Specification of DNA Binding Activity of NF-Kb Proteins

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Speciﬁcation of DNA Binding Activity of NF-kB Proteins

Fengyi Wan and Michael J. Lenardo

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Correspondence: [email protected]

Nuclear factor-kB (NF-kB) is a pleiotropic mediator of inducible and specific gene regulation involving diverse biological activities including immune response, inflammation, cell proliferation, and death. The fine-tuning of the NF-kB DNA binding activity is essential for its fundamental function as a transcription factor. An increasing body of literature illustrates that this process can be elegantly and specifically controlled at multiple levels by different protein subsets. In particular, the recent identification of a non-Rel subunit of NF-kB itself provides a new way to understand the selective high-affinity DNA binding specificity of NF-kB conferred by a synergistic interaction within the whole complex. Here, we review the mechanism of the specification of DNA binding activity of NF-kB complexes, one of the most important aspects of NF-kB transcriptional control.

uclear factor-kB (NF-kB), a collective term the phosphorylation and subsequent dispatch Nfor a family of transcription factors, was of the inhibitory IkBs to the proteasome for originally detected as a transcription-enhancing, protein degradation (Hacker and Karin 2006). DNA-binding complex governing the immuno- This cytoplasmic “switch” liberates NF-kB globulin (Ig) light chain gene intronic enhancer complexes for subsequent nuclear translocation (Sen and Baltimore 1986; Lenardo et al. 1987). and target gene transcription (Scheidereit NF-kB is evolutionarily and structurally con- 2006). It provides a pre-established genetic served and has representative members in a switch that is independent of new protein syn- wide range of species. In essentially all unstimu- thesis and triggered by a biochemical change lated nucleated cells, NF-kB complexes are in the cell. This adaptability and versatility no retained in latent cytoplasmic form through doubt underlies its broad use. A diverse spec- binding to a member of the inhibitor of trum of modulating stimuli can activate this NF-kB(IkB) proteins (Lenardo and Baltimore pleiotropic transcription factor; furthermore, 1989; Lenardo et al. 1989; Hayden and Ghosh the fundamental use of NF-kB has been high- 2004; Hayden and Ghosh 2008). NF-kB induc- lighted with an ever-increasing array of genetic tion typically occurs following the activation of targets, responsible for diverse biological activi- the IkB kinase (IKK) signalosome, resulting in ties including immune response, inﬂammation,

Editors: Louis M. Staudt and Michael Karin Additional Perspectives on NF-kB available at www.cshperspectives.org Copyright # 2009 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a000067 Cite this article as Cold Spring Harb Perspect Biol 2009;1:a000067

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F. Wan and M.J. Lenardo

cell proliferation, and death (Grilli et al. 1993) recognizes a small subset of relevant kB sites (also see http://www.nf-kb.org). from the large excess of potential binding sites The best known subunits of mammalian (about 1.4104 estimated in human genome) NF-kB consist of five proteins in the Rel is a critical step for stimulus-specific gene tran- family: RelA (p65), RelB, c-Rel, p50, and p52, scription. Increasing evidence suggests that which are capable forming homo- and hetero- specific chromatin modifications and configu- dimeric complexes in almost any combination rations are required for NF-kB proteins to (Hayden and Ghosh 2004). Each of these sub- access the chromosomally embedded cognate units harbors a prototypical amino-terminal kB motifs (Natoli et al. 2005; Natoli 2006). sequence of roughly 300 amino acids, termed The presence of kB sites, however, appears to the Rel homology domain (RHD), that mediates be a minimal requirement for NF-kB regulation dimerization, DNA-binding, nuclear locali- but not sufficient for gene induction (Wan et al. zation, and cytoplasmic retention by IkBs 2007). We will attempt to decipher the elegant (Rothwarf and Karin 1999; Chen and Greene but recondite control of DNA binding activity 2004). In contrast, the transcription activation of NF-kB proteins at multiple levels, which is domain (TAD) necessary for the target gene one of the most important, yet complex, expression is present only in the carboxyl termi- aspects of NF-kB function. This process, more nus of p65, c-Rel, and RelB subunits. NF-kB abstruse than initially considered, involves complexes have long been thought to function IkBs, Rel subunits, Rel-associating proteins, dimerically; but functional and biochemical and non-Rel subunits in both the cytoplasm information belied this simple conceptuali- and the nucleus, as well as complicated associ- zation. The native complex of NF-kBfrom ations in the nuclear chromatin. This synopsis nuclear extracts is more than 200 kDa, signifi- will highlight our current knowledge of the cantly higher than that reconstituted from pu- DNA binding activity of NF-kB complex, rified p50 and p65 proteins (115 kDa) (Urban focusing on its liberation from cytoplasmic et al. 1991). Moreover, native NF-kB complexes sequestration complexes to recruitment to have a .100-fold higher affinity for Ig kB motif cognate kB regulatory sites in the genome. DNA than reconstituted p65–p50 heterodimers (Phelps et al. 2000). A new study shows that IkB FAMILY PROTEINS another essential subunit of NF-kB complex, ribosomal protein S3 (RPS3), cooperates with The evidence that induction of NF-kB activity Rel dimers to achieve full binding and transcrip- does not require new protein synthesis, and tional activity (Wan et al. 2007). As an integral that the detergent deoxycholate liberated component, RPS3 plays a critical role in deter- active kB-site DNA binding activity in cytosolic mining the DNA binding affinity and specificity extracts from unstimulated cells, revealed a key of NF-kB, which will be discussed in more detail property of this gene regulatory system in the following discussion (Wan et al. 2007). (Baeuerle and Baltimore 1988). This established Therefore, the molecular machine known as a new regulatory paradigm involving the spe- NF-kB consists of both Rel and non-Rel sub- cific and reversible DNA binding of NF-kBpro- units that actually comprise multiple protein teins governed by NF-kB inhibitor protein(s), complexes with different gene activation speci- known as IkB. The IkB protein family currently ficities, masquerading as a single NF-kB com- consists of eight members: IkBa,IkBb,IkBg, plex in the nucleus. IkB1,IkBz, Bcl-3, and the Rel protein precur- NF-kB exerts its fundamental role as tran- sors p105 and p100, all of which possess a scription factor by binding to variations of the characteristic structural feature of ankyrin consensus DNA sequence of 50-GGGRNYY repeats. The centerpiece of both classical and YCC-30 (in which R is a purine, Y is a pyrimi- alternative NF-kB pathways is the IkBs, func- dine, and N is any nucleotide) known as kB tioning at the primary level to regulate the sites (Chen et al. 1998). How NF-kB selectively DNA binding activity of NF-kB proteins.

