From the Department of Biochemistry, University of Missouri at Columbia, Columbia, Missouri 65212
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ABSTRACT |
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The transcription factor GA-binding protein
(GABP) is composed of two subunits, GABP and GABP
. The
DNA-binding subunit, GABP
, is a member of the Ets family of
transcription factors, characterized by the conserved Ets-domain that
mediates DNA binding and associates with GABP
, which lacks a
discernible DNA binding domain, through ankyrin repeats in the
NH2 terminus of GABP
. We previously demonstrated
that GABP is subject to redox regulation in vitro and
in vivo through four COOH-terminal cysteines in GABP
. To
determine the roles of individual cysteines in GABP redox regulation, we generated a series of serine substitution mutants by site-directed mutagenesis and identified three redox-sensitive cysteine residues in
GABP
(Cys388, Cys401, and
Cys421). Sulfhydryl modification of Cys388 and
Cys401 inhibits DNA binding by GABP
, whereas,
modification of Cys421 has no effect on GABP
DNA binding
but inhibits dimerization with GABP
. The positions of
Cys388 and Cys401 within the known Ets-domain
structure suggest two very different mechanisms for redox regulation of
DNA binding. Sulfhydryl modification of Cys388 could
directly interfere with DNA binding or might alter the positioning of
the DNA-binding helix 3. Modification of Cys401 may inhibit
DNA binding through stabilization of an inhibitory helix similar to
that described in the Ets-1 protein. Thus, GABP is regulated through at
least two redox-sensitive activities, DNA binding and
heterodimerization.
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INTRODUCTION |
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Transcription in eukaryotes depends on at least two groups of proteins, general transcription factors and activator proteins (1, 2). While the former direct basal transcription, the later are responsible for cell-, tissue-, and gene-specific expression in response to various stimuli (3, 4). Many activator proteins are the end point of complex signal transduction pathways, and their activities are tightly regulated by various post-transcriptional modifications, including phosphorylation, glycosylation, and reduction/oxidation (redox)1 modification of cysteine residues (3, 5, 6).
The regulation of activator proteins by redox of reactive cysteine
residues has been demonstrated for members of several important transcription factor families including, NFB and AP-1 (for review, see Ref. 6). Both proteins require reducing conditions for DNA binding
in vitro; however, in vivo, these factors become
activated by oxidative stress-promoting agents (Ref. 6, and references therein). To explain this apparent contradiction, it has been proposed
that some effectors of pro-oxidant conditions may activate either the
expression or activity of specific enzymes (thioredoxin, Ref. 1) which
maintain NF
B and AP-1 in the reduced state (7, 8). Other pro-oxidant
effectors, such as reactive oxygen species (ROS), activate protein
kinases that 1) phosphorylate I
B leading to its inactivation and
concomitant activation of NF
B (6), and 2) phosphorylate Jun and Fos
proteins activating AP-1 activity (6, 9, 10).
Recently we demonstrated that murine GABP is redox-regulated both
in vitro and in vivo and that pro-oxidant
conditions, in contrast to NFB and AP-1, result in inhibition of
GABP DNA binding through COOH-terminal cysteine residues in the GABP
subunit (11). GABP is composed of two subunits, GABP
and GABP
,
and each subunit provides distinct functions to the complex (12, 13).
GABP
belongs to the Ets-protein family and is characterized by an
~85-amino acid region near the COOH terminus (Ets-domain) which is
necessary for DNA binding to the sequence (A/C)GGA(A/T)(A/G) (14, 15, 16). GABP
has no discernible DNA binding domain and is unable to
bind DNA on its own; however, GABP
contains at least one
transcription activation domain required for transcription activation
through the heteromeric complex (17-19). In addition, GABP
contains
two regions involved in protein-protein interactions, four ankyrin repeats at the NH2 terminus and a leucine-zipper at the
COOH terminus, which mediate GABP
-
and GABP
-
interactions,
respectively (12, 13, 17).
Transcription factors homologous to GABP have been discovered in
Xenopus laevis (20) and in human cells, including human nuclear respiratory factor 2 (NRF2) (21) and adenovirus E4 gene transcription factor 1 (E4TF1) (22). GABP/NRF2 has been shown to bind
to or regulate several promoters of genes encoding proteins involved in
oxidative phosphorylation, such as the ATP synthase subunit (23),
cytochrome oxidase subunit IV (COXIV) (24), cytochrome oxidase subunit
Vb (COXVb) (21), and the mitochondrial transcription factor 1 (mtTF-1)
(25). Since mtTF-1 in turn activates the transcription of
mitochondrial-encoded oxidative phosphorylation genes, a role for
GABP/NRF2 in coordination of nuclear and mitochondrial transcription of
oxidative phosphorylation genes has been proposed (25, 26).
