Identification of Redox-sensitive Cysteines in GA-binding Protein-alpha That Regulate DNA Binding and Heterodimerization*

Yurii Chinenov, Tonya Schmidt, Xiu-Ying Yang, and Mark E. MartinDagger

From the Department of Biochemistry, University of Missouri at Columbia, Columbia, Missouri 65212

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The transcription factor GA-binding protein (GABP) is composed of two subunits, GABPalpha and GABPbeta . The DNA-binding subunit, GABPalpha , is a member of the Ets family of transcription factors, characterized by the conserved Ets-domain that mediates DNA binding and associates with GABPbeta , which lacks a discernible DNA binding domain, through ankyrin repeats in the NH2 terminus of GABPbeta . We previously demonstrated that GABP is subject to redox regulation in vitro and in vivo through four COOH-terminal cysteines in GABPalpha . 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 GABPalpha (Cys388, Cys401, and Cys421). Sulfhydryl modification of Cys388 and Cys401 inhibits DNA binding by GABPalpha , whereas, modification of Cys421 has no effect on GABPalpha DNA binding but inhibits dimerization with GABPbeta . 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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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, NFkappa B 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 NFkappa 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 Ikappa B leading to its inactivation and concomitant activation of NFkappa 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 NFkappa B and AP-1, result in inhibition of GABP DNA binding through COOH-terminal cysteine residues in the GABPalpha subunit (11). GABP is composed of two subunits, GABPalpha and GABPbeta , and each subunit provides distinct functions to the complex (12, 13). GABPalpha 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). GABPbeta has no discernible DNA binding domain and is unable to bind DNA on its own; however, GABPbeta contains at least one transcription activation domain required for transcription activation through the heteromeric complex (17-19). In addition, GABPbeta 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 GABPalpha -beta and GABPbeta -beta 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 beta  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 GABPalpha and therefore inhibit GABPalpha DNA binding and GABPalpha -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 GABPalpha protein, we performed site directed mutagenesis of cysteine residues in the GABPalpha 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 GABPalpha DNA binding while Cys421 in the GABPalpha /GABPbeta dimerization domain confers redox sensitivity to GABPalpha -beta complex formation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning and Expression of Recombinant Proteins in Escherichia coli-- The nucleotide sequence coding for GABPalpha , GABPbeta , and GABPalpha c (C-terminal region containing the Ets and the GABPalpha /beta 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 [alpha -32P]dGTP and [alpha -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 GABPalpha (rGABPalpha ) and rGABPbeta were expressed and purified as described previously (11). The indicated amount of rGABPalpha proteins was prereduced (1 mM dithiotreitol (DTT)) and mixed with the indicated amount of rGABPbeta 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 epsilon -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).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Multiple Cysteines in the COOH Terminus of GABPalpha Are Involved in Redox Regulation of GABP DNA Binding-- Previously, we demonstrated that GABPalpha DNA binding and transcription activation functions are redox regulated both in vivo and in vitro and that COOH-terminal cysteine residues in GABPalpha are important for this regulation (11). We have shown that both full-length GABPalpha and a COOH-terminal truncation protein, GABPalpha c, containing only the DNA binding domain and the GABPalpha beta 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, GABPbeta was found to have no detectable role in redox regulation of GABP DNA-binding activity. GABPalpha 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 GABPalpha beta dimerization domain (Cys401 and Cys421) (Fig. 1). The latter four residues are present in GABPalpha c and are the putative targets for redox regulation of GABP.


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Fig. 1.   Structures of recombinant proteins, rGABPalpha and rGABPalpha c, and Cys right-arrow Ser substitution mutants. A, schematic of the known structural features of the GABPalpha protein. The position of cysteine residues in both GABPalpha and GABPalpha c proteins are indicated by the letter "C." The position of the Ets-domain (amino acids 316-400) and the GABPalpha /GABPbeta dimerization domain (amino acids 400-454) are indicated by solid and hatched boxes, respectively. B, GABPalpha and GABPalpha c Cys right-arrow Ser substitution mutants. The positions of the cysteine and substituted serine residues are indicated by the letters "C" and "S," both in GABPalpha and GABPalpha c. The quadruple mutants (SSSS), GABPalpha Q and GABPalpha cQ, lack all four cysteines in the COOH-terminal domains. WT, indicates the wild-type proteins.

