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

Yurii Chinenov,Tonya Schmidt,Xiu-Ying Yang,Mark E Martin

doi:10.1074/jbc.273.11.6203

Abstract

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.

Highlights

The regulation of activator proteins by redox of reactive cysteine residues has been demonstrated for members of several important transcription factor families including, NF␬B and AP-1
Multiple Cysteines in the COOH Terminus of GA-binding protein (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

Summary

The regulation of activator proteins by redox of reactive

Cysteine residues has been demonstrated for members of several important transcription factor families including, NF␬B and AP-1 (for review, see Ref. 6) Both proteins require reducing conditions for DNA binding in vitro; in vivo, these factors become activated by oxidative stress-promoting agents (Ref. 6, and references therein). We demonstrated that murine GABP is redox-regulated both in vitro and in vivo and that pro-oxidant conditions, in contrast to NF␬B and AP-1, result in inhibition of GABP DNA binding through COOH-terminal cysteine residues in the GABP␣ subunit [11]. Redox Regulation of GABP DNA Binding and Dimerization tion 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]. 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

EXPERIMENTAL PROCEDURES

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DISCUSSION