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Speciﬁcation of DNA Binding Activity of NF-kB Proteins

Specific modification and subsequent pathways converge on a tripartite IkB kinase degradation of IkBs is critical in regulating (IKK) complex consisting of two functionally NF-kB DNA binding activity, particularly in nonredundant kinases IKKa and IKKb,as the classic or canonical pathway (Table 1). In well as a regulatory subunit IKKg (Fig. 1A). unstimulated cells, NF-kB complexes are se- These mediate the phosphorylation of IkBs at questered with various binding preferences by specific amino acid residues (for instance, the IkBs in the cytoplasm because IkBs physically Ser-32 and Ser-36 in IkBa) predominantly mask the nuclear localization signal (NLS) through the action of the IKKb subunit in the of NF-kB. On stimulation, whether intra- or activated IKK complex. These phosphorylation extracellular, multiple intracellular signaling events are a prerequisite for their successive

Table 1. Post-translational modification of IkB family proteins IkB family proteins Target residues Enzymes References Phosphorylation IkBa S32 and S36 IKKb (Hayden and Ghosh 2004) IkBa S283, S289, T291, S293, CKII (Lin et al. 1996; McElhinny et al. and T299 1996; Schwarz et al. 1996) IkBa Y42 p56-lck, Syk, and (Koong et al. 1994) c-Src Y42 Unknown (Schoonbroodt et al. 2000) Y42 and Y305 Unknown (Waris et al. 2003) IkBa Unknown PI3K/Akt (Sizemore et al. 1999) IkBb S19 and S23 IKKb (Hayden and Ghosh 2004) IkBb S313 and S315 CKII (Chu et al. 1996) IkB1 S18 and S22 IKKb (Hayden and Ghosh 2004) Bcl-3 S394 and S398 GSK3b (Viatour et al. 2004) p100 S99, S108, S115, S123, S866, IKKa (Senftleben et al. 2001; Xiao et al. S870, and S872 2001; Xiao et al. 2004) p105 S927 and S932 IKKb (Lang et al. 2003) p105 S903 and S907 GSK3b (Demarchi et al. 2003) p105 S337 PKAc (Hou et al. 2003) Ubiquitination IkBa K21 and K22 bTrCP (Hayden and Ghosh 2004) IkBb K6 bTrCP (Hayden and Ghosh 2004) IkB1 K6 bTrCP (Hayden and Ghosh 2004) p100 K856 bTrCP (Amir et al. 2004) p105 Multiple Ks bTrCP (Cohen et al. 2004) Sumoylation IkBa K21 Unknown (Desterro et al. 1998) p100 K90, K298, K689, and K863 Ubc9 (Vatsyayan et al. 2008) Acetylation p100 Unknown P300 (Hu and Colburn 2005; Deng et al. 2006) p105 K431, K440, and K441 P300 (Furia et al. 2002; Deng and Wu 2003) S-nitrosylation p105 C62 (Matthews et al. 1996) IKKb,IkB kinase b; CKII, Casein kinase II; p56-lck, lymphocyte-specific protein tyrosine kinase; Syk, spleen tyrosine kinase; c-Src, normal cellular Src kinase; PI3K, phosphoinositide 3-kinases; GSK3b, glycogen synthase kinase 3 b; IKKa,IkB kinase a; PKAc, catalytic subunit of protein kinase A; bTrCP, b-transducin repeat-containing protein; Ubc9, E2 small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9; p300, E1A binding protein p300.

