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 o f transcription factors and protein kinases, which are believed to be respo nsible for the carcinogenic effects of excessive sun exposure. The present study investigated the effect of UV exposure on reactive oxygen species (RO S) generation and protein kinase B (Akt) phosphorylation in epidermal cells and determined if a relationship exists between these UV responses. Exposu re of mouse epidermal JB6 Cl 41 cells to UV radiation led to specific phosp horylation of Akt at Ser-473 and Thr-308 in a time-dependent manner. This p hosphorylation was confirmed by the observation that overexpression of Akt mutant, Akt-T308/S473A, attenuated phosphorylation of Akt at Ser-473 and Th r-308. UV radiation also generated ROS as measured by electron spin resonan ce (ESR) in JB6 Cl 41 cells. The generation of ROS by UV radiation was meas ured further by H2O2 and O . (-)(2) fluorescence staining assays. The mecha nism of ROS generation involved reduction of molecular oxygen to O . (-)(2) which generated H2O2 through dismutation. H2O2 produced . OH via a metal-i ndependent pathway. The scavenging of UV-generated H2O2 by N-acety-L-cytein e (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) inhi bited Akt phosphorylation at Ser-473 and Thr-308, whereas the pretreatment of cells with sodium formate (an (OH)-O-. radical scavenger) or superoxide dismutase (an O . (-)(2) radical scavenger) did not show any inhibitory eff ects. Furthermore, treatment of cells with H2O2 increased UV-induced phosph orylation of Akt at Ser-473 and Thr-308. These results demonstrate that UV radiation generates a whole spectrum of ROS including O . (-)(2), (OH)-O-., and H2O2 and induces phosphorylation of Akt at Ser-473. Among the various ROS, H2O2 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 carc inogenesis effects by UV radiation.