Oncogene (1999) 18, 6845 ± 6852 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Regulation of DNA binding by Rel/NF-kB transcription factors: structural views
Frances E Chen1 and Gourisankar Ghosh*,2
1Department of Biology, University of California ± San Diego, 9500 Gilman Drive, MC 0359, La Jolla, California, CA 92093-0359, USA; 2Department of Chemistry and Biochemistry, University of California ± San Diego, 9500 Gilman Drive, MC 0359, La Jolla, California, CA 92093-0359, USA
Rel/NF-kB transcription factors form homo- and nuclear localization and IkB binding. Additional Rel/ heterodimers with dierent DNA binding site speci®cities NF-kB family members include the v-Rel oncoprotein and DNA binding anities. Several intracellular path- of the avian Rev-T retrovirus, and the Drosophila ways evoked by a wide range of biological factors and Dorsal, Dif and Relish proteins. Rel/NF-kB proteins environmental conditions can lead to the activation of bind to DNA segments with a consensus sequence of Rel/NF-kB dimers by signaling degradation of the 5'-GGGRNYYYCC-3' collectively referred to as kB inhibitory IkB protein. In the nucleus Rel/NF-kB dimers elements or kB sites. For the NF-kB p50/RelA modulate the expression of a variety of genes including heterodimer alone, there are a large number of those encoding cytokines, growth factors, acute phase dierent kB sites that display varying degrees of response proteins, immunoreceptors, other transcription consensus (Figure 1). factors, cell adhesion molecules, viral proteins and Rel/NF-kB regulation can occur at multiple levels: regulators of apoptosis. The primary focus of this review (1) dimerization, (2) inhibition by IkB (and thus is on structural and functional aspects of Rel/NF- control of its nuclear translocation), (3) DNA kB:DNA complexes and their formation. The salient binding, (4) interaction with other transcriptional co- features of the Rel/NF-kB dimer:DNA structure are activators, and (5) interaction with the basal transcrip- described, as are modes of transcriptional regulation by tional machinery. This review describes common phosphorylation, altered DNA binding properties, vary- stereochemical features of several crystallographic ing protein conformations, and interactions with IkB Rel/NF-kB:DNA complexes, and, in the context of proteins. these models, provides some insights into how phosphorylation, protein sequence variation, DNA Keywords: Rel; NF-kB; IkB; DNA; structure; tran- sequence variation, and IkB modulation can aect scription the DNA binding characteristics of NF-kB. Other more general reviews of NF-kB are also available (Baeuerle and Henkel, 1994; Baldwin, 1996; Ghosh et al., 1998; Siebenlist et al., 1994). Introduction
Rel/NF-kB transcription factors form homo- and Structure of NF-kB/DNA complexes heterodimers with dierent DNA binding site specifi- cities and DNA binding anities. Various intracellular Three-dimensional structures have been solved of pathways evoked by a wide range of biological factors several Rel/NF-kB Rel homology (RH) domain and environmental conditions can activate the Rel/NF- dimers complexed to DNA, including those of the kB dimer by signaling degradation of the Rel/NF-kB p50, RelA, p52 and c-Rel homodimers, and the p50/ inhibitor IkB proteins. IkB degradation uncovers a RelA heterodimer (NF-kB) (Chen et al., 1998a,b; nuclear localization sequence (NLS) in each subunit of Cramer et al., 1997; Ghosh et al., 1995; MuÈ ller et al., the Rel/NF-kB dimer and allows the dimer to 1995). All models of Rel/NF-kB protein dimer translocate from its inactive cytoplasmic location into straddling DNA demonstrate a tertiary structure that the nucleus. In the nucleus it modulates the expression has been likened to that of a butter¯y and is unlike of a variety of genes including those encoding that of other observed transcription factors (Figure 2). cytokines, growth factors, acute phase response Each dimer subunit contains two sets of b sheet proteins, immunoreceptors, other transcription fac- immunoglobulin folds that form an N' terminal tors, cell adhesion molecules, viral proteins and domain (NTD) that contacts DNA both base regulators of apoptosis (see Pahl, 1999, this issue). speci®cally and backbone non-speci®cally, and a C' The mammalian Rel/NF-kB