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F. Wan and M.J. Lenardo

AB Immunoreceptor TNFa Ultraviolet light CD40L, LT-b, BAFF

Cytosol Cytosol

CARMA1-BCL10-MALT1 RIP1-TAB1-TAK1 p38 NIK -Caspase-8-CK1a

IKKg Proteasomal IKKb IKKa IKKa IKKa degradation CKII P P Processing PP

P P IkBa IkBa P P IKKg p100 NF-kB NF-kB ATM ReIB

PIDD-RIP1-IKKg

p52

IKKg IKKg ReIB NF-kB

DNA damage kB sites in target genes kB sites in target genes

Nucleus Nucleus

Figure 1. A schematic representation of how NF-kB DNA binding is regulated by IkB degradation and processing following activating stimuli. (A)IkB degradation mechanism that depends on site-speciﬁc phosphorylation of IkBs mediated by activated IKKb in the IKK complex as well as CKII. Unique intracellular signaling complexes (which contains more components as shown, e.g., CARMA1-BCL10-MALT1-Caspase-8-CK1a complex downstream of T cell receptor [TCR] ligation [Bidere et al. 2009]) phosphorylate and activate the IKK complex depending on the initial stimulus (e.g., TNF, immunoreceptors, and DNA damage). IKKb phosphorylates serine residues located in the amino-terminus of IkBs following TNF and TCR stimulation. In contrast, NF-kB induction by ultraviolet light and select chemotherapeutic agents requires the MAP kinase p38 and CKII. CKII phosphorylates serine residues located in the carboxyl terminus of IkBs. Phosphorylation of IkBs leads to successive ubiquitinylation and proteasomal degradation. The removal of IkBs liberates NF-kB complexes for nuclear translocation and binding to cognate regions in target genes. (B) The alternative pathway, which is induced by a few stimuli including CD40L, LT-b, and BAFF. NIK and IKKa are critical kinases for the phosphorylation of p100 at regulatory serine residues within its carboxyl terminus. This causes proteasomal degradation of the carboxyl terminus of the p100 molecule, generating a DNA binding NF-kB complex containing RelB and processed p52.

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Speciﬁcation of DNA Binding Activity of NF-kB Proteins

ubiquitinylation by b-transducin repeat- full degradation, of p100 represents another containing protein (bTrCP) and degradation mechanism by which IkB regulates the DNA in the 26S proteasome. The removal of IkBs lib- binding activity of NF-kB proteins. This was erates NF-kB for nuclear translocation and discovered recently in an alternative pathway binding to cognate sites in target genes. of NF-kB regulation involving RelB and p52 Additionally, various kinases other than the (Fig. 1B) (Hayden and Ghosh 2004). A few IKK complex may also mediate site-specific select stimuli including CD40 ligand (CD40L), modification of IkBs, contributing to the diver- lymphotoxin b (LT-b), and B-cell activating sity of signals regulating the latent DNA binding factor (BAFF) phosphorylate p100 on carboxy- activity of NF-kB. In ultraviolet (UV) light- terminal serine residues, triggering successive induced NF-kB activation, the phosphory- polyubiquitination and proteasomal degra- lation-dependent IkBa degradation process dation of the carboxyl terminus of the p100 does not rely on the IKK complex but the alter- molecule. The resulting complex, containing native serine/threonine kinase, casein kinase II RelB and processed p52, functions as the main (CKII) (Fig. 1A). Several studies have NF-kB species with selected DNA binding shown that CKII phosphorylates IkBa at a activity in the alternative pathway. Of interest, cluster of serine and threonine residues in the kinase signalosome mediating p100 phos- the carboxyl terminus through a p38-MAPK phorylation contains the NF-kB-inducing kinase-dependent pathway, which is critical kinase (NIK) and IKKa, and functions com- for degradation of IkBa and induced NF-kB pletely independently of IKKb and IKKg. DNA binding activity in cells exposed to UV Moreover, recent evidence suggests that IKKa light (Lin et al. 1996; McElhinny et al. 1996; phosphorylates p100 at several serine residues Schwarz et al. 1996). Moreover, tyrosine within the RHD of p52, and these modifications kinases p56-lck, Syk, and c-Src and the serine/ can augment p52 DNA binding, in addition to threonine kinase GSK3b have also been inducing the efficient processing of p105 (Xiao reported to phosphorylate IkBs and control et al. 2004). the DNA binding activity of NF-kB (Table 1) Besides their hallmark sequestration func- (Koong et al. 1994; Viatour et al. 2004). Along tion in cytoplasm, IkBs can also regulate the with phosphorylation, modifications such as DNA binding properties of NF-kB proteins in sumoylation, acetylation, and s-nitrosylation the nucleus. IkBz localizes to the nucleus fol- of the IkBs at specific residues can also trigger lowing its inducible expression (Yamamoto the degradation process (Table 1). However, et al. 2004) and Bcl-3 is also found in the nu- these modifications lead to the final common cleus associated with p50- and p52-containing pathway of ubiquitination of IkBs at specific NF-kB complexes (Cogswell et al. 2000). IkBs lysine residues as the essential requirement for such as IkBa,IkBb, and IkB1 are transcrip- IkB degradation and release of NF-kB for tional targets of NF-kB itself, creating a negative nuclear translocation (Table 1). feedback loop. This facilitates restoration of p100 and p105, the precursor proteins for latent NF-kB complexes in the cytoplasm and NF-kB subunits p52 and p50 respectively, maintains the cell’s responsiveness to sub- contain multiple ankyrin repeats (similar to sequent stimuli. With variant kinetics of degra- those of the IkB family) in their carboxy- dation and resynthesis, IkBs compete NF-kB terminal domains. In some circumstances, complexes off their chromosomal locations these keep NF-kB complexes cytoplasmically and export them back into cytoplasm. By affect- anchored. The regulation of NF-kBDNA ing the cytoplasmic–nuclear shuttling of binding activity by p105 mirrors IkBs of the NF-kB proteins, these titrate the occupancy of classical pathway by undergoing inducible, gene regulatory elements in nuclear DNA. complete degradation, particularly when it is Strikingly, IkBb may also function to directly bound to NF-kB complexes (Heissmeyer et al. regulate NF-kB DNA binding activity on rel- 2001). By contrast, partial processing, but not evant promoters or enhancers, as evident by

