©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Similar DNA-binding Motif in NFAT Family Proteins and the Rel Homology Region (*)

(Received for publication, August 12, 1994; and in revised form, November 7, 1994)

Jugnu Jain (1)(§)(¶) Emmanuel Burgeon (1)(§) Tina M. Badalian (1) Patrick G. Hogan (2) Anjana Rao (1)(**)

From the  (1)Dana-Farber Cancer Institute, Department of Pathology, and (2)Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cyclosporin-sensitive factor NFATp cooperates with Fos and Jun family proteins to regulate transcription of the interleukin 2 gene in activated T cells. We have defined a 187-amino-acid fragment of NFATp, located centrally within the protein sequence, as the minimal region required for DNA binding and for complex formation with Fos and Jun. The sequence of this region of NFATp shows a low degree of similarity to the Rel homology region. One specific short sequence in NFATp (RAHYETEG), located near the NH(2) terminus of the DNA-binding domain, resembles a highly conserved sequence (RFRYxCEG) that is located near the NH(2) terminus of the Rel homology region and that has been implicated in DNA binding by Rel family proteins. Mutational analysis demonstrates that the residues in this sequence that are identical in NFATp and Rel family proteins contribute to DNA binding by NFATp. Further, mutation of the threonine residue in this sequence to cysteine, as in Rel proteins, confers on NFATp a sensitivity to sulfhydryl modification similar to that of Rel family proteins. The results suggest that NFATp and Rel family proteins bind to DNA using similar structural motifs.


INTRODUCTION

The cyclosporin-sensitive factor NFAT (nuclear factor of activated T cells) is implicated in the inducible transcription of the interleukin 2 (IL2) (^1)gene, and possibly other cytokine genes, in activated T cells (reviewed in (1) ). The DNA binding specificity of NFAT is determined in part by NFATp, a 120-kDa phosphoprotein that is present in the cytosolic fraction of unstimulated T cells(2, 3) . Following T cell stimulation, NFATp appears in nuclear extracts via a calcium-dependent step that is inhibited by the immunosuppressive drugs cyclosporin A and FK506 (4, 5, 6) . (^2)These compounds act as a complex with their respective intracellular receptors to inhibit the enzymatic activity of the calcium- and calmodulin-dependent phosphatase calcineurin (7, reviewed in (8) ). Overexpression of calcineurin or expression of a constitutively active form of the calcineurin A chain in JURKAT T cells substitutes at least partially for the calcium signal required for NFAT-dependent reporter gene transcription(9, 10) , confirming that calcineurin plays a key role in the pathway of T cell activation. Together these observations have led to the hypothesis that calcineurin regulates cytokine gene transcription by influencing, directly or indirectly, the translocation of NFATp from the cytosol to the nucleus of stimulated T cells (reviewed in Refs. 1, 11).

The IL2 promoter contains two ``composite'' binding sites (12) for NFAT, which bind NFATp (or the related family member NFATc; 13) in association with members of the Fos and Jun families of transcription factors(14) . Participation of Fos and Jun may be required for transcription mediated by the distal site in transiently transfected cells(5, 15, 16) . (^3)Assembly of the active NFAT complex on the IL2 promoter NFAT site involves cooperative interactions among NFATp/c, Fos, and Jun. c-Fos (Fos) and c-Jun (Jun) show no binding to the site in the absence of NFATp(14) ; correspondingly, the DNAbulletprotein complex containing NFATp alone is far less stable than the complex containing all three proteins(6) . Assembly of the functional complex also requires site-specific interactions of all three proteins with each other and with DNA.^3 Using chemical cross-linkers, we have demonstrated direct protein-protein interaction between NFATp and Fos/Jun or Jun/Jun dimers in the presence of the appropriate DNA-binding site.^3 Additionally, mutation of the core NFATp binding sequence (GGAAAA) in the IL2 promoter NFAT site entirely eliminates the function of the site, as does mutation of an adjacent non-canonical AP-1 site that is not essential for NFATp binding but that is required for formation of the NFATp-Fos-Jun complex(6, 15) .^3 The minimal DNA binding regions of Fos and Jun, comprising their bZIP (basic region-leucine zipper) domains, are necessary and sufficient for the formation of a complex with NFATp(14) . However, the regions required for DNA binding and interaction with Fos and Jun have not been mapped for either NFATp or NFATc.

Analysis of cDNA clones encoding NFATp has indicated that NFATp is a member of a novel family of DNA-binding proteins, related by alternative splicing at their C termini (3) . (^4)The DNA-binding domain is contained within a central 464-amino-acid region of the protein that is common to all the alternatively spliced forms(3) . Within this region, there is a span of 300 amino acids that shows a low level of sequence identity with the Rel homology region (RHR) previously recognized in Rel family proteins. The sequence of this region of NFATp is well conserved in the newly identified family member NFATc(13, 17) . However, there has been no direct evidence that similar regions of NFATp and Rel proteins share corresponding functions.

