Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding.

Abstract

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue Modulating the level of expression of specific genes is a prerequisite for the control of cellular growth and differentiation. Gene expression is controlled by sequence-specific DNA binding proteins (transcription factors) which in certain cases are targets for signals transduced from cell surface receptors. The importance of this process for growth control is emphasized by the finding that several proto-oncogenes, including c-myc, c-myb, c-fos, and c-jun, encode sequencespecific transcription factors (reviewed in reference 20). Although the activity of these factors can be modulated by phosphorylation (4, 11), recent evidence has emerged for an additional and unusual form of regulation of DNA binding activity mediated by changes in reduction-oxidation (redox) status.

Human AP endonuclease 1 (HAP1) protein expression in breast cancer correlates with lymph node status and angiogenesis

“…To determine the cellular expression of HAP1 a standard avidin-biotin-peroxidase complex (ABC) technique was performed, using an anti-HAPI rabbit polyclonal antibody (Guedson et al, 1979). Polyclonal anti-HAPl antiserum (HAPI antibody 13) was obtained from rabbits after six injections of each of 100 gg of recombinant HAP1 protein (Walker et al, 1993). The antiserum was tested for specificity by Western blotting of whole-cell extracts from human HeLa cells (Kakolyris et al, submitted).…”

“…This reduction-oxidation (redox) activity is dependent on a cysteine residue located near the DNA binding domain of the factor and is structurally and functionally distinct from the repair activity of HAPI (Walker et al, 1993;. Despite the accumulating molecular and biochemical data about the function of this gene, our knowledge about the distribution of the protein product in normal and neoplastic tissues is particularly limited.…”

Human Apurinic/Apyrimidinic Endonuclease 1

“…In particular, none of the cysteine residues within APE1 are found in a C-X-X-C motif that is common to most redox regulatory factors, such as thioredoxin, a cellular component which appears to be involved with APE1 in a redox regulatory cascade (81). In addition, disulfide bond formation is considered a necessary step in the resolving activity of redox factors, and while most evidence supports a role for C65 in the thiol-mediated redox reaction (61,219), none of the other six cysteine residues in APE1 are located appropriately in the 3-dimensional structure to facilitate disulfide bond formation with C65. Finally, although some data imply a role for C93 in the redox reaction, both C65 and C93 are buried within the protein structure, while being positioned at a distance (9 Å ) that is not compatible with disulfide bond formation.…”

“…Since then, APE1 has been reported to modulate the redox status of both ubiquitous (e.g., AP-1, Egr-1, NF-jB, p53, CREB, and HIF-1a) and tissue-specific transcription factors (e.g., PEBP-2, Pax-5, and -8, TTF-1) with functions in stress responses and other cellular processes [reviewed in Kelley et al (101)]. Although the redox regulatory and DNA repair nuclease activities can be disabled separately by site-specific mutagenesis (219,234), the precise molecular details of the protein-facilitated activation step remain elusive (103). In particular, none of the cysteine residues within APE1 are found in a C-X-X-C motif that is common to most redox regulatory factors, such as thioredoxin, a cellular component which appears to be involved with APE1 in a redox regulatory cascade (81).…”

Redox factor-1 contributes to the regulation of progression from G0/G1 to S by PDGF in vascular smooth muscle cells

“…The redox activity of Ref-1 is regulated by chemical reduction and oxidation in vitro (8,46,47) and may involve Cys-65 and Cys-93 in the NH 2 -terminal region of the protein (43). In cultured cells, Ref-1 serves as an intermediary between thioredoxin and AP-1 in response to oxidant stress induced by PMA (19) or ionizing radiation (44) and mediates AP-1 activation by heat shock (8).…”