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F. Wan and M.J. Lenardo

its stable nuclear association with NF-kB com- themselves or to controlling their associations plexes that are already bound to kB sites in chro- with other proteins in transcriptional regu- matin (Thompson et al. 1995; Suyang et al. latory complexes. 1996). Moreover, both IkBz and Bcl-3 primarily The phosphorylation of p65 under basal interact with specific NF-kB complexes and and activated conditions is by far the best- subvert their DNA binding activity. IkBz was characterized modification among Rel proteins, reported to associate preferentially with p50 and p65 is the transcriptionally active com- homodimers and also negatively regulate ponent of the NF-kB species that is most abun- p65-containing NF-kB complexes (Kang et al. dant and has the broadest function. So far, nine 1992; Motoyama et al. 2005; Hayden and phosphorylation sites have been identified in Ghosh 2008), which suggests it may possess p65 located in both the RHD and TAD, with the capability to selectively inhibit or activate several sites remarkably critical for DNA specific NF-kB species. However, the role of binding capability. Ser-536 phosphorylation Bcl-3 in regulating DNA binding seems contro- by various kinases leads to a substantially versial. It has been proposed to remove the weaker interaction of p65 with newly syn- repressive p50 homodimers from kBDNA, thesized IkBa, leading to a dramatic decrease allowing transcriptionally active NF-kB species of nuclear p65 export, which enhances the dur- access to those cognate elements (Lenardo ation of kB DNA access (Table 2). Furthermore, and Siebenlist 1994; Hayden and Ghosh Ser-536-phosphorylated p65 does not interact 2008). However, recent evidence indicates that with cytosolic IkBa at all, which could acceler- Bcl-3 might also stabilize p50 homodimers ate p65 nuclear localization and attachment to and inhibit NF-kB activation by preventing DNA (Sasaki et al. 2005). Phosphorylation of transcriptionally active NF-kB complexes from p65 at Ser-276 and Ser-536 together with suc- binding to kB DNA (Carmody et al. 2007). cessive acetylation at Lys-310 alters its affinity Regardless of the inhibited or enhanced NF- to IkB proteins, resulting in different kinetics kB transactivation consequences, IkBs regulate of p65 cytoplasmic-nuclear shuttling and the DNA binding activity of NF-kB proteins subsequent DNA binding activity (Chen et al. by controlling the high-affinity interactions of 2005). Phosphorylation of Thr-254 by the NF-kB species to cognate DNA regulatory sites. nuclear peptidylprolyl isomerase Pin1, followed by isomerization of specific amino acid residues, also strongly increases p65 nuclear POST-TRANSLATIONAL MODIFICATIONS translocation and DNA binding activity. Pin1- OF REL PROTEINS induced conformational changes increase the Like IkB proteins, Rel subunits are subject structural stability of p65 with dramatic to numerous post-translational modifications, decrease in IkBa affinity (Ryo et al. 2003). which represents another mechanism of regu- Recently, phosphorylation of Ser-276 by the lating NF-kB activation that has been exten- mitogen- and stress-activated protein kinase-1 sively studied recently. These modifications, (MSK1) was shown to be essential for p65 within conserved RHD, TAD or linker se- binding to the kB intronic enhancer site of the quences, convey various physiological func- mast cell growth factor SCF (stem cell factor) tions of NF-kB (Viatour et al. 2005; Perkins gene in inflammation (Reber et al. 2009). Phos- 2006; Neumann and Naumann 2007; O’Shea phorylation of p65 can also repress the tran- and Perkins 2008). This includes control scriptional activity of other Rel subunits. For of NF-kB DNA binding activity through instance, Ser-536-phosphorylated p65 was modifications, particularly phosphorylation reported to specifically interact with RelB in and acetylation, of Rel subunits or adjusting the nucleus, forming a complex that cannot the association of NF-kB complexes to either bind to kB DNA (Jacque et al. 2005). IkBs or kB DNA (Table 2). This could be Post-translational modification of c-Rel and caused by steric alternations of the Rel proteins its effect on DNA binding activity is similar

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Speciﬁcation of DNA Binding Activity of NF-kB Proteins

Table 2. Post-translational modiﬁcations of Rel subunits that regulate NF-kB DNA binding activity Functional effect Rel family on DNA binding proteins Target residues Enzymes activity References Phosphorylation p65 T254 Unknown Enhancement (Ryo et al. 2003) S529 CKII Unknown (Bird et al. 1997; Wang et al. 2000; O’Mahony et al. 2004) Unknown PI3K/Akt Enhancement (Sizemore et al. 1999) S276 MSK1 Enhancement (Reber et al. 2009) S536 IKKa Enhancement (Jiang et al. 2003; O’Mahony et al. 2004) IKKb Enhancement (Sakurai et al. 1999) IKK1 Enhancement (Buss et al. 2004; Adli and Baldwin 2006; Mattioli et al. 2006) TBK1 Enhancement (Fujita et al. 2003; Buss et al. 2004) RSK1 Enhancement (Bohuslav et al. 2004) c-Rel Unknown TBK1 Enhancement (Harris et al. 2006) Unknown IKK1 Enhancement (Sanchez-Valdepenas et al. 2006) p50 S337 PKAc Enhancement (Guan et al. 2005) Acetylation p65 K218, K221, p300 Enhancement (Chen et al. 1998) and K310 K122 and K123 PCAF and Reduce (Kiernan et al. 2003) p300 p50 K431, K440, p300 Enhancement (Furia et al. 2002) and K441 CKII, Casein kinase II; MSK1, Mitogen- and stress-activated protein kinase-1; PI3K, Phosphoinositide 3-kinases; IKKa, IkB kinase a; IKKb,IkB kinase b; IKK1,IkB kinase epsilon; TBK1, TANK-binding kinase 1; RSK1, Ribosomal protein S6 kinase; 90 kDa, polypeptide 1; PKAc, catalytic subunit of protein kinase A; p300, E1A binding protein p300; PCAF, p300/ CREB binding protein-associated factor.