Our objective in these experiments was 2-fold: to identify the smallest stable DNA-binding fragment of NFATp for use in structural studies of NFATp-DNA and NFATp-Fos-JunbulletDNA complexes, and to explore more closely the correspondence of the NFATp DNA-binding domain and the Rel homology region. We show that a centrally located 187-amino-acid fragment of NFATp, contained within the portion of NFATp that is related to the Rel homology region, constitutes the minimal region required for specific binding to DNA and for interaction with Fos and Jun. The amino terminus of this fragment contains a sequence similar to a highly conserved sequence motif at the amino terminus of the RHR, and the residues of this motif that are common to NFATp and the RHR contribute to DNA binding by NFATp as well as Rel family proteins. The results suggest that NFATp and Rel family proteins use related structural motifs for binding to DNA.


EXPERIMENTAL PROCEDURES

Introduction of Deletions and Mutations into the NFATp cDNA

Sequential deletions were performed from the 5` and 3` ends of the NFATpXS(1-464) insert (3) in the bacterial expression vector pQE-31 (Qiagen), using exonuclease III (ExoIII) as recommended by the manufacturer (Erase-a-base kit, Promega). Deletions from the 5` end of the cDNA insert were performed using the KpnI site in the pQE-31 polylinker as the ExoIII-resistant site, and either the ApaI site or the BglII site in the XS(1-464) clone as the ExoIII-sensitive sites. For deletions from the 3` end of the insert, the plasmid was first digested with HindIII, the cleaved site was repaired with alpha-phosphorothioate dNTPs to render the repaired site ExoIII-resistant, and the plasmid was then digested with SalI to produce an ExoIII-sensitive end. The resulting linear DNAs were blunt-ended using S1 nuclease, recircularized, and used to transform competent Escherichia coli (strain DH5alpha; Stratagene). DNA from selected bacterial colonies was sequenced to determine the length and translation frame of the truncated cDNAs. Plasmids carrying suitable truncated cDNAs were used to transform E. coli strain M15(pREP4), and at least three independent colonies were picked for protein purification and further analysis (Qiagen).

Individual codons of NFATp were altered by oligonucleotide-directed mutagenesis(18) , using uracil-labeled single-stranded DNA of the NFATp cDNA clone in pBluescript SK (Stratagene) as template. For construction of NFATpKEB, the KpnI-MluI fragment of the wild-type or mutated NFATp cDNA was subcloned into NFATpXS(1-297), replacing a KpnI-MluI fragment that extends from the KpnI site of the pQE-31 polylinker to the MluI site of the insert. The resulting wild-type cDNA encodes a hexahistidine-tagged protein (NFATpKEB) with 25 additional amino acids of NFATp NH(2)-terminal to the sequence of NFATpXS(1-297). The presence of the desired mutation was confirmed by sequencing the entire KpnI-MluI fragment of each mutated NFATpKEB construct. In examining the effects of each mutation, proteins expressed by at least three independent bacterial colonies were purified and analyzed for binding to the NFAT oligonucleotide.

For insertion of the T C mutation into NFATpXS(1-297), the ApaI-MluI fragment of the mutated NFATp cDNA was used to replace the corresponding fragment of NFATpXS(1-297).

Expression and Purification of Recombinant Proteins

NFATpXS(1-464), other NFATp fragments, and NFATp fragments carrying single amino acid substitutions were expressed in bacteria as hexahistidine-tagged proteins. Typically, 100 ml of LB medium containing 100 µg/ml ampicillin and 25 µg/ml kanamycin was inoculated with 2 ml of an overnight culture and grown at 37 °C until the OD reached 0.7-0.9. The 100-ml culture was divided into two equal portions, isopropyl-beta-D-thiogalactopyranoside was added to a final concentration of 1-2 mM to one portion, and the bacteria were allowed to grow for another 3-4 h. Both uninduced and induced cultures were then harvested by centrifugation at 4000 times g for 10-20 min, and the bacterial pellets were either processed the same day or stored at -70 °C. Purifications were carried out by extracting bacterial proteins in 8 M urea, 5 mM 2-mercaptoethanol, 0.1 M sodium phosphate, 10 mM Tris-HCl pH 8.0 (urea-2ME), incubating the extract with nickel-chelate resin (Ni-NTA agarose; Qiagen) in the same buffer, washing the column with 10 mM imidazole in the urea-2ME buffer, and eluting specifically bound hexahistidine-tagged proteins with 100 mM imidazole in the urea-2ME buffer. An aliquot of the eluted proteins was analyzed by SDS-polyacrylamide gel electrophoresis, and the remainder was dialyzed overnight against a buffer containing 20 mM HEPES pH 7.4, 1 mM dithiothreitol, 100 mM NaCl, 2 mM EDTA, 20% glycerol, 0.01% sodium azide, and protease inhibitors (20 µM leupeptin, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride). After dialysis the proteins were stored in small aliquots at -70 °C or in liquid nitrogen. Protein concentrations were determined using the Lowry et al.(19) protocol. In some of the early experiments, the purification was performed in the absence of any reducing agents: this procedure significantly decreased the specific activity of the purified proteins in the DNA binding assay, but did not alter the conclusions about the ability of specific fragments to bind to the NFAT oligonucleotide.