To identify which of the four COOH-terminal cysteines are involved in GABP redox regulation, a set of GABPalpha c mutants, each with a single Cys right-arrow Ser substitution was constructed, and the sensitivity of each mutant protein to NEM treatment was determined. The mutant proteins designated alpha cSCCC, alpha cCSCC, alpha cCCSC, and alpha cCCCS (described in Fig. 1) were expressed in E. coli, purified as described under "Experimental Procedures," and their requirement for DTT and sensitivity to sulfhydryl modifiers was tested by EMSA assays. If only a single cysteine residue was necessary for redox regulation, the substitution of this residue to serine would be expected to render the mutant protein redox insensitive and resistant to sulfhydryl modification. However, all of the singly substituted GABPalpha c mutants bound DNA as heterotetramers in the presence of rGABPbeta with comparable activity (Fig. 2) and all required reduction with DTT for DNA binding.2 DNA-binding activity of the mutant proteins in the absence of rGABPbeta was nearly undetectable, consistent with our previous observations in which wild-type GABPalpha c also bound DNA poorly in the absence of rGABPbeta (11). Treatment with NEM inhibited DNA binding of all four singly substituted mutant proteins although alpha cCCSC consistently retained some residual DNA-binding activity following NEM treatment (Fig. 2, lanes 7 and 8). These results suggest that multiple cysteines are redox sensitive and participate in regulation of GABPalpha DNA binding.


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Fig. 2.   The effect of NEM treatment on GABPalpha c single Cys right-arrow Ser substitution mutants. Pre-reduced rGABPalpha c (lanes 1 and 2), rGABPalpha cQ (lanes 3 and 4), rGABPalpha cSCCC (lanes 5 and 6), rGABPalpha cCSCC (lanes 7 and 8), rGABPalpha cCCSC (lanes 9 and 10), and rGABPalpha cCCCS (lanes 11 and 12) proteins (0.01 µg each) were treated with 250 µM NEM as described under "Experimental Procedures." A slight excess of rGABPbeta 1 protein (0.015 µg) was then added, and DNA binding of the heteromeric complexes containing treated and untreated rGABPalpha c proteins was measured by EMSA analysis using the dPEA3-0 probe containing two adjacent PEA3/EBS sites. NEM treatment is indicated by +. The (alpha c)2beta 2 tetramer complex is the predominant complex formed with all rGABPalpha c proteins bound to the dPEA3-0 probe.

Cys388, Cys401, and Cys421 Are Targets for Redox Regulation of rGABPalpha c DNA Binding in Vitro-- Since single Cys right-arrow Ser substitutions of COOH-terminal cysteines in GABPalpha c failed to confer complete resistance to sulfhydryl modifiers, we generated a set of GABPalpha c triple Cys right-arrow Ser substitution mutants, resulting in mutant GABPalpha c proteins containing only one of the four cysteine residues designated alpha cCSSS, alpha cSCSS, alpha cSSCS, and alpha cSSSC (Fig. 1). In addition, a mutant lacking all four cysteines, GABPalpha cQ (alpha cSSSS), was also generated. GABPalpha 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 rGABPbeta (Fig. 3). As expected, the alpha cCCCC protein was completely inhibited by NEM treatment (Fig. 3, lanes 1 and 2), whereas the alpha cSSSS (Q) mutant, lacking any cysteine residues was unaffected by NEM treatment (Fig. 3, lanes 3 and 4). Treatment of alpha cSCSS (Fig. 3, lanes 7 and 8) and alpha 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 GABPalpha c DNA binding. Treatment of alpha 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 GABPalpha redox sensitivity. Surprisingly, although Cys421 lies outside the known DNA binding domain, NEM modification partially inhibited alpha cSSSC DNA binding (Fig. 3, lanes 11 and 12). Since DNA binding of the truncated rGABPalpha c proteins can be detected by EMSA only when complexed with rGABPbeta , interference with the GABPalpha c-GABPbeta interaction would affect the apparent DNA binding of the GABPalpha c mutant proteins. Thus, it is possible that modification of Cys421 prevents alpha cSSSC/GABPbeta complex formation rather than affecting the intrinsic DNA-binding activity.