to p65, but with fewer characterized kinase(s) for kB motifs (Starczynowski et al. 2007). or target residue(s). c-Rel can be specifically These data show that the carboxy-terminal phosphorylated by the serine/threonine kinases modifications, especially in the TAD, are vital NIK, TBK1, and IKK1 on its carboxyl terminus, in regulating c-Rel DNA binding activity. which dissociates the IkBa-c-Rel complex and The RelB monomer is easily degraded and enhances binding to cognate DNA (Harris preferentially binds to p52 and p50, making it et al. 2006; Sanchez-Valdepenas et al. 2006). an unusual NF-kB subunit (Hayden and Furthermore, tyrosine phosphorylation of Ghosh 2004). Although the kinases have not c-Rel within its carboxyl terminus also been identified yet, phosphorylation of RelB at increased its ability to bind kB sites in human multiple residues including Thr-84, Ser-254, neutrophils stimulated with granulocyte- Ser-368, and Ser-552, have been reported as colony-stimulating factor (Druker et al. 1994). essential for its dimerization and degradation More importantly, dysregulated Ser-525 phos- (Viatour et al. 2005; Neumann and Naumann phorylation located within c-Rel TAD, caused 2007). It certainly deserves further investigation by a serine to proline point mutation dis- whether these or other unknown modifications covered in two patients with follicular and of RelB regulate DNA binding, given the mediastinal B-cell lymphoma, might slightly crucial role of RelB in the alternative pathway enhance c-Rel DNA binding activity, at least of NF-kB activation. Although inducible

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F. Wan and M.J. Lenardo

phosphorylation of p50, similar to its precursor pull-down assays using kB oligonucleotides p105, has been noticed in various cell types (Furia et al. 2002). Collectively, site-specific (Neumann and Naumann 2007), site-specific acetylation of Rel proteins, at least p65 and modifications of transactivation repressive p50 p50, augments their intrinsic DNA binding and p52 have been rarely reported. One recent activity, as shown for other transcription study showed the catalytic subunit of PKA factors (Boyes et al. 1998). (PKAc) specifically phosphorylated mature p50 at Ser-337 located within RHD and aug- REL SUBUNIT-ASSOCIATING PROTEINS mented DNA binding activity, which may be important for maintaining stable negative regu- For full NF-kB transactivation to occurat a given lation of NF-kB gene expression in unstimulated target DNA locus, a successive enhanceosome cells (Guan et al. 2005). complex needs to be assembled by multiple Acetylation is another key modification of coactivators, corepressors, other transcription Rel subunits that regulates their association factors, and basal transcription machinery pro- with IkBs and direct binding to cognate DNA teinsthrough protein–protein interactions with (Table 2). Five residues, Lys-122, Lys-123, Lys- Rel subunits (Hayden and Ghosh 2004; Hayden 218, Lys-221, and Lys-310, can be specifically and Ghosh 2008). For instance, over 100 acetylated in p65 in response to TNFa stimu- proteins can modulate NF-kB association with lation. Acetylation at Lys-218 and Lys-221 chromatin or the assembly of an NF-kB enhan- markedly diminishes the binding of p65 to ceosome via their interaction with full length, IkBa thereby increasing its DNA binding RHD, TAD, or the central region of p65 (Chen et al. 2002). In particular, Lys-221 of (O’Shea and Perkins 2008). Until a short time p65 directly interacts with Met-279 of IkBa in ago, Rel subunits were widely regarded as the the crystal structure of p65-p50-IkBa complex only executers to recognize and bind kBDNA. (Huxford et al. 1998); therefore, acetylation of However, several Rel-associating proteins, Lys-221 may result in a conformational change albeit with no kB DNA binding capability them- in p65 and lessen its interaction with IkBa selves, have recently been illustrated to be essen- (Chen et al. 2002). In contrast, acetylation of tial in regulating NF-kB binding to cognate p65 at Lys-122 and Lys-123 accelerates its dis- DNA (Table 3). These Rel-associating proteins sociation from DNA and successive nuclear feature restricted tissue distribution, specific export by IkBa, attenuating NF-kB transactiva- expression profiles, or unique pathophysio- tion (Kiernan et al. 2003). In addition, acety- logical circumstances and inhibit, as currently lation of Rel subunits may modulate their reported, the DNA binding activity of NF-kB binding with DNA directly. Acetylation of p65 proteins. This establishes Rel-associating pro- at Lys-221 apparently causes a conformation teins as a group of tissue- or cell context-specific change favoring kB DNA binding. This is sup- negative regulators of NF-kB beyond the IkB ported by the crystal structure of p65-p50-kB family, adding another level of complexity to DNA complex illustrating that Lys-221 directly the exquisite spatiotemporal control of DNA contacts the DNA backbone (Chen et al. binding activity of NF-kB proteins. 1998). Various mutational analyses strengthen Some Rel-associating proteins attenuate the concept that this acetylation influences NF-kB DNA binding in cotransfection exper- DNA-binding properties. Mutation of Lys-221 iments. RelA-associated inhibitor (RAI), iso- produces a sharp decline in the DNA binding lated in a yeast two-hybrid screen utilizing the affinity of p65 homodimers and a substantial central region of p65 as bait, interacts with decrease in that of p65-p50 heterodimers p65 in vitro and in vivo, and specifically inhibits (Chen et al. 2002). Strikingly, p50 can be acety- p65 binding to the cognate DNAwhen cotrans- lated in vitro by p300/CBP at Lys-431, Lys-440, fected in human embryonic kidney 293 cells and Lys-441, and this modification augments (Yang et al. 1999). A similar function was its DNA binding properties, as evidenced by ascribed to the aryl hydrocarbon receptor