Recombinant c-Fos (Fos(118-211)C154S, C204S) and c-Jun (Jun(186-334)), expressed in E. coli and purified from bacterial lysates, were a kind gift of Drs. T. Kerppola and T. Curran (Roche Institute, Nutley, NJ). These recombinant proteins have been described previously(14, 20) .

Electrophoretic Mobility Shift Assays

Binding reactions (15 µl) contained 0.1 ng-0.1 µg of purified recombinant NFATp, 17 µg/ml poly(dI)bulletpoly(dC), 12 µg of bovine serum albumin, 6 µl of binding buffer (20 mM HEPES pH 7.5, 250 mM NaCl, 20% glycerol, 0.5 mM dithiothreitol), and 10,000 counts/min (0.2-0.5 ng) of labeled oligonucleotide corresponding to the distal NFAT site of the murine IL2 promoter. The higher concentrations of NFATp proteins were required if the proteins had been purified in the absence of reducing agents. Where indicated, a 200-fold excess of unlabeled oligonucleotide was included in the binding reaction. After incubation for 20 min at room temperature, free and bound probe were separated by electrophoresis under non-denaturing conditions on a 4% polyacrylamide gel at room temperature(21) . Association of recombinant NFATp proteins with Fos and Jun was tested by inclusion of 50 nM c-Jun and/or c-Fos in the binding reaction as described previously(3, 14) .

When testing the sensitivity to alkylation and oxidation, wild-type and mutant NFATp proteins (0.1-1 ng) were diluted in binding buffer and incubated on ice for 10 min. Diamide or iodoacetamide were then added to final concentrations of 0.39-25 mM from stock solutions that had been freshly prepared in water. After reaction for 10 min at room temperature, the radiolabeled probe was added and allowed to bind for a further 10 min before electrophoresis. The concentrations shown in Fig. 7, B and D, are the final concentrations of diamide and iodoacetamide without adjustment for the 0.2 mM dithiothreitol present in the binding reaction. These results were quantified using a Betagen Betascope (Waltham, MA). For each lane, the radioactivity in DNAbulletprotein complexes was divided by the total input radioactivity (the sum of the radioactivity in the DNAbulletprotein complexes and in the free probe) to obtain a value for the fraction of protein-bound oligonucleotide, and within each experiment these values were normalized relative to the fraction bound in the untreated control (100%).


Figure 7: Effect of sulfydryl modifying agents on DNA binding by NFATpXS(1-297) (WT) and its T C variant. A, electrophoretic mobility shift assay of proteins treated with increasing concentrations of iodoacetamide (IAM). Lanes 1-5 and 6-8 show two independent preparations of the T C variant, and lanes 9-11 show NFATpXS(1-297). B, diagram summarizing the effect of iodoacetamide on DNA binding. Each point is the average of three to seven experiments (bars indicate standard deviations), except for the wild-type protein treated with 25 mM IAM, which gives the mean and range of two experiments. C, electrophoretic mobility shift assay of proteins treated with increasing concentrations of diamide. Lanes 1-5 show wild-type NFATpXS(1-297), and lanes 6-10 show the T C variant. D, diagram summarizing the effect of diamide on DNA binding. Each point is an average of five or six experiments, except the point for 0.39 mM diamide, which is an average of three experiments. The bars indicate standard deviations.



Sequence Alignment

The amino acid sequences of Rel family proteins were obtained from the literature or from the GenBank data base. In addition to murine Rel(22) , the proteins and their GenBank accession numbers were human Rel (X75042), chicken Rel (X52193), turkey Rel (K02455), v-Rel (K00555), Xenopus laevis Xrel1 (M60785), murine RelA (M61909), human RelA (M62399), chicken RelA (D13721), murine NF-kappaB1 (M57999), human NF-kappaB1 (M55643), chicken NF-kappaB1 (M86930), human NF-kappaB2 (X61499), chicken NF-kappaB2 (U00111), murine RelB (M83380), human RelB (M83221), Drosophila melanogaster dorsal (M23702), and D. melanogaster Dif (L29015). The highly similar Rel protein sequences were aligned by visual inspection, and consensus residues were defined as those residues that were identical in at least 16 of the 18 Rel proteins. In each case the residue present in murine Rel matches the consensus.

An initial alignment of NFATpXS(1-297) with murine Rel(8-296), the Rel homology region, was made based on the correspondences (NFATp/Rel) RAHYETEG/RFRYKCEG near the NH(2) terminus, and VRLVFRVHVP/VRLCFQVFLP immediately after the variable segment of the Rel homology region. The alignment of NFATp and Rel was then further adjusted to preserve alignment of identical residues in NFATp with identified consensus residues in Rel. Specifically, the following sequences were brought into alignment (NFATp/Rel/consensus): IEVQP/IIEQP/IxEQP, HRITGK/HDLVGK/HxLVGK, SNPI/SNPI/SxxI, WE/WE/Wx, LFVEIPEY/IVFKTPPY/IVFxTPxx, PVKV/PVTV/PxxV, and SQPQHFTYHP/SESMDFRYLP/SxxxxFxYxP. Thus, for example, a single-residue gap was introduced to align an SxxxxFxYxP motif in NFATp with the same motif at the end of the Rel homology region. The consensus residues in this motif are invariant in the 18 Rel proteins. These further adjustments resulted in the introduction of four short gaps in the alignment (in either the NFATp sequence or the Rel sequence) that correspond to gaps introduced in aligning murine Rel with certain other Rel family proteins, and four other single-residue gaps including the example cited.


