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

Abstract

Reactive oxygen species (ROS) arise through normal cellular aerobic respiration, and, in combination with external sources such as ionizing radiation, cigarette tar and smoke, and particulate matter generated by combustion, can have a profound negative effect on cellular macromolecules such as DNA that may lead to a number of human pathological disorders including accelerated aging and cancer. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidinic (AP) sites, which without removal are known to halt mRNA and DNA synthesis, or act as non-coding lesions resulting in the increased generation of DNA mutations. In human cells, the major enzyme in correcting the deleterious effects of AP sites in DNA is through the participation 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 out such disparate roles are unknown. The presence of a number of phosphorylation sites scattered throughout both functional domains of APE/Ref-1 however offered one possible mechanism that we reasoned could play a role in dictating 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 corresponded to an increase in the phosphorylation status of APE/Ref-1. Importantly, human cells exposed to the oxidizing agent hypochlorite, followed by methyl methanesulfanate, responded with an increase in redox activity by APE/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 the ability of APE/Ref-1 to perform its in vivo redox function is correlated to its susceptibility to PKC phosphorylation that notably occurs in response to DNA damaging agents.

Figure 1

Potential PKC phosphorylation sites possessed by APE/Ref-1.

Figure 2

In vitro phosphorylation of GST–APE/Ref-1 by PKC increases redox activity as shown by EMSA. An extract of wild-type K562 cells (10 µg) was used in combination with 1.5 µg of purified GST–APE/Ref-1 or PKC-phosphorylated GST–APE/Ref-1. ATP (Promega) was 500 µM, and where indicated 125 µM of phosphotidyl serine was used. Lane 1, oligo alone; lane 2, K562 cell lysate, which was also present in the reactions shown in lanes 3–9; lane 3, purified GST–APE/Ref-1; lane 4, purified GST–APE/Ref-1 and ATP; lane 5, phosphorylated GST–APE/Ref-1 and ATP; lane 6, dialyzed, phosphorylated GST–APE/Ref-1; lane 7, dialyzed GST–APE/Ref-1 phosporylated in the presence of phosphotidyl serine; lane 8, reaction in lane 6, plus phosphatase; lane 9, reaction in lane 7, plus phosphatase

Figure 3

PMA treatment of APE/Ref-1 stably-transfected K562 cells results in increased amounts of APE/Ref-1 phosphorylation and enhanced redox activity. (A) A monoclonal antibody prepared against APE/Ref-1 was used to immunoprecipitate (IP) the overexpressed protein (and the endogenous expressed APE/Ref-1 as well), and to detect the amount of APE/Ref-1 by western blot as described in Materials and Methods. PKC activity was measured as described in Materials and Methods. (B) The methods for PMA exposure and detection of redox activity by EMSA are provided in the Materials and Methods section. Lane 1, cells exposed to PMA for 2 h; lane 2, immunodepletion of APE/Ref-1 from lysates of cells exposed to PMA for 2 h; lane 3, unexposed cells; and lane 4, immunodepletion of APE/Ref-1 in unexposed cells. The fold increase was quantitated by a densitometer.

Figure 4

Exposure to hypochlorite and MMS leads to an increase in PKC activity, phosphorylation (as dewtermined by IP of APE/Ref-1), and redox activity possessed by APE/Ref-1. (A) The conditions for exposure of HA–APE/Ref-1 transfected K562 cells to hypochlorite and MMS are described in Materials and Methods. Redox activity was measured by EMSA. (B) The techniques used here are described in Materials and Methods.

Figure 5

Bar diagram summarizing the results presented in Figure 4 showing that increases in PKC activity are correlated with increases in both the phosphorylation and redox activity of APE/Ref-1.