Identification of redox-sensitive cysteines in GA binding protein-α that regulate DNA binding and heterodimerization

The transcription factor GA-binding protein (GABP) is composed of two subunits, GABP alpha and GABP beta. The DNA-binding subunit, GABP alpha, is a member of the Ets family of transcription factors, characterized by the conserved Ets-domain that mediates DNA binding and associates with GABP beta, which lacks a discernible DNA binding domain, through ankyrin repeats in the NH, terminus of GABP beta. We previously demonstrated that GABP is subject to redox regulation in vitro and in vivo through four COOH-terminal cysteines in GABP alpha. 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 alpha (Cys(388), Cys(401), and Cys(421)). Sulfhydryl modification of Cys(388) and Cys(401) inhibits DNA binding by GABP alpha whereas, modification of Cys(421) has no effect on GABP alpha DNA binding but inhibits dimerization with GABP beta. The positions of Cys(388) and Cys(401) within the known Ets-domain structure suggest two very different mechanisms for redox regulation of DNA binding. Sulfhydryl modification of Cys(388) could directly interfere with DNA binding or might alter the positioning of the DNA-binding helix 3. Modification of Cys(401) 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.