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Fig. 3.   The effect of NEM treatment on GABPalpha c Cys right-arrow Ser mutants containing only one of the four COOH-terminal cysteine residues. Pre-reduced rGABPalpha cWT (lanes 1 and 2), rGABPalpha cQ (lanes 3 and 4), rGABPalpha cCSSS (lanes 5 and 6), rGABPalpha cSCSS (lanes 7 and 8), rGABPalpha cSSCS (lanes 9 and 10), and rGABPalpha cSSSC (lanes 11 and 12) proteins (0.01 µg each) were treated with 250 µM NEM as described under "Experimental Procedures." A slight excess of rGABPbeta 1 protein (0.015 µg) was then added, and DNA binding of heteromeric complexes containing treated and untreated rGABPalpha c proteins was measured by EMSA analysis using the dPEA3-0 probe. Treatment with NEM is indicated by +. The position of the predominant (alpha c)2(beta )2 complexes are indicated by an arrow on the left.

Alkylation of Cys388 and Cys401 Inhibits the Intrinsic DNA-binding Activity of Full-length GABPalpha -- Because full-length rGABPalpha is able to bind DNA in the absence of GABPbeta , we generated a set of triply substituted proteins in the context of the full-length GABPalpha protein, as well as a mutant (GABPalpha Q) lacking all four COOH-terminal cysteines (Fig. 1). All full-length triple mutants, designated alpha CSSS, alpha SCSS, alpha SSCS, and alpha SSSC, and GABPalpha Q (SSSS) were able to bind DNA in the absence of GABPbeta , although the DNA-binding activity of most of the mutant proteins was reduced (10-40%) relative to the wild-type protein.2

Since the full-length GABPalpha protein contains five additional cysteines, the wild-type and GABPalpha Q proteins were treated with DTNB to confirm that redox sensitivity of the full-length GABPalpha protein was exclusively mediated through the four COOH-terminal cysteines (Fig. 4). DTNB is widely used as a highly specific, reversible, sulfhydryl modifier, which can be removed from conjugated cysteines by reduction with DTT (30, 31). DTNB treatment completely inhibited GABPalpha DNA binding (Fig. 4, lane 2), whereas GABPalpha Q was totally unaffected (Fig. 4, lane 5). The inhibitory effect of DTNB on GABPalpha was completely reversed by the subsequent addition excess of DTT (Fig. 4, lane 3) demonstrating that DTNB inhibition of GABPalpha DNA binding was sulfhydryl-specific. These experiments demonstrate that the COOH-terminal cysteine residues (Cys338, Cys388, Cys401, and Cys421) are necessary and sufficient for redox regulation of GABP DNA binding in vitro and confirm that the remaining NH2-terminal cysteine residues play no detectable role in such regulation.


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Fig. 4.   Redox sensitivity of GABP DNA binding is mediated solely by the four cysteine residues in the COOH-terminal DNA binding and dimerization domains of GABPalpha . Prereduced, full-length recombinant proteins, rGABPalpha (CCCC, lanes 1-3) (0.012 µg) and rGABPalpha Q (SSSS, lanes 4-6) (0.1 µg), were treated with 1 mM DTNB alone or with 1 mM DTNB followed by 5 mM DTT as described under "Experimental Procedures." DNA-binding activity was measured by EMSA analysis using the PEA3m probe containing a single PEA3/EBS. Treatment by DTNB or DTT is indicated by +. The protein-DNA complexes formed by rGABPalpha and rGABPalpha Q proteins are indicated by arrows.

