Identification of redox-sensitive cysteines in GA-binding protein-alpha that regulate DNA binding and heterodimerization.
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.
Chinenov
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Y
Department of Biochemistry, University of Missouri at Columbia, Columbia, Missouri 65212, USA.
Schmidt
T
T
Yang
X Y
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Martin
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ME
D000478:Alkylation; D000595:Amino Acid Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005033:Ethylmaleimide / Q000494:pharmacology; D051268:GA-Binding Protein Transcription Factor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000235:genetics; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins; D014157:Transcription Factors; D003545:Cysteine; D005033:Ethylmaleimide