RESULTS

We have previously shown that a 464-amino-acid fragment of murine NFATp (NFATpXS(1-464), Fig. 1, center bar) binds specifically to the IL2 promoter NFAT site, alone or as a complex with recombinant Fos and Jun(3) . To map the regions of NFATp required for DNA binding and association with Fos and Jun proteins, we generated a series of truncated derivatives of NFATpXS(1-464). The hexahistidine-tagged proteins were expressed in bacteria and purified using a nickel-chelate column. A Coomassie Blue-stained SDS gel of selected purified proteins is shown in Fig. 2A. Proteins shorter than 40 kDa appeared homogeneous (lanes 4-9), whereas proteins longer than XS(1-297) or XS(151-464) showed evidence of partial degradation, which may have occurred either within the bacteria or during purification (lanes 2, 3, 10, and 11).


Figure 1: Schematic representation of a region of the NFATp cDNA and of key protein fragments aligned with the segments of the cDNA that encode them. The line at the top represents a 1.5-kilobase KpnI-SmaI fragment of the murine NFATp cDNA (3) with relevant restriction sites indicated. NFATpXS(1-464) is a DNA-binding fragment comprising 464 amino acid residues of NFATp(3) . NFATpKEB is a DNA-binding fragment consisting of 322 residues of NFATp, with its COOH terminus at position 297 of NFATpXS(1-464). The sequence RAHYETEG is present near the NH(2) terminus of both protein fragments. Residues analyzed by replacement with alanine (or, in one case, with cysteine) are marked by circles.




Figure 2: Properties of purified NFATpXS(1-464) and its COOH terminally and NH(2) terminally truncated derivatives. A, migration of the proteins on an SDS-polyacrylamide gel. Proteins were visualized by staining with Coomassie Brilliant Blue. Lane 1 contains Bio-Rad low molecular weight markers, and lane 12 contains Rainbow markers (Amersham Corp.). The molecular masses of the markers (kDa) are indicated. B, binding of purified NFATpXS(1-464) and COOH terminally truncated proteins to the IL2 promoter NFAT site, assessed by electrophoretic mobility shift assay. Arrows indicate the DNAbulletprotein complex of NFATpXS(1-464) and the free probe. The binding of recombinant dihydrofolate reductase (DHFR), expressed and purified in the same manner, was assessed as a negative control (lane 14).



The ability of the purified proteins to bind to the IL2 promoter NFAT site was assessed in an electrophoretic mobility shift assay (Fig. 2B). Residues near the COOH-terminal end of NFATpXS(1-464) are not required for specific DNA binding, since the COOH terminally truncated proteins XS(1-376), XS(1-329), XS(1-297), and XS(1-187) bound DNA efficiently (Fig. 2B, lanes 1-3, and 9). In contrast, the NH(2)-terminal region appeared essential for DNA binding, since deletion of as few as 29 amino acids from the NH(2) terminus of NFATpXS(1-464) abrogated DNA binding (data not shown). The XS(1-187) protein represented the minimal DNA-binding fragment of NFATp, since further COOH-terminal truncation of this protein yielded proteins that were no longer capable of binding DNA (Fig. 2B, lanes 10-12). As previously shown, purified NFATpXS(1-464) bound to the NFAT site (lane 13) whereas control extracts from bacteria expressing a hexahistidine-tagged dihydrofolate reductase, purified by the same method, did not bind (lane 14). Proteins intermediate in length between XS(1-297) and XS(1-187) showed a decreased ability to bind DNA independently in an electrophoretic mobility shift assay (Fig. 2B, lanes 4-8). This change reflects a lower binding affinity rather than a loss of ability to recognize the NFAT site, since these proteins could bind efficiently to the NFAT site in the presence of Fos and Jun (see below).

The specificity of DNA recognition by the truncated NFATp proteins was established by competition with excess unlabeled wild-type or mutated oligonucleotides (see Fig. 3A for sequences of the oligonucleotides). In each case, formation of the proteinbulletDNA complex was efficiently competed by the wild-type oligonucleotide and the M1 and M4 mutant oligonucleotides (Fig. 3B, lanes 2, 3, and 6; lanes 8, 9, and 12; and data not shown). The M2 mutant oligonucleotide competed partially (lanes 4, 10, and data not shown), whereas the M3 oligonucleotide, which bears a mutation in the essential ``GGAA'' core sequence, did not compete at all (lanes 5, 11, and data not shown). This pattern of competition is diagnostic for specific binding of NFATp, the NFATp-Fos-Jun complex, and recombinant NFATpXS(1-464) to the NFAT site of the IL2 promoter(3, 5, 6, 14) .