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Speciﬁcation of DNA Binding Activity of NF-kB Proteins

Table 3. Rel-associating proteins that regulate NF-kB DNA binding activity Domain/portion Evidence for Functional Associating/ of Rel proteins integration into effect on NF-kB interacting sufﬁcient for DNA binding DNA binding subunits proteins binding complexes activity References p65 RAI RHD, aa 176–405 No Inhibition (Yang et al. 1999) AhR Not mapped No Inhibition (Tian et al. 1999; Kim et al. 2000; Ruby et al. 2002) b-Catenin Not mapped No Inhibition (Deng et al. 2002) Cdk9 Not mapped No Inhibition (Amini et al. 2002) PIAS1 TAD, aa 299–551 No Inhibition (Liu et al. 2005) VP1686 Not mapped No Inhibition (Bhattacharjee et al. 2006) Myocardin RHD, aa 1–276 No Inhibition (Tang et al. 2008) RPS3a RHD, aa 21–186 Yes Enhancement (Wan et al. 2007) p50 b3-Endonexin Not mapped No Inhibition (Besta et al. 2002) Cdk9 Not mapped No Inhibition (Amini et al. 2002) b-Catenin Not mapped No Inhibition (Deng et al. 2002) RAI, RelA associated inhibitor; AhR, Aryl hydrocarbon receptor; Cdk9, Cyclin-dependent kinase 9; PIAS1, Protein inhibitor of activated STAT1; VP1686, Vibrio parahaemolyticus type III secretion protein; RPS3, Ribosomal protein S3; b3-Endonexin, Integrin b 3 binding protein. aRPS3, an integral subunit of certain NF-kB DNA binding complex that also physically interacts with p65, is listed here to compare with Rel-associating proteins in their DNA-binding regulatory functions.

(AhR), a ligand-activated transcription factor. antiapoptosis-related NF-kB target genes When cotransfected in 293 cells, AhR specifi- (Bhattacharjee et al. 2006). cally inhibited the DNA binding activity of The regulatory effects of Rel-associating p65, but not the p50–p50 complex (Tian et al. proteins have been studied not only in the cellu- 1999; Ruby et al. 2002), suggesting that synergic lar context of NF-kB signaling, but also in interaction between AhR and NF-kB, mainly in vitro biochemical investigations with puri- the p65 subunit, is critical for suppression of fied proteins. One of the first studies described immune responses and xenobiotic metabolism. a surprising cross-regulation of NF-kBby Some pathogen-encoded proteins also possess b-catenin (Deng et al. 2002). Both p65 and the ability to regulate host NF-kB binding to p50 could complex with b-catenin indepen- cognate DNA, hampering immune responses dently of IkBa, but seemed to require directed against the microbes that express additional cellular factors. b-catenin markedly them. For instance, Vibrio parahaemolyticus is attenuated the DNA binding of both p50–p65 a causative agent of human gastrointestinal dis- and p50–p50 complexes as shown by electro- eases and significantly suppresses the induction phoretic mobility shift assays (EMSA), causing of the DNA binding activity of NF-kB via the reduced NF-kB target gene expression. Of physical interaction between its effector protein note, b-catenin is not directly integrated into VP1686 and p65. Such attenuation of NF-kB the NF-kB-DNA complex in spite of its strong DNA binding activity by VP1686 is sufficient association with NF-kB subunits, suggesting it to sensitize infected-macrophages for death, may interact with NF-kB proteins to disrupt because of the diminished expression of many their DNA binding ability (Deng et al. 2002).