To determine the effect of sulfhydryl modification of individual cysteines on the intrinsic DNA-binding activity of GABPalpha , the wild-type and mutant proteins were treated with NEM and analyzed by EMSA assays in the presence and absence of GABPbeta . As expected, NEM treatment of wild-type GABPalpha nearly completely inhibited its DNA-binding activity in the presence and absence of GABPbeta (Fig. 5A, lanes 1-4). In contrast, NEM treatment of GABPalpha Q had no effect on DNA binding (Fig. 5A, lanes 5-8), and the presence of GABPbeta had no effect on DNA binding of NEM-treated GABPalpha or GABPalpha Q proteins (Fig. 5A, lanes 4 and 8). The consistency of the results of NEM and DTNB modification and the resistance of GABPalpha Q to NEM treatment (Fig. 5, lanes 5-8), even at high concentrations (5 mM)2, demonstrate that inhibition of GABPalpha DNA binding by these reagents is not the result of nonspecific reactions of NEM (or DTNB) with other amino acids such as lysine or histidine.


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Fig. 5.   The effect of NEM treatment on DNA-binding activities of full-length GABPalpha Cys right-arrow Ser substitution mutants containing only one of the four COOH-terminal cysteine residues. Prereduced rGABPalpha WT(CCCC) (0.012 µg) (lanes 1-4), rGABPalpha Q(SSSS) (0.06 µg) (lanes 5-8), rGABPalpha CSSS (0.012 µg) (lanes 9-12), rGABPalpha SCSS (0.06 µg) (lanes 13-16), rGABPalpha SSCS (0.012 µg) (lanes 17-20), and rGABPalpha SSSC (0.06 µg) (lanes 21-24) proteins were treated with 500 µM NEM as described under "Experimental Procedures," and incubated with PEA3m probe in the presence and absence of limiting quantities (GABPalpha /GABPbeta  = 5/1) of rGABPbeta 1 protein (0.004-0.01 µg). The presence of rGABPbeta 1 and treatment with NEM are indicated by +. Because the DNA-binding activities of the GABPalpha mutant proteins differed by up to 4-fold, the amount of each protein used was normalized to give equivalent DNA binding in these experiments.

Modification of the alpha CSSS protein with NEM had little or no effect on DNA-binding activity (Fig. 5B, lanes 1-4), which is consistent with the results obtained with the alpha cCSSS protein (Fig. 3), and suggests that Cys338 plays no detectable role in redox sensitivity of GABPalpha DNA binding. The DNA-binding activities of the alpha SCSS (Fig. 5B, lanes 5-8) and alpha SSCS proteins (Fig. 5C, lanes 1-4), were inhibited by treatment with NEM, also consistent with our results obtained with the truncated GABPalpha c mutant proteins. Similar results were obtained with DTNB treatment.2 Therefore, modification of either Cys388 or Cys401 is sufficient to inhibit DNA binding, either directly or by affecting the conformation of the GABPalpha DNA binding domain.