Figure 3: Specificity of binding of truncated NFATpXS proteins to the IL2 promoter NFAT site. A, nucleotide sequence of the murine NFAT oligonucleotide, and the base substitutions in the M1-M4 mutant oligonucleotides, which are otherwise identical to the NFAT oligonucleotide. The filled circles indicate the 2 guanine residues whose methylation interferes strongly with binding of NFATp, the open circle represents the guanine residue whose methylation interferes only partially with the binding of NFATp. The M1-M3 mutant oligonucleotides have been described(5) . The M3 mutation destroys the binding site for NFATp whereas the M4 mutation alters all the residues of the adjacent, noncanonical AP-1 site. B, electrophoretic mobility shift assays. Binding reactions contained radiolabeled NFAT oligonucleotide, purified NFATpXS(1-297) or NFATpXS(1-187), and a 200-fold excess of unlabeled competitor oligonucleotides as indicated. The portion of the gel containing free probe is not shown.



The truncated NFATp proteins were also evaluated for their ability to interact with Fos and Jun on the IL2 promoter NFAT site. A representative experiment using XS(1-297), XS(1-223), and XS(1-187) with truncated c-Fos and c-Jun proteins is shown in Fig. 4. All the NFATp fragments that were capable of binding independently to the NFAT site (see Fig. 2B) were also able to interact with Fos and Jun, as indicated by the appearance of DNAbulletprotein complexes (containing NFATp-Jun-Jun and NFATp-Fos-Jun) of reduced electrophoretic mobility (Fig. 4, lanes 3, 4 and 13, 14; and data not shown). Although proteins intermediate between XS(1-297) and XS(1-187) showed a pronounced decrease in independent DNA binding (Fig. 2B, lanes 4-8), they were able to form DNAbulletprotein complexes containing Fos and Jun (Fig. 4, lanes 8 and 9; and data not shown). However, the COOH terminally truncated proteins shorter than XS(1-187) and all the NH(2) terminally truncated proteins were incapable of DNA binding, even in the presence of Fos and Jun (data not shown). As expected from previous results(3, 14) , Fos alone was not able to interact with NFATp proteins to form DNAbulletprotein complexes (lanes 2 and 12), and there was no detectable binding of Fos and Jun to the IL2 promoter NFAT site in the absence of NFATp (lanes 5, 10, and 15). The NFATp-Jun-Jun and NFATp-Fos-Jun complexes bound with the correct specificity to the IL2 promoter NFAT site, as judged by competition with the panel of wild-type and mutated NFAT oligonucleotides shown in Fig. 3(data not shown). Fig. 5summarizes the properties of all the truncated NFATp proteins examined in this study.


Figure 4: Association of truncated NFATpXS proteins with Fos and Jun on the IL2 promoter NFAT site. Recombinant fragments encompassing the DNA-binding domains of Fos and Jun were added as indicated to binding reactions containing NFATpXS(1-297), NFATpXS(1-223), or NFATpXS(1-187), and radiolabeled NFAT oligonucleotide, and DNAbulletprotein complexes were analyzed by electrophoretic mobility shift assay. The difference in the intensity of the DNAbulletprotein complexes seen in lanes 1 and 2 is due to the use of a lower amount of NFATpXS(1-297) in lane 1; addition of Fos does not enhance the binding of NFATp.




Figure 5: Summary of the properties of bacterially expressed NFATp fragments. The shaded bars represent the regions of NFATp present in the recombinant proteins. To show precisely the position of each truncation in the NFATp sequence, the terminal amino acids of NFATp included in the recombinant proteins are indicated (in one-letter code) by letters within the bars, whereas vector-encoded amino acids are indicated by letters outside the bars. Only the last 3-5 vector-encoded amino acids are shown for the NH(2) terminally truncated proteins and for NFATpKEB; the complete vector-encoded sequence of NFATpXS(1-464) is MRGSHHHHHHTAPHASSV. A + in the DNA binding column indicates that the protein can bind to DNA but does not imply that all proteins bind with similar affinities. ± indicates that the protein binds weakly in the absence of Fos and Jun (Fig. 2B, lanes 4-8; Fig. 4, lane 6), whereas - indicates no detectable binding. A, COOH-terminal truncations. B, NH(2)-terminal truncations. C, NFATpKEB (also see Fig. 1).



NFATpXS(1-297) corresponds almost exactly to the region of NFATp that has been aligned with the Rel homology region(3, 17) . Moreover, the NH(2)-terminal region of NFATpXS, like the NH(2)-terminal region of the RHR, is essential for DNA binding. An RAHYETEG sequence in this region of NFATp resembles a sequence at the NH(2) terminus of the Rel homology region, RFRYxCEG, that is highly conserved among Rel family proteins (Fig. 6A) and that has been implicated in DNA binding(23, 24, 25, 26, 27, 28) .


