Reactive oxygen species (ROS) control the expression of Bcl-2 family proteins by regulating their phosphorylation and ubiquitination

We examined the influence of ROS on the phosphorylation and complex formation of Bcl-2 family proteins in Mn-superoxide dismutase (SOD) antisense-transfected squamous cell carcinoma cells, OSC-4 cells. The increase of intracellular ROS level induced by cis-diamminedichloroplatinum (CDDP) and gamma-ray treatment was greater in antisense-transfected cells than in control vector-transfected cells, and apoptosis was more extensively induced in the former. Antisense-transfected cells expressed high levels of Bax and Bak, but low levels of Bcl-2 and Bcl-X-L when treated with CDDP, peplomycin, 5-fluorouracil or gamma-rays. After treatment with these agents, the phosphorylation of protein kinase A, Bcl-2 (Thr56) and Bad (Ser155) was increased, especially in antioxidant (N-acetylcysteine and pyrrolidine dithiocarbamate)-pretreated control cells, but the phosphorylation levels were very low in the antisense-transfected cells. Bcl-2 ubiquitination was increased, but ubiquitination of Bad and Bax was decreased in the antisense-transfected cells, although their ubiquitination was increased by the antioxidants. These results reveal that ROS induce apoptosis by regulating the phosphorylation and ubiquitination of Bcl-2 family proteins, resulting in increased proapoptotic protein levels and decreased antiapoptotic protein expression.