The DNA-binding activity of the alpha SSSC monomer complex was not affected by NEM treatment (Fig. 5C, lanes 5-8), even at high concentrations (up to 5 mM),2 demonstrating that Cys421 plays no role in redox sensitivity of the intrinsic DNA-binding activity of GABPalpha . However, in the presence of GABPbeta , NEM-treated alpha SSSC protein failed to form the GABPalpha /GABPbeta dimer complex (Fig. 5C, lane 8), even when GABPbeta was added in excess.2 Cys421 lies within the region of GABPalpha previously implicated in GABPalpha -beta dimerization (17). Modulation of the ability of the alpha SSSC protein to form GABPalpha -beta complexes by sulfhydryl modifiers is consistent with the notion that Cys421 lies within the GABPalpha /GABPbeta dimerization interface. Thus, these observations demonstrate that redox regulation of GABP may occur on at least two levels, 1) the intrinsic GABPalpha DNA-binding activity and 2) GABPalpha -beta dimerization.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In this report, we have investigated the roles of the four COOH-terminal cysteine residues of GABPalpha in redox sensitivity of GABP DNA binding in vitro. Using Cys right-arrow Ser substitution mutants in the context of the full-length protein and a truncated protein (GABPalpha c) containing only the DNA binding and dimerization domains, we demonstrate that redox regulation of GABP occurs at two levels, the activity of the GABPalpha DNA binding domain and heterodimerization of GABPalpha with the GABPbeta subunit.

GABPalpha Cys338 Is Insensitive to Redox Modification-- Sulfhydryl modifiers have little or no effect on the DNA-binding activity of alpha 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 GABPalpha ) in redox sensitivity of the v-Ets DNA binding domain (32). While the three-dimensional structure of the GABPalpha 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 GABPalpha . 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 GABPalpha and may explain the observed difference in their redox sensitivity. In Fli-1 (33, 34), the carbonyl oxygen of Cys299 (Cys338 in GABPalpha ) forms a hydrogen bond with the amide proton of Asp313 (Gln352 in GABPalpha ), stabilizing the beta 1-beta 2 beta -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 alpha -helix 1 and beta -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 GABPalpha , 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 GABPalpha is at least partially buried within this hydrophobic core and is inaccessible to modification.


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Fig. 6.   Sequence comparison of GABPalpha cysteines with sequences of known Ets-domain structures. A, sequence alignment of the region of GABPalpha Cys338 and the corresponding regions of the Ets-1 (35-36), Fli-1 (33, 34), and PU.1 (37, 38) proteins. B, sequence alignment of the region of GABPalpha Cys388 and the corresponding sequences of the Ets-1, Fli-1, PU.1, and Elk-1 (41) proteins. C, sequence alignment of GABPalpha Cys401 and the corresponding region of the Ets-1 protein, including the sequence of the putative alpha -helix 4 inhibitory domain.

Modification of GABPalpha Cys388 Directly Interferes with DNA Binding-- GABPalpha Cys388 is located in the region corresponding to beta -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 GABPalpha ) to aspartic acid results in a virus whose transforming activity is temperature-sensitive (40). Similarly, substitution of Arg74 in Elk-1 (Cys388 in GABPalpha ) 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 GABPalpha , completely abolished DNA binding of these proteins (40, 42). Lysine in the position corresponding to Lys389 in GABPalpha (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 alpha -helix 3, both of which are directly involved in DNA binding.

Modification of GABPalpha Cys401 Inhibits DNA Binding Indirectly-- GABPalpha Cys401 lies outside the ETS domain and is not required for DNA binding (43)2. In Ets-1, this region forms a loop linking beta -strand 4 of the Ets-domain and a COOH-terminal alpha -helix 4 (36). Based on the published NMR structure (35, 36), in Ets-1, Cys416 (Cys401 in GABPalpha ) 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 beta -strand 4 abolishes DNA binding (42). Residues analogous to GABPalpha 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 GABPalpha protein truncated at residue 400 is competent for DNA binding, suggesting that residues beyond Val400, including Cys401, are not directly involved in GABPalpha 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.