Figure 6: Effect of substitutions in the RAHYETEG motif on DNA binding by NFATp. A, alignment of the sequence around the RAHYETEG motif, located near the NH(2) terminus of the DNA-binding domain of NFATp, with the corresponding sequences around the conserved RFRYxCEG motif in representative Rel family proteins. Citations for the Rel protein sequences are given under ``Experimental Procedures.'' B, migration of purified mutant proteins on an SDS-polyacrylamide gel. NFATpXS(1-297), NFATpKEB (the parent protein into which substitutions were introduced), and the mutant proteins were separated on a 15% SDS-polyacrylamide gel and visualized by staining with Coomassie Brilliant Blue. Protein markers are shown in lane 1, and their molecular masses (kDa) are indicated. C, binding of the purified wild-type and mutant proteins to the IL2 promoter NFAT site, assessed by electrophoretic mobility shift assay. The arrow indicates the DNAbulletprotein complexes. The faint slower-migrating complexes in lanes 3, 5, and 8 are nonspecifically bound, as judged by competition with the panel of unlabeled oligonucleotides shown in Fig. 3.



We examined by site-directed mutagenesis whether amino acid residues in the RAHYETEG sequence of NFATp contributed to DNA binding. Individual residues in the RAHYETEG sequence were replaced with alanine, to minimize the possibility of causing global alterations in protein structure. Among the residues mutated, Arg, Tyr, and Glu in NFATp (numbering based on NFATpXS) are identical to the residues at the corresponding positions in Rel family proteins, while the residues at positions corresponding to His and Thr in NFATp are conserved among Rel family proteins, but differ from the residues in NFATp (Fig. 6A). Residue Glu, at a position corresponding to the variable residue in the Rel motif RFRYxCEG, was omitted from this analysis, as were the 2 residues (Ala and Gly) for which mutation to alanine was not appropriate. These mutations were introduced into NFATpKEB (Fig. 1, lower bar), an NFATp fragment that contains at its amino terminus an additional 25 amino acids of NFATp not present in XS(1-297), and the proteins were expressed in bacteria and purified as already described (Fig. 6B). In electrophoretic mobility shift assays, NFATpKEB proteins in which the Arg, Tyr, or Glu residues had been mutated to alanine showed a striking decrease in their ability to bind to DNA (Fig. 6C, lanes 3, 5, and 8). Binding of these proteins to the NFAT site was undetectable even when binding was assayed in the presence of Fos and Jun (data not shown). On the other hand, proteins in which the His and Thr residues had been mutated to alanine were still capable of binding to DNA (Fig. 6C, lanes 4 and 6). The specificity of DNA binding of the latter two mutant proteins was established by competition with wild-type and mutated NFAT oligonucleotides (data not shown), exactly as shown for the truncated proteins in Fig. 3B.

Thr in the RAHYETEG sequence of NFATpXS corresponds to the conserved cysteine residue in the RFRYxCEG sequence of Rel family proteins (Cys in human NF-kappaB1, Cys in RelA; see Fig. 6A). When wild-type Rel family proteins, which contain cysteine at this position, are alkylated with N-ethylmaleimide or iodoacetate, or oxidized with diamide, they show a severe decrease in their ability to bind to DNA; in contrast, alkylation of proteins in which this residue has been mutated to serine has much less effect(23, 24, 26) . We therefore mutated Thr in both NFATpXS(1-297) and NFATpKEB to cysteine and compared the wild-type and mutated (T C) proteins for DNA binding and sensitivity to sulfhydryl modification. The T C mutant of each protein bound DNA at least as well as the wild-type protein (Fig. 6C, lane 7). When binding of the two proteins to the IL2 promoter NFAT site was compared over a range of concentrations, the T C mutant required 5-fold lower concentrations than the wild-type protein for equivalent binding (data not shown). However DNA binding of the T C mutant was strikingly sensitive to alkylation with iodoacetamide (Fig. 7A, lanes 1-8; Fig. 7B), whereas DNA binding of wild-type NFATp was barely affected by iodoacetamide at concentrations up to 25 mM (Fig. 7A, lanes 9-11; Fig. 7B). Similarly, the DNA binding of the T C mutant was effectively abolished by the lowest concentration (0.4 mM) of diamide tested (Fig. 7C, lanes 7-10); in contrast, much higher concentrations (3 mM) of this nonspecific oxidizing agent were required to affect the DNA binding of the wild-type protein (Fig. 7C, lanes 2-5; Fig. 7D). The loss of activity of the wild-type protein following treatment with diamide but not iodoacetamide probably reflects variable formation of intra- and intermolecular disulfide bonds with this oxidizing agent.


DISCUSSION

Definition of the Minimal DNA-binding Domain of NFATp

We have identified the minimal DNA-binding domain of NFATp as a 187-amino-acid region located centrally within the protein. Small deletions (20-30 amino acids) at either end of this minimal region eliminate the ability of NFATp to bind the IL2 promoter NFAT site, either independently or in the presence of Fos and Jun, suggesting that residues near both ends of the minimal fragment contribute to DNA binding. We have confirmed by site-directed mutagenesis that residues near the NH(2) terminus of this minimal fragment are important for DNA binding. Further examination of residues located near the COOH terminus of the minimal fragment is needed to establish whether they are involved directly in DNA binding, or whether they have other functions such as maintaining the correct DNA binding conformation of the protein. While residues 188-297 of NFATpXS are not essential for high affinity DNA binding, it remains possible that this region makes an additional weaker contribution to DNA binding.