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F. Wan and M.J. Lenardo

Inhibition of NF-kB DNA binding activity by INTEGRAL NON-REL SUBUNITS IN NF-kB b-catenin could be important in oncogenesis, COMPLEXES as hinted at by an inverse correlation between the b-catenin expression levels and levels of the As for the unresolved and important question of NF-kB target gene Fas in colon and breast how regulatory specificity of NF-kB is achieved, tumor tissues (Deng et al. 2002). Furthermore, it has long been regarded that the variability of the inhibitory effects on NF-kB DNA binding kB sequences may govern the usage of certain by Cyclin-dependent kinase 9 (Cdk9), the Rel dimers at specific promoters. Principally, PIAS1 (protein inhibitor of activated STAT each kB site variant could preferentially recruit [signal transducers and activator of transcrip- one type of Rel dimer over other species tion] 1) protein, and myocardin were also (Natoli et al. 2005; Natoli 2006). Selective shown using EMSA, in which these proteins NF-kB gene expression, however, cannot be significantly reduced the DNA binding activity completely explained by a simple correlation of NF-kB (Amini et al. 2002; Liu et al. between the sequence of kB sites in target 2005; Tang et al. 2008). Cdk9 markedly sup- genes and the requirement for a specific Rel pressed the association of the p50–p50, p65– dimer (Hoffmann et al. 2003). However, kB p65, and p50–p65 complexes to kB DNA, and sites may still impart a specific configuration the ability of NF-kB to modulate HIV-1 gene for NF-kB binding, because a single nucleotide transcription was controlled by this inhibitory change in an NF-kB binding site affected the for- function (Amini et al. 2002). Both PIAS1 and mation of productive interactions between Rel myocardin specifically inhibited the DNA dimer and coactivators (Leung et al. 2004). binding activity of p65-containing complexes, Therefore, other protein components beyond serving as negative regulators of NF-kBin Rel subunits could form integral parts of the certain conditions. PIAS1 was originally NF-kB binding complex, thus controlling its identified in the Jak/STAT signaling pathway, recognition and action on target genes. whereas myocardin is expressed specifically in Support for this hypothesis comes from obser- cardiac and smooth muscle cells (Liu et al. vations made in seminal previous studies. 2005; Tang et al. 2008). Of interest, their effects First, the contradiction in the size of native on NF-kB DNA binding appear essential for NF-kB from nuclear extracts (. 200 kDa) and tuning cytokine-induced NF-kB target gene that reconstituted from purified p50 and p65 expression and cardiomyocyte proliferation proteins (115 kDa) implies the presence of and differentiation, respectively (Liu et al. other proteins in the native complex (Urban 2005; Tang et al. 2008). et al. 1991). Second, reconstituted p65–p50 As described above, b-catenin and Cdk9 heterodimers from purified proteins have a both complex with p50 and dramatically .100-fold lower affinity for DNA than native inhibit its DNA binding activity (Amini et al. NF-kB complexes, at least for binding to the Ig 2002; Deng et al. 2002). b3-endonexin is kB motif (Phelps et al. 2000). Third, distinct var- another p50-interacting molecule that inhibits iants of the kB motifs display different responses p50–p65 complex binding to kBDNA. to various NF-kB inducers in different cell types Moreover, binding of b3-endonexin to p50 implying selective regulation. Finally, a large was inhibited in the presence of wild-type but number of NF-kB binding motifs beyond not mutated kB oligonucleotides, suggesting a either canonical or variant kB site sequences steric competition between b3-endonexin and were revealed throughout the human genome kB DNA for the p50–p65 complex. Despite by mapping with chromatin immunopre- the association with the transcription repressive cipitation-coupled microarray or sequencing p50 subunit, b3-endonexin negatively regulates (Martone et al. 2003; Schreiber et al. 2006; expression of the urokinase-type plasminogen Lim et al. 2007). Collectively, these findings activator receptor that is essential for endo- strongly suggested that other non-Rel proteins thelial migration (Besta et al. 2002). could not only regulate NF-kB DNA binding

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Speciﬁcation of DNA Binding Activity of NF-kB Proteins

activity, but also participate in DNA binding as activity. These observations suggest a new regu- an essential component. latory paradigm in which DNA binding activity This hypothesis was confirmed by a recent could be regulated within NF-kB complexes study showing that RPS3, an integral non-Rel through synergistic interactions between Rel subunit in certain NF-kB DNA binding com- and non-Rel subunits (Fig. 2). plexes, is essential for the recruitment of RPS3 was found to physically interact with NF-kB p65 to selected kB sites (Wan et al. p65 in a proteomic screen, and shown to be criti- 2007). RPS3 prominently features a hetero- cal in NF-kB transactivation. Under conditions geneous nuclear protein K (hnRNP K) ho- of reduced RPS3, the DNA binding activity of mology (KH) domain, a structural motif that NF-kB complexes was significantly attenuated. binds single-stranded RNA and DNA with Knockdown of RPS3 resulted in failed recruit- some sequence specificity (Siomi et al. 1993). ment of p65 to selected endogenous gene regu- Indeed, the KH domain within RPS3 is essential latory sites and abortive induction, despite for association with p65 (Wan et al. 2007). This normal p65 nuclear translocation. Therefore, study underscored the inherent complexity of RPS3 facilitates p65 binding to cognate DNA, NF-kB binding to kB sites by demonstrating which is essential for normal expression of that DNA binding capability is not conferred specific NF-kB target genes involved in key phys- strictly by Rel subunits, as has been long iological processes. This was dramaticallyshown assumed. Rather, the integral non-Rel subunit for the induction of immunoglobulin k light RPS3 represents a newly recognized subunit chain gene expression in B cells and cell prolifer- that potently contributes to DNA binding ation and cytokine secretion in T cells that were

P U S IkBa

IKKg IkBa P A IKKb IKKa p65 p50 p65 p50 P A P P S3 S3 IkBa

PIAS1 P A p65 p50 b-Catenin P A Stimuli S3

VP1686

RAI

P A Nucleus IkBa p65 p50 Cytosol P A S3 Selective kB site in target genes

1kB degradation or processing

Post-translational modifications of Rel subunits

Rel-associating proteins

Integral non-Rel subunit Cytosol Nucleus

Figure 2. Complex regulation of NF-kB DNA binding activity. As illustrated in one of the most abundant NF-kB complexes, the DNA binding activity of NF-kB is controlled in both the cytoplasm and nucleus at multiple levels: involving degradation or processing of inhibitory IkBs; the post-translational modiﬁcation of Rel subunits; Rel-associating proteins that modulate NF-kB DNA binding potential; and an integral non-Rel subunit RPS3 required for selective NF-kB target gene transcription based on enhanced DNA binding afﬁnity. The bars represent the intracellular locations where the DNA binding activity of NF-kB proteins is regulated by indicated proteins.

