Activation of APE/Ref-1 redox activity is mediated by reactive oxygen species and PKC phosphorylation

Reactive oxygen species (ROS) arise through normal cellular aerobic respira tion, and, in combination with external sources such as ionizing radiation, cigarette tar and smoke, and particulate matter generated by combustion, c an have a profound negative effect on cellular macromolecules such as DNA t hat may lead to a number of human pathological disorders including accelera ted aging and cancer. A major end product of ROS damage to DNA is the forma tion of apurinic/apyrimidinic (AP) sites, which without removal are known t o halt mRNA and DNA synthesis, or act as non-coding lesions resulting in th e increased generation of DNA mutations. In human cells, the major enzyme i n correcting the deleterious effects of AP sites in DNA is through the part icipation of AP endonuclease (APE), which initiates the removal of baseless sites in DNA through the catalytic scission of the phosphodiester bond 5' and adjacent to an AP site. Interestingly, APE also possesses an activity ( Ref-1) that controls the redox status of a number of transcription factors including Fos and Jun. The means by which APE/Ref-1 is directed to carry ou t such disparate roles are unknown. The presence of a number of phosphoryla tion sites scattered throughout both functional domains of APE/Ref-1 howeve r offered one possible mechanism that we reasoned could play a role in dict ating how this protein responds to different stimuli. Here we show that the in vitro redox activity of APE/Ref-1 is stimulated by PKC phosphorylation. Furthermore, when human cells were exposed to the PKC activator phorbol 12 -myristate 13-acetate, an increase in redox activity was observed that corr esponded to an increase in the phosphorylation status of APE/Ref-1- Importa ntly, human cells exposed to the oxidizing agent hypochlorite, followed by methyl methanesulfanate, responded with an increase in redox activity by AP E/Ref-1 that also involved an increase in PKC activity and a corresponding increase in the phosphorylation of APE/Ref-1. These results suggest that th e ability of APE/Ref-1 to perform its in vivo redox function is correlated to its susceptibility to PKC phosphorylation that notably occurs in respons e to DNA damaging agents.