The COOH-terminal 24 residues of the Ets-1 protein, including alpha -helix 4, have been implicated in the regulation of DNA binding through interaction with an NH2-terminal inhibitory domain (44). In the Ets-1 NMR structure, alpha -helix 4 lies anti-parallel to and forms several contacts with alpha -helix 1 from the Ets-domain and forms additional contacts with the NH2-terminal inhibitory domain. The Ets-1 alpha -helix 4 is well defined locally although it is not positioned precisely within the tertiary fold (36), suggesting significant conformational mobility of this putative inhibitory helix. Therefore, mutations or modifications in the loop between beta -strand 4 and alpha -helix 4, or in alpha -helix 4 itself, that increase the stability of interactions with the Ets-domain may result in inhibition of DNA binding. This putative inhibitory domain is highly conserved in GABPalpha (59% identity, 88% similarity) (Fig. 6C), and Cys401 is located within the loop region between beta -strand 4 and alpha -helix 4 (45, 46, 47). Our data suggest that redox modification of Cys401 inhibits GABPalpha DNA binding, possibly by facilitating the interaction between the inhibitory alpha -helix 4 and the DNA binding domain. The Ets-2, PEA3, and ERM proteins also contain cysteines in the position analogous to Cys401 in GABPalpha , suggesting that these Ets proteins may also be redox-sensitive.

Modification of GABPalpha Cys421 Inhibits GABPalpha -beta Dimerization-- Deletion of the 54 COOH-terminal residues of GABPalpha abolishes GABPalpha -GABPbeta dimerization while not affecting DNA binding (17, 48).2 Consistent with this result, the modification of Cys421 in the COOH-terminal portion of GABPalpha does not directly affect the DNA binding of monomeric GABPalpha ; however, modification of Cys421 inhibits GABPalpha -GABPbeta dimerization. This result strongly suggests that Cys421 lies within the GABPalpha -GABPbeta dimerization interface and that modification of this residue prevents interaction with GABPbeta . 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 GABPalpha -GABPbeta complexes does not significantly diminish DNA binding of the GABPalpha subunit. Since GABPalpha lacks any discernible transactivation domain (50), transcription activation by GABP depends on the transactivation domain provided by GABPbeta (50). Thus, GABPalpha bound to DNA in the absence of GABPbeta may function as a low-affinity repressor.

GABP has now been demonstrated to be regulated through at least three pathways: by phosphorylation (51), by redox modification of DNA binding (Ref. 11, and this study), and by redox modification of inter-subunit interactions (this study). The importance of GABP(NRF2) for the regulation of genes involved in energy production (cytochrome oxidase subunits IV, Vb, and VII, and ATPase beta  subunit) and control of mitochondrial transcription and replication (mitochondrial transcription factor 1), together with the wide distribution of this factor, suggests that GABP may function as a transcriptional sensor of the redox and energy states of mammalian cells. Stable expression of a non-redox-sensitive mutant, such as GABPalpha Q, in a mammalian cell line will be useful to further characterize redox regulation of GABP and establish its importance in cellular homeostasis.

    ACKNOWLEDGEMENTS

We thank Dr. Tom Quinn for many helpful suggestions concerning molecular modeling of the Ets domain structure of GABPalpha and for critical reading of the manuscript. We thank Dr. Mark Hannink for many helpful suggestions during the course of these studies.

    FOOTNOTES

* 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.