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F. Wan and M.J. Lenardo

markedly impaired by RPS3 knockdown (Wan a subset of TCR ligation-induced NF-kB genes et al. 2007). Strikingly, purified RPS3 protein (Wan et al. 2007). Whether the RPS3 subunit exerted a dramatic synergistic effect on the is essential in other stimuli-induced NF-kB DNA binding activity of both p65–p65 and signal pathways and whether it targets certain p50–p65, but not p50–p50 complexes in NF-kB complexes to specific kB sites in differ- EMSA (Wan et al. 2007). As a DNA-binding ent cell types certainly deserves further investi- facilitator, RPS3 dramatically stabilizes NF-kB gation. Because RPS3 is only required for association with certain cognate sites. This selected particular genomic kB sites to be acti- could explain the extremely high affinity of vated under certain conditions and preferen- semipurified NF-kB complexes for DNA, tially directs binding to kB sites with some which is not manifested by complexes formed sequence specificity, we call it a “specifier” solely of purified p50 and p65 subunits. By subunit of NF-kB. This also lends credence to contrast, none of the aforementioned Rel- the idea that there are actually multiple molecu- associating proteins has been shown to integrate lar complexes containing RPS3-like “specifier” into NF-kB DNA binding complexes, and all of subunits with different gene activation speci- them inhibit the p65 DNA binding activity, ficities that all masquerade as single NF-kB although several were assessed in vitro using complex in the nucleus. Indeed, another KH recombinant proteins in EMSA (Amini et al. domain protein, Src-associated in mitosis, 2002; Deng et al. 2002; Liu et al. 2005; Tang 68 kDa (Sam68), was found to be essential for et al. 2008). some NF-kB gene transcription where RPS3 is It is important to recognize that RPS3 is not not required (F. Wan and M. Lenardo, an NF-kB-associated transcriptional coactiva- unpubl.). Sequence specificity preferred by tor, which by definition reorganizes chromatin various KH domains could confer different templates and recruits the basal transcriptional gene activation patterns via diverse NF-kB machinery to the promoter region. RPS3 pos- complexes. These findings may unveil a novel sesses little, if any, intrinsic transcriptional acti- regulatory paradigm in which KH domain pro- vating ability in a standard coactivation assay teins serve as essential functional components (Wan et al. 2007). Furthermore, the ability of in regulating the DNA binding activity of not RPS3-specific antibodies to dramatically super- only NF-kB, but also other transcription shift or diminish p65-containing DNA com- factors, because several other KH domain pro- plexes in EMSAs strongly suggests that RPS3 is teins have been shown to bind to DNA rec- an integral part of NF-kB DNA binding com- ognition motifs and to promote transcription plexes (Wan et al. 2007). By contrast, adminis- (Tomonaga and Levens 1996; Ostrowski et al. tration of a specific antibody against p300, one 2003; Moumen et al. 2005). of well-characterized transcriptional coactivators that complex with NF-kB, did not alter CONCLUDING REMARKS NF-kB-DNA complexes, suggesting that p300 is not incorporated into the DNA binding Since it was originally identified as a regula- complex (Deng et al. 2002). Further lines of tor of k light chain expression in B cells over evidence support the notion that RPS3 is an 20 years ago, NF-kB has served as a paradigm integral subunit of NF-kB: RPS3 physically for signaling associated with inflammation, associates with p65, p50, and IkBa in resting autoimmunity, and cancer. Recruitment of cells (guided through its interaction with NF-kB proteins to regulatory DNA sites p65); RPS3 can specifically translocate to the within the chromatin is fundamental and nucleus in response to T cell receptor (TCR) crucial for their target gene transcription. The and TNFa stimulation; RPS3 is recruited to complexity inherent in the DNA binding kB sites in a large number of NF-kB-driven activity of NF-kB proteins is essential for genes in vivo upon stimulation; and RPS3 and achieving a fine-tuned regulatory specificity. p65 are significantly correlated in transcribing An increasing body of literature illustrates that

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Specification of DNA Binding Activity of NF-κB Proteins

Fengyi Wan and Michael J. Lenardo

Cold Spring Harb Perspect Biol 2009; doi: 10.1101/cshperspect.a000067 originally published online September 9, 2009

Subject Collection NF-kB

Use of Forward Genetics to Discover Novel Oncogenic Activation of NF-κB Regulators of NF- κB Louis M. Staudt Tao Lu and George R. Stark Selectivity of the NF-κB Response The Regulatory Logic of the NF-κB Signaling Ranjan Sen and Stephen T. Smale System Ellen O'Dea and Alexander Hoffmann NF-κB in the Nervous System Roles of the NF-κB Pathway in Lymphocyte Barbara Kaltschmidt and Christian Kaltschmidt Development and Function Steve Gerondakis and Ulrich Siebenlist Signaling to NF-κB: Regulation by Ubiquitination The IKK Complex, a Central Regulator of NF-κB Ingrid E. Wertz and Vishva M. Dixit Activation Alain Israël Ubiquitination and Degradation of the Inhibitors of NF-κB in the Nervous System NF- κB Barbara Kaltschmidt and Christian Kaltschmidt Naama Kanarek, Nir London, Ora Schueler-Furman, et al. A Structural Guide to Proteins of the NF-κB The Nuclear Factor NF-κB Pathway in Signaling Module Inflammation Tom Huxford and Gourisankar Ghosh Toby Lawrence NF-κB in the Immune Response of Drosophila NF-κB as a Critical Link Between Inflammation Charles Hetru and Jules A. Hoffmann and Cancer Michael Karin Control of NF-κB-dependent Transcriptional Specification of DNA Binding Activity of NF-κB Responses by Chromatin Organization Proteins Gioacchino Natoli Fengyi Wan and Michael J. Lenardo

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