protein family mem- terminal domain (CTD) that mediates dimerization bers, which include p50, p52, c-Rel, RelA (p65), and and non-speci®c DNA contacts. Unlike most tran- RelB, share an N-terminal domain called the Rel scription factors which use alpha helices to bind homology (RH) domain. The RH domain contains DNA, the Rel/NF-kB proteins use ten ¯exible loops sequences responsible for dimerization, DNA binding, extending from the secondary structure of these immunoglobulin folds to mediate DNA contacts. These structures support numerous biochemical results that indicate the N' region of the RH domain is important for DNA binding (Coleman et al., 1993; *Correspondence: G Ghosh Toledano et al., 1993). Rel/NF-kBstructure FE Chen and G Ghosh 6846
Figure 1 Various kB sites
DNA binding on kB site DNA occurs via loops on lysine electrostatically interacts with the pyrimidines the edges of immunoglobulin folds and loosely re¯ects opposite of the guanine-rich strand. The nonspeci®c the twofold symmetry of the subunits in the dimer. phosphate backbone contacts, which contribute to the These contacts delineate two subsites within a kB site. high overall DNA anity of the Rel/NF-kB dimer, The numerous base-speci®c contacts on a subsite are are mediated by residues in all ®ve loops of each made via residues in the long loop L1 of the protein subunit. which reaches into the major groove of the DNA and After solving both the p50 and the RelA homodimer contacts the distal end of the subsites. Two arginine crystal structures (Chen et al., 1998b; Ghosh et al., residues in this loop make bidentate contacts with 1995; MuÈ ller et al., 1995), it was determined that kB conserved guanines and this is stabilized by hydrogen sequences are pseudosymmetric with a 5 base pair long bonds from a glutamate that binds the cytosines in the p50 subsite, a 4 base pair long RelA subsite, and a 1 opposite strand. A tyrosine residue makes van der base pair spacer between them. The structure of the Waals contacts while a positively-charged arginine or DNA-bound p50/RelA heterodimer con®rms this Rel/NF-kB structure FE Chen and G Ghosh 6847
Figure 2 The secondary structure of a Rel/NF-kB dimer bound to a space ®lling model of the target DNA
model and exhibits the expected directional binding in a biphasic manner, with the protein either bound or light of the subsites de®ned above and the observed unbound and transcription on or o, but in a ®nely biochemical experiments (Chen et al., 1998a; Zabel et tuned manner that can incorporate dierent levels of al., 1991). However, other experiments have indicated regulation. Four points of regulation that aect Rel/ that there is some ¯exibility in the size of the subsites NF-kB proteins include phosphorylation, variations in especially if it is localized to one side. Chen et al. the primary sequence, tertiary conformational changes, (1998b) showed that a RelA dimer given only one and interaction with IkB proteins. optimal subsite can still bind to the length of the kB site DNA by making base-speci®c contacts to that Phosphorylation subsite and only maintaining phosphate backbone contacts to the other. Additional work on the RelA Rel/NF-kB proteins have been observed to be homodimer also shows that given longer optimal positively regulated by phosphorylation events both subsites, a RelA subunit can utilize additional residues constitutively and inducibly in vivo (Li et al., 1994b). to bind a 5 base pair subsite in a manner similar to Hyperphosphorylation of p50 is required for phorbol that of p50 (Y-Q Chen, unpublished). ester and hemagglutinin induced nuclear translocation in Jurkat cells, and phosphorylated p50 accounts for most of the observed nuclear p50 population (Li et Regulation of DNA binding by Rel/NF-kB transcription al., 1994a). Phosphorylation of p50 has also been factors shown to increase the anity of formation and stability of p50:DNA complexes in vitro (Li et al., There is much experimental evidence to support the 1994a), whereas hypophosphorylation has been found hypothesis that transcriptional activation occurs not in to decrease stability of p50:DNA complexes in Ad12 Rel/NF-kBstructure FE Chen and G Ghosh 6848 E1-transformed Hooded Lister rat cells (Kushner and in determining dimerization anity. The particularly