The nature of signaling between mitochondria and the nucleus affecting
the regulation of nuclear-encoded mitochondrial genes is not known, but
likely candidates include reactive oxygen species (for review, see Ref.
27). Approximately 1-2% of the oxygen consumed during respiration is
only partially reduced, forming reactive oxygen species including
superoxide, hydrogen peroxide, and hydroxyl radicals (27, 28). The
inhibition of the terminal steps of the respiratory chain, including
cytochrome C oxidase and ATPase (complexes IV and V), by drugs or due
to mutation, leads to increased production of superoxide and hydrogen
peroxide (27, 28). ROSs may directly oxidize redox-sensitive sulfhydryl groups in the COOH-terminal portion of GABP and therefore inhibit GABP
DNA binding and GABP
-dependent transcription,
providing a mechanism linking the activity of the respiratory chain
with the expression of its components.
To characterize the mechanisms of redox sensitivity of the GABP
protein, we performed site directed mutagenesis of cysteine residues in
the GABP
DNA-binding and dimerization domains. We demonstrated that
two cysteine residues in the Ets DNA binding domain, Cys388
and Cys401, are sensitive to redox changes affecting
GABP
DNA binding while Cys421 in the GABP
/GABP
dimerization domain confers redox sensitivity to GABP
-
complex
formation.
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EXPERIMENTAL PROCEDURES |
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Cloning and Expression of Recombinant Proteins in Escherichia
coli--
The nucleotide sequence coding for GABP, GABP
, and
GABP
c (C-terminal region containing the Ets and the
GABP
/
dimerization domains) were amplified by PCR from the
cDNAs kindly provided by C. C. Thompson, Carnegie Institute of
Washington (17), and cloned into pET15b (Novagen) as described
previously (11). The individual rGABP proteins were expressed in
E. coli BL21 strain and purified (
90% purity estimated by
Coomassie Brilliant Blue R-250 staining) by nickel chelating
chromatography as described previously (11).
Site Directed Mutagenesis-- Site-directed mutagenesis was performed using the Transformer site directed mutagenesis kit (CLONTECH) according to the manufacturer recommendations or as described by Kunkel, et al. (29). The following primers were synthesized by the University of Missouri DNA Core Facility on an Applied Biosystems DNA Synthesizer Model 380B and used to substitute the corresponding cysteines to serines (substituted serine codons are underlined): 5'-GCTCGAGACTCGATATCTTGGGTT for Cys338, 5'-GGACATGATTTCGAAA GTTCAAGG for Cys388, 5'-TACAAATTTGTTTCTGACTTGAAGACT for Cys401, 5'-CTGGTCATAGAGTCTGAACAGAAGAAA for Cys421. The sequences of all mutants were confirmed by restriction analysis as well as by DNA sequencing (Sequenase 2.0 kit, U. S. Biochemical Corp.).
Electrophoretic Mobility Shift Assay (EMSA)--
For EMSA
analysis, double-stranded oligonucleotide probes were end-labeled with
[-32P]dGTP and [
-32P]dCTP (NEN Life
Science Products) by incubation at 25 °C with the Klenow fragment of
E. coli DNA polymerase I (New England Biolabs) and were
subsequently purified using the Mermaid Kit (Bio 101). The sequences of
the probes are: PEA3m, 5'-TCGAGCACCTTGAGGAAGTCTCGA, and dPEA3-0, 5'-TCGAGCAGGAAGAGGAAGTCTCGA (only the
upper strand is shown). Recombinant GABP
(rGABP
) and rGABP
were expressed and purified as described previously (11). The indicated
amount of rGABP
proteins was prereduced (1 mM
dithiotreitol (DTT)) and mixed with the indicated amount of rGABP
in
20 µl of EMSA buffer (20 mM HEPES, pH 7.1, 50 mM KCl, 1 mM benzamidine, 20% glycerol), containing 100 ng of poly(dI-dC)·poly(dI-dC) and bovine serum albumin
(0.5 mg/ml), and subjected to EMSA analysis as described previously
(11).
