disappears approximately every 3060 min; thesecycles are due to repeated degradation and re-synthesisof I kB and the consequent activation and inactivation of NF- kB, respectively (Hoffmann et al ., 2006).

Organization of this collection of reviews

As with the 1999 issue of Oncogene Reviews on NF- kB(Gilmore, 1999), the choice of subjects to review wasdifcult. Because Oncogene is a journal dedicatedprimarily to the control of cell growth and oncogenesis,I decided to make the role of NF- kB in these processesthe focus of this issue. Nevertheless, to set the stage, itwas necessary to include several papers on the regula-tion of NF- kB: the regulation of NF- kB by upstreamIKK pathways (Scheidereit, 2006); the dynamics anddirect mechanisms of gene regulation by NF- kB(Hoffmann et al ., 2006); post-translational modica-tions that regulate components of the NF- kB pathway(Perkins, 2006); and the controversial role of reactiveoxygen species in the regulation of NF- kB activity(Bubici et al ., 2006). These papers are followed by threepapers in which the normal physiological roles of NF- kB are discussed. Minakhina and Steward describethe role of NF- kB in Drosophila development andimmunity; Hayden et al . describe what is known aboutthe extensive role that NF- kB plays in the mammalianimmune system; and Gerondakis et al . discuss what hasbeen learned about the physiological roles of NF- kBsignaling components through the use of mouse knock-outs and transgenics. The next four papers focus onsubjects related to NF- kB and pathogenesis/oncoge-nesis, describing the role of NF- kB in apoptosis (Duttaet al ., 2006) and cancer (Basseres and Baldwin, 2006),how mutations in NF- kB pathway genes are related tohuman disease (Courtois and Gilmore, 2006) and howviruses affect NF- kB signaling for their pathogenesis,their replication, or as part of detection by the hostorganism (Hiscott et al ., 2006). The nal two papersdescribe ways that the NF- kB pathway can be modu-lated, in some cases for possible therapeutic purposes:De Bosscher et al . describe the extensive literature oninhibitory crosstalk between the important steroidreceptor pathways and NF- kB, and Gilmoreand Herscovitch have unearthed an extensive collection

of inhibitors of NF- kB signaling and discuss their

RHD TAD

RHD

RelARelBc-RelDorsalDif

p50/p105

p52/p100Relish

Rel

NF- B

, , , Bcl-3, I BCactus

I B

LZ NBDHLHKinase, IKK

CC1 CC2 LZ ZF()NEMO

SS

SS

Figure 1 Structures of core NF- kB signaling proteins. Thegeneralized structures of the two subfamilies (Rel and NF- kB) of NF- kB transcription factors are shown at the top. All have aconserved DNA-binding/dimerization domain called the Relhomology domain (RHD), which also has sequences importantfor nuclear localization and I kB inhibitor binding. The C-terminalhalves of the Rel proteins have transcriptional activation domains(TAD). The C-terminal halves of the NF- kB subfamily proteinshave ankyrin repeat-containing inhibitory domains (red bars),which can be removed by proteasome-mediated proteolysis. Aswith the C-terminal domains of the NF- kB proteins, theindependent I kB proteins consist mainly of ankyrin repeats, andseveral (I kBa , IkBb , IkBe, IkBg) have two N-terminal serineresidues (S) that serve as IKK phosphorylation sites, which signalthe protein for ubiquitination and degradation. The generalizedstructures of IKK a and b (kinase domain; HLH, helix-loop-helix;LZ, leucine zipper; NBD, NEMO binding domain) and of NEMO

(CC, coiled coil; LZ, leucine zipper; ZF, zinc nger) are also shown.

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mechanisms of action and how some may proveimportant for the treatment of human diseases.

Future perspectives

Even with the many advances since 1999, there continueto be several unsolved mysteries in the saga of NF- kB. If I use my crystal ball to predict what the next 7 yearshave in store, I suspect that progress will be made in thefollowing four general areas: basic biochemistry of the

signaling pathway, physiology and disease, therapy andsimple model systems.