Ricciardi, 1999). The adenovirus Ad12 exploits favorable juxtaposition in the p50/RelA heterodimer of hypophosphorylation of p50 to downregulate the Asp-254 in the p50 subunit and Asn-200 in RelA helps transcription of the major histocompatability complex to overcome the loss of hydrogen bonds resulting from I gene allowing the cell to evade cytotoxic T cell- juxtaposing Tyr-267 in p50 with its analog, Phe-214, in mediated lysis. While preliminary phosphoamino RelA. The RelA homodimer, however, forgoes analysis indicates that p50 is phosphorylated on favorable hydrogen bonding interactions by facing serine residues (Hayashi et al., 1993; Li et al., two Phe-214 residues and also by not providing 1994b), it still remains to be determined which additional energy with two opposing Asn-200. phosphorylated residues of p50 are responsible for The dierential and oncogenic activity of v-Rel the observed eects on DNA binding. Nevertheless, it arises, at least in part, from an altered DNA-binding is apparent that phosphorylation cannot be enhancing anity (see Gilmore, 1999, this issue). The cause of this DNA binding by a direct mechanism since the altered anity originates from numerous mutations additional negative charge resulting from a phosphor- that v-Rel has acquired, which include substitution of ylation can only provide repulsive forces when binding the ®rst two N-terminal and last 118 C-terminal to a negatively-charged DNA molecule. Therefore, residues with virus-encoded Env residues, and numer- phosphorylation must indirectly aect DNA binding ous single amino acid substitutions, insertions and by p50, perhaps by stabilizing a residue or a segment deletions within the coding sequence. It is important to of residues that modulate DNA binding. note that these mutations in v-Rel are located in Phosphorylation has also been demonstrated to residues that are not directly involved in DNA binding, positively aect the transcriptional activity of RelA. as such mutations would no doubt abrogate DNA As compared to the isolated RH domain, nonpho- binding and activation altogether instead of modifying sphorylated full-length RelA does not bind well to it. The exact mutations responsible for the altered DNA in EMSA experiments (Zhong et al., 1998). DNA-binding anity of v-Rel have not been precisely Phosphorylation, however, at serine 276 of RelA by the determined, but several possibilities exist. Hrdlickova et catalytic domain of PKA increases its DNA anity. al. (1995) have mapped the altered DNA-binding GST pulldown experiments suggest a model in which speci®city to the mutations (from c-Rel to v-Rel) at the NTD of RelA interacts with the transactivation Met20Thr, Asp82Gly, and Arg92Glu. Previous experi- domain (TAD) and masks sites that interact with the mental evidence has shown that Met20Thr is one of transcriptional co-activator CBP/p300. Phosphoryla- four residues which are responsible for dierent DNA- tion abrogates this masking and allows these interac- binding speci®cities between p50 and RelA (Coleman et tions and transcriptional activation to occur. al., 1993). Furthermore, Nehyba et al. (1997) have also shown that Met20Thr in v-Rel alters DNA-binding speci®cities and have provided additional evidence that Protein sequence variation the Arg250Gly and Glu269Ala mutations in v-Rel Although the NF-kB p50/RelA heterodimer bound to cause promiscuous kB site binding. Regarding phos- the mouse immunoglobulin kB site was the ®rst Rel/ phorylation, it is unclear what eect, if any, results NF-kB discovered and often serves as a canonical from the mutation or loss of phosphorylatable residues protein:DNA complex, it is also important realize that in c-Rel, namely the change of a serine 285 in c-Rel to there are many other kB sites (Figure 1). The dierent a proline in v-Rel, and the deletion of Ser-352 from c- Rel/NFkB dimer species each have dierent anities Rel in v-Rel. Perhaps these mutations instead aect the for these kB sites as revealed by EMSA (Fujita et al., energetic conformations of a protein region which 1992; Kunsch et al., 1992). One reason for the observed indirectly aects DNA binding. At least it is known varying anities is the primary sequence dierences that the mutation Ser286Tyr within the RH domain is between family members. For