Treatment of Recombinant GABP Proteins with Sulfhydryl Modifying
Reagents--
The sulfhydryl modifiers N-ethylmaleimide
(NEM) (Sigma) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's
reagent) (Pierce) (31) were added to concentrated solutions of
pre-reduced (1.0 mM DTT) rGABP proteins (~0.3 mg/ml) and
then diluted to a final DTT concentration of 20 µM. The
prereduced proteins were then treated with NEM (250-500
µM) or DTNB (1 mM) for 20 min on ice, and the
DNA-binding properties of the treated proteins were analyzed by EMSA.
To prevent possible nonspecific modification of -amino group of
lysines or imidazole rings of histidine residue by NEM, all
modification experiments were performed at low NEM concentrations, at
low temperature, and at pH 7.1, conditions which are unfavorable to the
modification of lysines and histidines (30).
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RESULTS |
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Multiple Cysteines in the COOH Terminus of GABP Are Involved in
Redox Regulation of GABP DNA Binding--
Previously, we demonstrated
that GABP
DNA binding and transcription activation functions are
redox regulated both in vivo and in vitro and
that COOH-terminal cysteine residues in GABP
are important for this
regulation (11). We have shown that both full-length GABP
and a
COOH-terminal truncation protein, GABP
c, containing only
the DNA binding domain and the GABP
dimerization domain, require
the presence of reducing agents (DTT) for DNA binding and are inhibited
by treatment with oxidized glutathione (GSSG) or NEM (11), implicating
the COOH-terminal cysteine residues in redox regulation of GABP
DNA-binding. In contrast, GABP
was found to have no detectable role
in redox regulation of GABP DNA-binding activity. GABP
contains nine
cysteine residues, five in the NH2-terminal two thirds of
the protein (Cys22, Cys37, Cys61,
Cys69, and Cys223), two in the Ets domain
(Cys338 and Cys388), and two in the GABP
dimerization domain (Cys401 and Cys421) (Fig.
1). The latter four residues are present
in GABP
c and are the putative targets for redox
regulation of GABP.
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Cys388, Cys401, and Cys421 Are
Targets for Redox Regulation of rGABPc DNA Binding in
Vitro--
Since single Cys
Ser substitutions of COOH-terminal
cysteines in GABP
c failed to confer complete resistance
to sulfhydryl modifiers, we generated a set of GABP
c
triple Cys
Ser substitution mutants, resulting in mutant
GABP
c proteins containing only one of the four cysteine
residues designated
cCSSS,
cSCSS,
cSSCS, and
cSSSC (Fig. 1). In addition, a
mutant lacking all four cysteines, GABP
cQ
(
cSSSS), was also generated. GABP
c and
the mutant proteins were treated with NEM or DTNB, and the DNA-binding
activity of the modified proteins was measured by EMSA assay in the
presence of rGABP
(Fig. 3). As
expected, the
cCCCC protein was completely inhibited by
NEM treatment (Fig. 3, lanes 1 and 2), whereas
the
cSSSS (Q) mutant, lacking any cysteine residues was
unaffected by NEM treatment (Fig. 3, lanes 3 and
4). Treatment of
cSCSS (Fig. 3, lanes
7 and 8) and
cSSCS (Fig. 3, lanes
9 and 10) with NEM, or DTNB,2 nearly
completely abolished DNA binding, demonstrating the importance of
Cys388 and Cys401 in redox sensitivity of
GABP
c DNA binding. Treatment of
cCSSS (Fig. 3, lanes 5 and 6) with NEM had no
significant effect on DNA binding, suggesting that Cys338
is not likely to be important in GABP
redox sensitivity.
Surprisingly, although Cys421 lies outside the known DNA
binding domain, NEM modification partially inhibited
cSSSC DNA binding (Fig. 3, lanes 11 and
12). Since DNA binding of the truncated
rGABP
c proteins can be detected by EMSA only when
complexed with rGABP
, interference with the GABP
c-GABP
interaction would affect the apparent DNA
binding of the GABP
c mutant proteins. Thus, it is
possible that modification of Cys421 prevents
cSSSC/GABP
complex formation rather than affecting the intrinsic DNA-binding activity.
