UV Induces Phosphorylation of Protein Kinase B (Akt) at Ser-473 and Thr-308 in Mouse Epidermal Cl 41 Cells through Hydrogen Peroxide

The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors and protein kinases, which are believed to be responsible for the carcinogenic effects of excessive sun exposure. The present study investigated the effect of UV exposure on reactive oxygen species (ROS) generation and protein kinase B (Akt) phosphorylation in epidermal cells and determined if a relationship exists between these UV responses. Exposure of mouse epidermal JB6 Cl 41 cells to UV radiation led to specific phosphorylation of Akt at Ser-473 and Thr-308 in a time-dependent manner. This phosphorylation was confirmed by the observation that overexpression of Akt mutant, Akt-T308/S473A, attenuated phosphorylation of Akt at Ser-473 and Thr-308. UV radiation also generated ROS as measured by electron spin resonance (ESR) in JB6 Cl 41 cells. The generation of ROS by UV radiation was measured further by H₂O₂ and O₂^.- fluorescence staining assays. The mechanism of ROS generation involved reduction of molecular oxygen to O₂^.-, which generated H₂O₂ through dismutation. H₂O₂ produced ^.OH via a metal-independent pathway. The scavenging of UV-generated H₂O ₂ by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H₂O₂ inhibitor) inhibited Akt phosphorylation at Ser-473 and Thr-308, whereas the pretreatment of cells with sodium formate (an ^.OH radical scavenger) or superoxide dismutase (an O₂ ^.- radical scavenger) did not show any inhibitory effects. Furthermore, treatment of cells with H₂O₂ increased UV-induced phosphorylation of Akt at Ser-473 and Thr-308. These results demonstrate that UV radiation generates a whole spectrum of ROS including O ₂^.-, ^.OH, and H₂O₂ and induces phosphorylation of Akt at Ser-473. Among the various ROS, H ₂O₂ seems most potent in mediating UV-induced phosphorylation of Akt at Ser-473 and Thr-308. It is possible that Akt may play a role in the carcinogenesis effects by UV radiation.