(Received for publication, August 12, 1994; and in revised form, November 7, 1994)
From the
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 terminus of the
DNA-binding domain, resembles a highly conserved sequence
(RFRYxCEG) that is located near the NH
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.
The cyclosporin-sensitive factor NFAT (nuclear factor of
activated T cells) is implicated in the inducible transcription of the
interleukin 2 (IL2) ()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) . (
)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) . ()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 DNA
protein
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.
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.
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) .
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) . ()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-JunDNA
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.
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
-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).
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) .
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 DNAprotein complexes was divided by the total
input radioactivity (the sum of the radioactivity in the
DNA
protein 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.
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 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.
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
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 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 DNA
protein 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-terminal region appeared
essential for DNA binding, since deletion of as few as 29 amino acids
from the NH
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
proteinDNA 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
DNAprotein 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 DNA
protein 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
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 DNA
protein
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 DNAprotein complexes were analyzed by
electrophoretic mobility shift assay. The difference in the intensity
of the DNA
protein 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 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
-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-terminal
region of NFATpXS, like the NH
-terminal region of the RHR,
is essential for DNA binding. An RAHYETEG sequence in this region of
NFATp resembles a sequence at the NH
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 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 DNA
protein 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-
B1, 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.
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-JunDNA 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-
B1 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) ,
and the DNA
protein 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.
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-B1, and RelA to
DNA(23, 25, 26, 27, 28) .
Single amino acid substitutions at Arg
, Phe
,
Arg
, Tyr
, or Glu
of human
NF-
B1, 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-
B1 with a glutamate residue increases binding to a
B oligonucleotide(26) . Rel proteins with a substitution
of serine for cysteine in the RFRYxCEG motif (at Cys
of NF-
B1, 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-
B1, contribute to DNA
binding(26, 27, 28) . Consistent with a
direct role in DNA recognition, His
of NF-
B1 and
other variable residues within this region influence the specificity of
Rel proteins for
B sites. Thus an H
A mutant at position 67
of NF-
B1 shows 50-fold reduced affinity for the major
histocompatibility complex
B site, considered to be a NF-
B1
selective site, but only 2-fold-reduced affinity for the
-interferon
B site(28) . Conversely, concerted
replacement of the variable residues Met
,
Lys
, Arg
, and Ala
of RelA with
the corresponding residues from NF-
B1 converts the site preference
of the altered protein to resemble that of NF-
B1(27) .
Cross-linking and covalent modification provide complementary
evidence that this region of Rel proteins is close to DNA. In
NF-B1, residues Tyr
and His
can be
cross-linked to BrdU bases inserted at specific positions into the
-interferon site(28) . In NF-
B1 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-
B1 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-
B1 was
assayed using the immunoglobulin enhancer
B 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-
B1, 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-
B1 with a
glutamate residue increases binding to a
B
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 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-
B sites in the immunoglobulin
enhancer (37) and the tumor necrosis factor
promoter(38, 39) .
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.