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Alkylation of Cys388 and Cys401 Inhibits
the Intrinsic DNA-binding Activity of Full-length GABP--
Because
full-length rGABP
is able to bind DNA in the absence of GABP
, we
generated a set of triply substituted proteins in the context of the
full-length GABP
protein, as well as a mutant (GABP
Q) lacking all
four COOH-terminal cysteines (Fig. 1). All full-length triple mutants,
designated
CSSS,
SCSS,
SSCS, and
SSSC, and GABP
Q (SSSS)
were able to bind DNA in the absence of GABP
, although the
DNA-binding activity of most of the mutant proteins was reduced
(10-40%) relative to the wild-type protein.2
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DISCUSSION |
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In this report, we have investigated the roles of the four
COOH-terminal cysteine residues of GABP in redox sensitivity of GABP
DNA binding in vitro. Using Cys
Ser substitution mutants in the context of the full-length protein and a truncated protein (GABP
c) containing only the DNA binding and dimerization
domains, we demonstrate that redox regulation of GABP occurs at two
levels, the activity of the GABP
DNA binding domain and
heterodimerization of GABP
with the GABP
subunit.
GABP Cys338 Is Insensitive to Redox
Modification--
Sulfhydryl modifiers have little or no effect on the
DNA-binding activity of
CSSS, suggesting that Cys338 is
insensitive to redox changes and is not involved in redox regulation of
GABP DNA binding. These findings are in contrast with published reports
implicating Cys394 (Ala335 in GABP
) in redox
sensitivity of the v-Ets DNA binding domain (32). While the
three-dimensional structure of the GABP
Ets-domain has not been
reported, the high level of homology in primary structure and high
similarity of the published three-dimensional structures of PU.1,
Ets-1, and Fli-1 proteins allow us to use these structures to estimate
the molecular environment of residues of interest in GABP
. Analysis
of the tertiary structures of several Ets-proteins (Ets-1, Fli-1, and
PU.1) (33-38) revealed that the local environment of
Cys350 in Ets-1 (Cys394 in vEts) (Fig.
6A) is likely to be very
different from that of Cys338 in GABP
and may explain
the observed difference in their redox sensitivity. In Fli-1 (33, 34),
the carbonyl oxygen of Cys299 (Cys338 in
GABP
) forms a hydrogen bond with the amide proton of
Asp313 (Gln352 in GABP
), stabilizing the
1-
2
-sheet and restricting the movement of this residue. In
addition, Fli-1 Cys299 is shielded by several hydrophobic
residues (Phe286, Leu287, Leu290,
Ile300, Met311, Val317,
Asp313, and Glu316 in Fli-1), which contribute
to the central hydrophobic core of the ETS domain. In contrast, the
position of Cys394 in vEts (Cys350 in Ets-1) is
within the loop between
-helix 1 and
-strand 1, is not involved
in formation of the central hydrophobic core, and is likely to be
solvent-exposed (Fig. 6A) (33-38). Most Ets proteins
contain either a hydrophobic or an aromatic residue in the position
that is equivalent to Cys338 in GABP
, suggesting the
involvement of residues in this position in formation of the central
hydrophobic core. Thus, it is likely that the sulfhydryl of
Cys338 in GABP
is at least partially buried within this
hydrophobic core and is inaccessible to modification.
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Modification of GABP Cys388 Directly Interferes with
DNA Binding--
GABP
Cys388 is located in the region
corresponding to
-strand 3 of the Ets-1 (35, 36), Fli-1 (33, 34),
and PU.1 Ets domains (37, 38) (Fig. 6B). This residue is not
highly conserved in ETS proteins; although, neither bulky aromatic nor
negatively charged residues have been found in this position in any of
the known ETS proteins (16, 38, 39). Substitution in v-Ets of the
histidine equivalent to His403 in human Ets-1
(Cys388 in GABP
) to aspartic acid results in a virus
whose transforming activity is temperature-sensitive (40). Similarly,
substitution of Arg74 in Elk-1 (Cys388 in
GABP
) to aspartic acid leads to the inactivation of DNA binding (41). Furthermore, substitution of Lys404 in Ets-1 or
Lys245 in PU.1, which correspond to Lys389
adjacent to Cys388 in GABP
, completely abolished DNA
binding of these proteins (40, 42). Lysine in the position
corresponding to Lys389 in GABP
(Lys245 in
PU.1) is absolutely conserved in all known Ets-proteins and is directly
involved in contacts with the phosphate backbone 5' to the core GGAA
sequence in the PU.1-DNA complex crystal structure (Fig. 6B)
(37, 38). Similarly, in Fli-1, the equivalent residue was determined by
NMR to be within 4 Å of the bound DNA by intermolecular nuclear
Overhauser effects (33). Therefore, since Cys388 is located
adjacent to Lys389, it is likely to be in close proximity
to bound DNA, suggesting that sulfhydryl oxidation to sulfenic (RSOH)
or sulfinic (RSO2H) acids at this site could directly
interfere with DNA binding through electrostatic repulsion as has been
proposed for redox regulation of Fos and Jun proteins (Ref. 6, and
references therein). This is consistent with the fact that, as
described above, substitution to aspartic acid of residues in v-Ets and
Elk-1 proteins analogous to Cys388 inhibits transformation
or DNA binding, respectively. Alternatively, modification by
glutathione disulfide (GSSG), which we previously demonstrated to
inhibit GABP DNA binding (11) to form a GABP-glutathione conjugate at
Cys388, could inhibit DNA binding by sterically altering
the position of Lys389 or
-helix 3, both of which are
directly involved in DNA binding.