Basic biochemistryAlthough we have learned a great deal aboutthe intracellular NF- kB signaling pathway, we stillhave very rudimentary knowledge about the dynamicsof the NF- kB pathway in cells in tissue culture andknow essentially nothing about these dynamics in wholeorganisms. In most cell types and signaling conditions,it is still not known what is the contribution of specic NF- kB complexes (e.g., p50-RelA vs p52-c-Rel

vs c-Rel-c-Rel) to given physiological responses or how

Canonical Pathway Non-canonical Pathway Pathway 3

Nucleus

P

p50

p50

l Proteasomaldegradation

IKKNIK

p 5 2

RelA

RelA

NEM

p 5 0p100

processing

p105processing

P

PP

P p 1 0 0

RelB

RelB

P

NEMONEM

?

p 1 0 5

p50

p50

Bcl-3

B

B

B B

Nucleus Nucleus

Figure 2 NF- kB signal transduction pathways. In the canonical (or classical) NF- kB pathway, NF- kB dimers such as p50/RelA aremaintained in the cytoplasm by interaction with an independent I kB molecule (often I kBa). In many cases, the binding of a ligand to a

cell surface receptor (e.g., tumor necrosis factor-receptor (TNF-R) or a Toll-like receptor) recruits adaptors (e.g., TRAFs and RIP) tothe cytoplasmic domain of the receptor. In turn, these adaptors often recruit an IKK complex (containing the a and b catalytic subunitsand two molecules of the regulatory scaffold NEMO) directly onto the cytoplasmic adaptors (e.g., by virtue of the K63-ubiquitin-binding activity of NEMO). This clustering of molecules at the receptor activates the IKK complex. IKK then phosphorylates I kB attwo serine residues, which leads to its K48 ubiquitination and degradation by the proteasome. NF- kB then enters the nucleus to turnon target genes. The auto-regulatory aspect of the canonical pathway, wherein NF- kB activates expression of the I kBa gene that leadsto resequestration of the complex in the cytoplasm by the newly synthesized I kB protein is not shown. The non-canonical (oralternative) pathway is largely for activation of p100/RelB complexes during B- and T-cell organ development. This pathway differsfrom the canonical pathway in that only certain receptor signals (e.g., Lymphotoxin B (LT b), B-cell activating factor (BAFF), CD40)activate this pathway and because it proceeds through an IKK complex that contains two IKK a subunits (but not NEMO). In the non-canonical pathway, receptor binding leads to activation of the NF- kB-inducing kinase NIK, which phosphorylates and activates anIKK a complex, which in turn phosphorylates two serine residues adjacent to the ankyrin repeat C-terminal I kB domain of p100,leading to its partial proteolysis and liberation of the p52/RelB complex. Other distinct NF- kB pathways no doubt exist. For example,in Pathway 3, p50 (or p52) homodimers enter the nucleus, where they become transcriptional activators by virtue of interaction withthe I kB-like co-activator Bcl-3 (or I kBz). How Pathway 3 is regulated is not known. In all three pathways, various post-translationalmodications (e.g., phosphorylation, acetylation and prolyl isomerization) of the NF- kB subunits can modulate their transcriptionalactivity (see Perkins, 2006, this issue).


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different NF- kB dimers are targeted to specic promo-ters. I suspect that we will have tables of genes, listingthe NF- kB dimers that control them under specicconditions. Undoubtedly, there is also much more to belearned about how post-translational modications andproteinprotein interactions modulate and/or specifyactivated NF- kB responses.

Despite the numerous papers on the IKK complex,there is still much to be uncovered about its compositionand regulation. What is the precise stoichiometry of corecomponents in the IKK complex, especially, how manymolecules of NEMO are in the complex in uninduced vsinduced states? What are the three-dimensional struc-tures of components of the IKK complex? How does thestructure (and composition) of the IKK complex changeupon activation? I suspect that there are dynamicchanges in the complex that have not yet beenaddressed. For example, what are the roles of thevarious non-core components that have been reported

to associate with the IKK complex (e.g., ELKS, heat-shock proteins, etc)? What is the nature of the IKK a-dependent IKK complex used in the non-canonicalpathway? What are the biological roles of pathwaysother than the canonical and non-canonical NF- kBpathways (e.g., Pathway 3 in Figure 2, or constitutiveturnover or tyrosine-induced release of I kBa)? Whatother cellular pathways are regulated by IKK?