The residues of NFATp required for Fos-Jun interaction are contained within its minimal DNA-binding domain, since truncated proteins capable of binding DNA but unable to interact with Fos and Jun were not obtained. Similarly, the minimal DNA-binding domains of Fos and Jun are in general sufficient for assembly of the multimeric NFATp-Fos-JunbulletDNA complex(14) , although an acidic region of Fos may be required under certain conditions(29) . Given the resemblance between NFATp and Rel family proteins (see below), it is interesting that RelA and NF-kappaB1 can bind to and cooperate functionally with proteins of the C/EBP family, which like Fos and Jun bind to DNA via a basic region-leucine zipper (bZIP) motif(30, 31, 32) , and that RelA can also interact with Fos and Jun(33) . As with the NFATp-Fos-Jun interaction, the Rel-bZIP interaction requires the DNA binding and dimerization domains of the transcription factors involved(30, 31, 33) . There is also evidence that DNA itself plays a central role in the formation of the NFATp-Fos-Jun complex. Mutation of either the core NFATp-binding site (GGAAAA) or the adjacent non-consensus AP-1 site abrogates assembly of the complex(14) ,^3 and the DNAbulletprotein complex containing all three proteins is much more stable than the complex containing NFATp alone(6) . In fact, we have not detected interactions between NFATp and Fos-Jun (or Jun-Jun) dimers in the absence of DNA.^3

A DNA Binding Motif (RFRYxCEG/RAHYETEG) shared by NFATp and Rel Family Proteins

Analysis of DNA binding by the truncated proteins focused attention on the NH(2) terminus of NFATpXS(1-464), where the removal of 29 amino acids converted this DNA-binding fragment of NFATp to a fragment incapable of binding DNA. Fig. 6A and Fig. 8show that this region at the NH(2) terminus of the DNA-binding domain of NFATp aligns with a region critical for DNA binding in NF-kappaB1, RelA, and v-Rel, that is correspondingly located at the NH(2) terminus of the Rel homology region.


Figure 8: Comparison of the amino acid sequences of murine NFATpXS(1-297) (3) and the Rel homology region of murine Rel(22) . Identities between NFATp (upper sequence) and Rel (lower sequence) are indicated by vertical lines. Residues in Rel that are consensus residues for the Rel family (see ``Experimental Procedures'') are marked with arrowheads. Portions of the two sequences delimited by double dashes (=) correspond to a previously recognized segment within the Rel homology region whose length and sequence are variable among Rel family proteins. There are 48 identities between NFATp and Rel among 289 Rel residues shown, and 30 of 94 Rel consensus residues are conserved.



There is strong evidence that this region in Rel proteins is a site of DNA contact. Mutations within or immediately adjacent to this region affect binding of v-Rel, NF-kappaB1, and RelA to DNA(23, 25, 26, 27, 28) . Single amino acid substitutions at Arg, Phe, Arg, Tyr, or Glu of human NF-kappaB1, or at Tyr or Glu of RelA, reduce binding to a labeled oligonucleotide probe (26, 28; see Fig. 6A for numbering). Replacement of Val in NF-kappaB1 with a glutamate residue increases binding to a kappaB oligonucleotide(26) . Rel proteins with a substitution of serine for cysteine in the RFRYxCEG motif (at Cys of NF-kappaB1, or at Cys of v-Rel) are able to bind to their specific sites, but have lost their sensitivity to sulfhydryl reagents (23, 24, 26) . Additional residues flanking the central motif, including for example His and Leu of NF-kappaB1, contribute to DNA binding(26, 27, 28) . Consistent with a direct role in DNA recognition, His of NF-kappaB1 and other variable residues within this region influence the specificity of Rel proteins for kappaB sites. Thus an H A mutant at position 67 of NF-kappaB1 shows 50-fold reduced affinity for the major histocompatibility complex kappaB site, considered to be a NF-kappaB1 selective site, but only 2-fold-reduced affinity for the beta-interferon kappaB site(28) . Conversely, concerted replacement of the variable residues Met, Lys, Arg, and Ala of RelA with the corresponding residues from NF-kappaB1 converts the site preference of the altered protein to resemble that of NF-kappaB1(27) .

Cross-linking and covalent modification provide complementary evidence that this region of Rel proteins is close to DNA. In NF-kappaB1, residues Tyr and His can be cross-linked to BrdU bases inserted at specific positions into the beta-interferon site(28) . In NF-kappaB1 and v-Rel, chemical modifications of the conserved cysteine residue, including reaction with iodoacetate, reaction with N-ethylmaleimide, and oxidation of sulfydryl groups, inhibit DNA binding(23, 24, 26) . Conversely, the essential cysteine residue in the RFRYxCEG motif is protected from modification with N-ethylmaleimide or with iodoacetate in the presence of oligonucleotide(23, 24) , consistent with the interpretation that this region of the protein is closely apposed to DNA.