Modification of GABP Cys401 Inhibits DNA Binding
Indirectly--
GABP
Cys401 lies outside the ETS domain
and is not required for DNA binding (43)2. In Ets-1, this
region forms a loop linking
-strand 4 of the Ets-domain and a
COOH-terminal
-helix 4 (36). Based on the published NMR structure
(35, 36), in Ets-1, Cys416 (Cys401 in GABP
)
is solvent-exposed and accessible for modification. The precise role of
this region in DNA binding is not known. However, substitution of
either of the adjacent residues in Ets-1 (Val415 or
Asp417) significantly decreases DNA binding, and
substitution of the conserved residue, Phe414 to leucine,
in
-strand 4 abolishes DNA binding (42). Residues analogous to
GABP
Cys401 in Ets-1, PU.1, and Fli-1 do not form any direct
contacts with DNA in the three-dimensional structures of these
proteins. Furthermore, a mutant GABP
protein truncated at residue
400 is competent for DNA binding, suggesting that residues beyond
Val400, including Cys401, are not directly
involved in GABP
DNA binding.2 Thus, it is likely that
mutations or modification of residues in this region results in some
structural alteration rather than directly interfering with DNA
binding.
Modification of GABP Cys421 Inhibits GABP
-
Dimerization--
Deletion of the 54 COOH-terminal residues of GABP
abolishes GABP
-GABP
dimerization while not affecting DNA binding
(17, 48).2 Consistent with this result, the modification of
Cys421 in the COOH-terminal portion of GABP
does not
directly affect the DNA binding of monomeric GABP
; however,
modification of Cys421 inhibits GABP
-GABP
dimerization. This result strongly suggests that Cys421
lies within the GABP
-GABP
dimerization interface and that
modification of this residue prevents interaction with GABP
.
Recently the redox regulation of intersubunit interactions in the
CAAT-binding protein, NF-Y, has been reported (49). Under non-reducing
conditions, the NF-YB subunit fails to associate with NF-YC, which
causes a substantial decrease in the DNA-binding activity of NF-Y. In contrast, disruption of GABP
-GABP
complexes does not
significantly diminish DNA binding of the GABP
subunit. Since
GABP
lacks any discernible transactivation domain (50),
transcription activation by GABP depends on the transactivation domain
provided by GABP
(50). Thus, GABP
bound to DNA in the absence of
GABP
may function as a low-affinity repressor.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tom Quinn for many helpful
suggestions concerning molecular modeling of the Ets domain structure
of GABP and for critical reading of the manuscript. We thank Dr.
Mark Hannink for many helpful suggestions during the course of these
studies.
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FOOTNOTES |
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* This work was done during the tenure of an Established Investigatorship from the American Heart Association (to M. E. M.) and was supported in part by Grant RB-95-010 from the University of Missouri System Research Board.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Missouri-Columbia, Columbia, MO 65212. Tel.: 573-882-5654; Fax: 573-884-4597.
1 The abbreviations used are: redox, reduction/oxidation; GABP, GA-binding protein; NRF2, nuclear respiratory factor 2; EMSA, electrophoretic mobility shift assay; rGABP; recombinant GABP; DTT, dithiotreitol; NEM, N-ethylmaleimide; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid).
2 Y. Chinenov, L. Harrison, and M. E. Martin, unpublished observations.
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REFERENCES |
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