example, the preference not responsible for DNA binding or nuclear transloca- of the p50 protein for a 5 base pair long subsite can be tion in that it has been shown to be required for PEST attributed to a histidine in the L1 loop that makes sequence-dependent IkBa association and cytoplasmic base-speci®c contacts to the purine outside of the retention (Rottjakob et al., 1996). conserved guanines. In RelA the homologous residue is instead a non-base-interacting alanine and thus RelA DNA sequence variations has a preference for 4 base pair subsites. Dierences in the primary sequences of the various Another interesting form of regulation can be observed Rel/NF-kB family members also provide an explana- among the dierent Rel/NF-kB crystal structures and tion for the observed dierential dimer formation. results from the variable conformations of the proteins Although there are ®ve dierent mammalian Rel/NF- and their target DNAs. This eect was ®rst noted in kB proteins, not all 15 dierent combinatorial dimers the structure of the RelA homodimer (Chen et al., are observed in vivo or in vitro. Not only are some 1998b) and was the result of a fortuitous, albeit at the dimers not observed, but the remainder form with time incorrect, assumption that kB sites were sym- various anities. For example, the p50/RelA hetero- metric with two 5-base-pair subsites. This assumption dimer forms preferentially over the p50 homodimer, eectively produced a DNA target with one subsite which forms with greater anity than the RelA that was shifted relative to the other and which homodimer (Ganchi et al., 1993; Kunsch et al., mutated out a crucial base required by Glu-39 to 1992). A current model (Huang et al., 1997; anchor the network of hydrogen bonds supporting Sengchanthalangsy et al., 1999) proposes that amino most of the base-speci®c contacts mediated by that acids Asp-254 and Tyr-267 in p50 and their amino acid subunit. The result is a subunit with a large rotation analogs in the other Rel family members play key roles and translation in the NTD, that makes no base- Rel/NF-kB structure FE Chen and G Ghosh 6849 speci®c contacts, yet still allows the overall complex to eect a pronounced kink that precludes protein bind the DNA. Biochemical experiments also demon- binding (Tisne et al., 1999). Indeed, in vitro DNA strated a correlation between the transcriptional bending experiments show that the various kB sites do activity of a p50 homodimer and a conformational have inherently dierent bend angles (Schreck et al., change as detected by a protease protection assay 1990). The intrinsic bend found in various kB sites (Fujita et al., 1992). Subsequent crystal structure dictates via indirect readout what type of protein determination of a target DNA containing pseudosym- conformation is required for binding and thus aects metric subsites of the correct size produced a the resulting transcriptional activity (Figure 3). symmetric protein conformation with base-speci®c contacts on both subunits. The variability in tertiary IkB modulation conformation of the NTD observed in the RelA homodimer, however, has also been observed, albeit As described above, IkB proteins act as direct to lesser degrees, in other structures such as the p52 inhibitors of the Rel/NF-kB complexes by obscuring homodimer and the p50/RelA heterodimer (Chen et al., the nuclear localization sequence (NLS) of the dimer. 1998a; Cramer et al., 1997). Even between models of One of the major and most-studied IkB proteins is p50/RelA heterodimer bound to dierent DNAs this IkBa. Interaction of IkBa with NF-kB results in the eect is observed. Structures have been solved of the cytoplasmic sequestration of NF-kB until the activa- NF-kB heterodimer (p50-RelA) bound to the kB site of tion of a signaling pathway that ultimately phosphor- the immunoglobulin light chain gene enhancer (NF- ylates, ubiquitinates, and degrades IkBa (Karin, 1999, kB:Ig) (Chen et al., 1998a), kB site in the PRD-II from this issue). These events unveil the NLSs and enable the IFNb promoter (NF-kB:PRD2) (AK Aggarwal, the nuclear translocation of the NF-kB dimer where it unpublished results), and a kB element of the human binds to DNA and activates transcription. The gene urokinase plasminogen activator promoter (NF- encoding IkBa