The DNA binding motif RFRYxCEG in Rel proteins aligns with the sequence RAHYETEG in NFATp, and our data suggest a close similarity in function. Single residue changes at Arg, Tyr, and Glu in the RAHYETEG sequence of NFATpXS interfere with DNA binding. These residues in NFATp are identical to the conserved residues at the corresponding positions in the Rel motif. NFATp with an H A substitution binds with high affinity to DNA, and thus differs from NF-kappaB1 with an R I substitution at the corresponding position(26) . This finding does not rule out the use of a similar binding motif by NFATp and Rel proteins, since binding of mutated NFATp was assessed using the IL2 NFAT site, while binding of the R I mutant of NF-kappaB1 was assayed using the immunoglobulin enhancer kappaB site. The available data on Rel proteins are not extensive, and it is also conceivable that some amino acid substitutions, but not R I, can be accommodated at this position in Rel proteins without a loss of function.

Consistent with the findings for Rel proteins, the Thr residue in NFATpKEB may be mutated, to an alanine or cysteine residue in our experiments, without loss of DNA binding. The T C substitution in NFATpKEB or NFATpXS(1-297) renders DNA binding sensitive to sulfhydryl reagents, extending the similarity between NFATp and Rel proteins. Even the acidic residue Glu of NFATpXS, which differs most strikingly from the corresponding residues in NF-kappaB1, RelA, and Rel, can be accommodated in the context of Rel proteins, since the conserved motif in murine Rel B, human Rel B, and dorsal is RFRYECEG (34-36; Fig. 6A), and since, as noted above, replacement of Val in NF-kappaB1 with a glutamate residue increases binding to a kappaB oligonucleotide(26) .

Our data suggest that the sequence RAHYETEG is a site of DNA contact for NFAT family proteins. An identical sequence is present in NFATc (13) , near the NH(2) terminus of a 270 amino acid region that shows 75% identity with NFATp. The effects of amino acid substitutions and covalent modifications provide evidence of a functional correspondence between NFAT family and Rel family members, extending the observation that there is a weak resemblance of a segment of NFATp and NFATc to the Rel homology region (3, 13, 17) and that NFATp can bind to NF-kappaB sites in the immunoglobulin kappa enhancer (37) and the tumor necrosis factor alpha promoter(38, 39) .

The Extended Alignment of NFATp and the Rel Homology Region

The alignment of the DNA-binding fragment NFATpXS(1-297) with c-Rel and other Rel proteins extends over the entire Rel homology region, and portions with conserved residues are interspersed with regions in which the sequences have little identity (Fig. 8). Our evidence indicates that the correspondence between the sequences RAHYETEG and RFRYxCEG may reflect a similar DNA-binding site. The question of whether the other limited sequence similarities reflect similar protein folding, or conserved protein-protein or protein-DNA interactions, will be resolved by further functional mapping of these proteins.

A documented protein-protein interaction of the Rel proteins is dimerization, and some of the residues essential for dimerization are in the COOH-terminal part of the RHR(25, 40, 41) . The corresponding region of NFATp is apparently dispensable for DNA binding as well as for interaction with Fos and Jun. A simple interpretation would be that NFATp can bind to some sites in DNA as a monomer, possibly stabilizing the binding of the monomer by making additional contacts on DNA outside the RAHYETEG sequence. In fact, cross-linking studies indicate that there is no obligate requirement for dimerization of NFATp before DNA binding.^3

Conclusion

This study has analyzed the DNA-binding domain of NFATp by a series of nested truncations of the protein, by amino acid substitutions, and by covalent modification. The results define a minimal DNA-binding domain of 187 amino acids that is located centrally in the protein. Identification of this functional fragment of NFATp provides the appropriate starting material for structural studies on the NFATpbulletDNA complex and on the NFATp-Fos-JunbulletDNA complex. The detailed examination of one region that appears critical for DNA binding has raised the possibility that protein-DNA contacts made by NFATp and other NFAT family proteins resemble those made by Rel family proteins.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants CA42471 and GM46227 (to A. R.) and grants from Hoffmann-La Roche, Inc. (to A. R. and P. G. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

Supported by a Medical Foundation fellowship.

**
Scholar of the Leukemia Society of America. To whom correspondence should be addressed: B465, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-375-8265; Fax: 617-375-8303.

(^1)
The abbreviations used are: IL2, interleukin 2; RHR, Rel homology region.

(^2)
K. T-Y. Shaw, A. M. Ho, A. Raghavan, A. Rao, and P. G. Hogan, manuscript in preparation.

(^3)
L. Chen, J. Jain, M. G. Oakley, P. B. Dervan, A. Rao, P. G. Hogan, and G. L. Verdine, manuscript in preparation.

(^4)
C. Luo, E. Burgeon, J. Carew, T. Badalian, P. G. Hogan, and A. Rao, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Tom Curran and Tom Kerppola for recombinant Fos and Jun proteins; Peter Cockerill for suggesting a modification to the procedure for purifying recombinant proteins; and Lin Chen, Greg Verdine and Ulrich Siebenlist for discussions.


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