Dagger 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Buratowski, S. (1994) Cell 77, 1-3[Medline] [Order article via Infotrieve]
  2. Ptashne, M., and Gann, A. A. F. (1990) Nature 346, 329-331[CrossRef][Medline] [Order article via Infotrieve]
  3. Clakhoven, F., and Geert, A. B. (1996) Biochem. J. 317, 329-342[Medline] [Order article via Infotrieve]
  4. Kornberg, R. D. (1996) TIBS 21, 325-326 [Medline] [Order article via Infotrieve]
  5. Reason, A. J., Morris, H. R., Panico, M., Marais, R., Treisman, R. H., Haltiwanger, R. S., Hart, G. W., Kelly, W. G., Dell, A. (1992) J. Biol. Chem. 267, 16911-16921[Abstract/Free Full Text]
  6. Sen, C. K., and Packer, L. (1996) FASEB J. 10, 707-720
  7. Schenk, H., Klein, M., Erdbrugger, W., Droge, W., and Schulze-Osthoff, K. (1994) Proc. Natl. Acad. Sci. 91, 1672-1676[Abstract]
  8. Xanthoudakis, S., Miao, G., Wang, F., Pan, Y. C., Curran, T. (1992) EMBO J. 11, 3323-3335[Abstract]
  9. Knight, R. J., and Buxton, D. B. (1996) Biochem. Biophys. Res. Commun. 218, 83-88[CrossRef][Medline] [Order article via Infotrieve]
  10. Laderoute, K. R., and Webster, K. A. (1997) Circulation Research 80, 336-344[Abstract/Free Full Text]
  11. Martin, M. E., Chinenov, Y., Yu, M., Schmidt, T. K., Yang, X.-Y. (1996) J. Biol. Chem. 271, 25617-25623[Abstract/Free Full Text]
  12. LaMarco, K., and McKnight, S. T. (1989) Genes Dev. 3, 1372-1382[Abstract]
  13. LaMarco, K., Thompson, C. C., Byers, B. P., Walton, E. M., McKnight, S. T. (1991) Science 253, 789-792[Medline] [Order article via Infotrieve]
  14. Seth, A., Ascione, R., Fisher, R. J., Mavrothalassitis, G. J., Bhat, N. K., Papas, T. S. (1992) Cell Growth Differ. 3, 327-334[Medline] [Order article via Infotrieve]
  15. Wang, C-Y., Petryniak, B., Ho, I-C., Thompson, C. B., Leiden, J. M. (1992) J. Exp. Med. 175, 1391-1399[Abstract]
  16. Hromas, R., and Klemsz, M. (1994) Int. J. Hematol. 59, 257-265[Medline] [Order article via Infotrieve]
  17. Thompson, C. C., Brown, T. A., and McKnight, S. L. (1991) Science 253, 762-768[Medline] [Order article via Infotrieve]
  18. Gugneja, S., Virbasius, J. V., and Scarpulla, C. (1995) Mol. Cell. Biol. 15, 102-111[Abstract]
  19. Sawada, J., Goto, M., Sawa, C., Watanabe, H., and Handa, H. (1994) EMBO J. 13, 1396-1402[Abstract]
  20. Marchioni, M., Morabito, S., Salvati, A. L., Beccari, E., Carnevali, F. (1993) Mol. Cell. Biol. 13, 6479-6489[Abstract]
  21. Virbassius, J. V., Virbassius, C. A., Scarpulla, R. C. (1993) Genes Dev. 7, 380-392[Abstract]
  22. Watanabe, H., Sawada, J., Yano, K., Yamaguchi, K., Goto, M., and Handa, H. (1993) Mol. Cell. Biol. 13, 1385-1391[Abstract]
  23. Villena, J. A., Martin, I., Vinas, O., Cormand, B., Iglesias, R., Mampel, T., Giralt, M., and Villarroya, F. (1994) J. Biol. Chem. 269, 32649-32654[Abstract/Free Full Text]
  24. Carter, R. S., and Avadhani, N. G. (1994) J. Biol. Chem. 269, 4381-4387[Abstract/Free Full Text]
  25. Virbasius, J. V., and Scarpulla, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1309-1313[Abstract]
  26. Scarpulla, R. C. (1996) Trends Cardiovasc. Med. 6, 39-45 [CrossRef]
  27. Poyton, R. O., and McEwen, J. E. (1996) Annu. Rev. Biochem. 65, 563-607[CrossRef][Medline] [Order article via Infotrieve]