itself has kB sites in its promoter that kB:uPA) (Chen and Ghosh, unpublished results). The allows the synthesis of new IkBa molecules. These NTDs of all three structures also demonstrate varying newly-formed IkBa molecules enter the nucleus and degrees of rotation and translation. remove the Rel/NF-kB complexes from DNA and re- The bound target DNAs in the NF-kB crystal direct them to the cytoplasm. structure models also display varying degrees of The three-dimensional structures of the IkBa:NF- bending: the IFNb DNA being the straightest (&38), kB RH domain protein complex has been determined uPAkB DNA is the most bent (4208), and IgkB DNA (Huxford et al., 1998; Jacobs and Harrison, 1998). is intermediate (&148). NMR studies have been done The six ankyrin repeats of IkBa bind asymmetrically on the HIV-1 LTR 5'-GGGACTTTC-3' and a mutated to the p50-RelA heterodimer and each ankyrin repeat kB DNA sequence (Tisne et al., 1998, 1999). The wild- contains an outer and inner helix and a loop of type kB site exists in an equilibrium between being variable sequence, followed by a short beta hairpin. inherently straight or bent 208 towards the major This is consistent with other ankyrin-repeat domain groove while the mutations, biochemically observed to proteins such as GABP (Batchelor et al., 1998) and abrogate protein binding (Nabel and Baltimore, 1987), p53BP (Gorina and Pavletich, 1996). The helices
Figure 3 Variable Rel/NF-kB conformations in the N' terminal domains as a result of DNAs with diering global conformations Rel/NF-kBstructure FE Chen and G Ghosh 6850 stack on one another and form a framework from Future directions which the loop and beta turn `®ngers' extend and mediate most of the contacts with the RelA protein There are still numerous questions that need to be (Figure 4). The length of variable protein between answered about the Rel/NF-kB proteins 13 years after each ankyrin repeat also makes contacts to RelA. their ®rst discovery (Sen and Baltimore, 1986). In light These contacts neatly explain the observed preference of the role that phosphorylation plays in protein of IkBa for RelA-containing homo- and heterodi- regulation, additional biochemical and structural work mers. Also observed in the structure are electrostatic with phosphorylated Rel/NF-kB and IkB proteins will interactions between the sixth ankyrin repeat and the be useful. It will be interesting to observe the eect of acidic/PEST tail with the p65 NTD that eects a Rel/NF-kB and protein conformation by testing the large rotation away from the DNA-binding cleft and transcriptional activity of a kB site in the context of buries key DNA-binding residues and, thus, prohibits dierent promoters. The mechanism of v-Rel oncogen- DNA binding. Phosphorylation also plays a likely esis remains to be fully elucidated and requires role in the regulation of these interactions as the biochemical experiments to thoroughly explain the PEST sequence is phosphorylated by casein kinase II eects of the v-Rel mutations on DNA binding and (McElhinny et al., 1996). IkB interaction. Also of particular use will be
a Rel/NF-kB structure FE Chen and G Ghosh 6851 b
Figure 4 (a) Ribbon diagram of IkB with the sixth ankyrin repeat and PEST sequence labeled in gray. (b) Schematic of IkB interaction with heterodimer and the movement of the p65 NTD
biochemical and structural work on multiprotein:DNA Acknowledgements complexes to further enumerate what dierent inter- The authors thank AK Aggarwal for generously providing protein forces are present and how they ultimately vary the coordinates of the NF-kB:IFN kBDNAcomplex. the levels of transcription. There are many such Thanks are also extended to T Huxford for the ®gure of systems to investigate such as the NF-kB hetero- IkBa. This work was supported by fellowships from the NSF and the Lucille P Markey Charitable Trust to F dimer:Sp1 complex on the HIV-1 long terminal repeat Chen. This work was also supported by a NCI research promoter or the much larger IFNb enhanceosome. grant (CA71871) to G Ghosh. With the ever-increasing understanding of the roles Rel/NF-kB proteins play in various physiological processes, it is apparent that its mediation of transcription is both layered and complex.
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