Physiology and diseaseThere is no question that the use of additional and morenely tuned mouse genetic model systems (e.g., condi-tional knockouts) will reveal novel and sometimes

unexpected roles for NF- kB in normal physiology. Forexample, by the time of the next review series, there islikely to be a full paper on the role of NF- kB in learning,memory and behavior. Furthermore, it is likely thatpolymorphisms that play a role in inter-individualsusceptibilities to diseases, especially ones involvingpathogens, will be identied in components or targetgenes of NF- kB signaling.

Anti-NF- kB therapyWhether or not its patent stands the test of time, NF- kBwill no doubt continue to occupy a central focus of therapeutic intervention. By the time of the next reviewissue, I expect that clear and focused modulators of NF- kB signaling will be in use in humans. I speculatethat such inhibitors will rst show efcacy in unusualand accessible diseases, which have NF- kB dependency(e.g., cylindromatosis). Although there has been greatfocus on potent single-step NF- kB signaling inhibitors, Isuspect that low-dose, multi-step inhibition of NF- kB(e.g., compounds or combinations of compounds thatact at more than one step) will be more effective.Perhaps, we will also have an appreciation of howchronic use of natural product-derived NF- kB inhibitors(e.g., curcumin, green tea and antioxidants) contributeto human health.

NF- kB in simple organismsAmong simpler organisms, we will begin to have anappreciation for the role that NF- kB plays in insectvectors for human and animal diseases (e.g., inmosquitoes carrying disease-causing microbes), andhow we might genetically modify components of NF-kB signaling in these organisms to decrease their vectoreffectiveness. Similarly, I suspect that our recentdiscovery of a primitive NF- kB system in the phylumCnidaria will lead to insights into the role that NF- kBplays in the response of simple marine organisms andecosystems to environmental stresses, be they man-made, physical or biotic.

An updated nomenclature for components of the NF- j Bsignal-transduction pathway

Among the many publications on this topic, there areinconsistencies in the naming of genes and proteins inthe NF- kB pathway. In this collection of reviews, wehave used what I believe is a simple nomenclature(Table 1). The revised nomenclature reects the new

members of the pathway, common usage over the pastseveral years, and at times my own judgment.

Acknowledgements

I thank Melissa Chin for help with artwork in the gures. Ialso thank D Kalaitzidis, D Starczynowski and members of mylab for comments on the manuscript. I especially thank theauthors for their contributions to this review issue, and col-lectively, we apologize to any researchers who feel that theirwork was overlooked. Research in my laboratory on NF- kBis currently supported by the National Institutes of Health.For additional, detailed information on NF- kB, the reader isdirected to our lab website at www.nf-kb.org.

Table 1 Nomenclature for NF- kB core signaling proteins

Gene (human gene) Protein

Dorsal Dorsalcactus Cactusdif Dif Relish Relishv-rel v-Relc-rel (REL ) c-Relrelb (RELB ) RelBrela (RELA ) RelA (p65)nfkb1 (NFKB1 ) p50, p105 or p50/p105nfkb2 (NFKB2 ) p52, p100 or p52/p100ikba (IKBA ,NFKBA1 ) IkBaikbb (IKBB ) IkBbikbe (IKBE ) IkBenfkb1 (NFKB1 ) IkBgikbz (IKBZ ) IkBzbcl-3 (BCL3 ) Bcl-3ikka (IKKA,IKBKA,CHUK ) IKK aikkb (IKKB,IKBKB ) IKK bikke (IKKE,IKBKE ) IKK enemo (NEMO ) NEMO (IKK g)

Abbreviations: IKK, I kB kinase; NEMO, NF- kB essential modulator;NF- kB, nuclear factor-kappaB.


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Introduction to NF-jB Players, Pathways, Perspectives