  28. Bandy, B., and Davidson, A. J. (1990) Free Radical Biol. Med. 8, 523-539[CrossRef][Medline] [Order article via Infotrieve]
  29. Kunkel, T. A., Roberts, J., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-374[Medline] [Order article via Infotrieve]
  30. Torchinskii, Yu. M. (1974) Sulfhydryl and Disulfide Groups of Proteins, pp. 23-27, Consultants Bureau, New York
  31. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77[Medline] [Order article via Infotrieve]
  32. Wasylyk, C., and Wasylyk, B. (1993) Nucleic Acids Res. 21, 523-529[Abstract]
  33. Liang, H., Mao, X., Olejniczak, E. T., Nettesheim, D. G., Yu, L., Meadows, R. P., Thompson, C. B., Fesik, S. W. (1994) Nat. Struct. Biol. 1, 871-876[Medline] [Order article via Infotrieve]
  34. Liang, H., Olejniczak, E. T., Mao, X., Nettesheim, D. G., Yu, L., Thompson, C. B., Fesik, S. W. (1994) Proc. Natl. Acad. Sci. 91, 11655-11659[Abstract/Free Full Text]
  35. Werner, M. H., Clore, G. M., Fisher, C. L., Fisher, R. J., Trinh, L., Shiloach, J., Gronenborn, A. M. (1995) Cell 83, 761-771[Medline] [Order article via Infotrieve]
  36. Donaldson, L. W., Petersen, J. M., Graves, B. J., McIntosh, L. P. (1996) EMBO J. 15, 125-134[Abstract]
  37. Kodandapani, R., Pio, F., Ni, C.-Z., Piccialli, G., Klemsz, M., S. R., Maki, R. A., Ely, K. R. (1996) Nature 380, 456-460[CrossRef][Medline] [Order article via Infotrieve]
  38. Pio, F., Kodandapani, R., Ni, C.-Z., Shepard, W., Klemzs, M., McKercher, S. R., Maki, R. A., Ely, K. R. (1996) J. Biol. Chem. 271, 23329-23337[Abstract/Free Full Text]
  39. Laudet, V., Niel, C., Duteque-Coquillaud, M., Leprince, D., and Stehelin, D. (1992) Biochem. Biophys. Res. Commun. 190, 8-14[CrossRef]
  40. Golay, J., Introna, M., and Graf, T. (1988) Cell 55, 1147-1158[Medline] [Order article via Infotrieve]
  41. Janknecht, R., Zinck, R., Ernst, W. H., Nordheim, A. (1994) Oncogene 9, 1273-1278[Medline] [Order article via Infotrieve]
  42. Mavrothalassitis, G., Fisher, R., Smyth, F., Watson, D. K., Papas, T. S. (1994) Oncogene 9, 425-435[Medline] [Order article via Infotrieve]
  43. Reddy, E. S., and Rao, V. N. (1990) Cancer Res. 50, 5013-5016[Abstract]
  44. Petersen, J. M., Donaldson, J. J, McIntosh, C. P., Alber, T., Graves, B. J. (1995) Science 269, 1866-1869[Medline] [Order article via Infotrieve]
  45. Watson, D. K., McWilliams-Smith, M. J., Nunn, M. F., Duesberg, P. H., O'Brien, S. J., Papas, T. S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7294-7298[Abstract]
  46. Xin, J. H., Cowie, A., Lachance, P., and Hassell, J. A. (1992) Genes Dev. 6, 481-496[Abstract]
  47. Monte, D., Baert, J.-L., Defossez, P.-A., De Launoit, Y., Stehelin, D. (1994) Oncogene 9, 1397-1406[Medline] [Order article via Infotrieve]
  48. Sawada, J., Goto, M., Sawa, C., Watanabe, H., and Handa, H. (1994) EMBO J. 13, 1396-1402[Abstract]
  49. Nakshatri, H., Bhat-Nakshatri, P., and Currie, A. (1996) J. Biol. Chem. 271, 28784-28791[Abstract/Free Full Text]
  50. Gugneja, S., Virbasius, J. V., and Scarpulla, R. C. (1995) Mol. Cell. Biol. 15, 102-111[Abstract]
  51. Flory, E., Hoffmeyer, A., Smola, U., Rapp, U. R, Bruder, J. T. (1996) J. Virol. 70, 2260-2268[Abstract]


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