Ultraviolet-induced Phosphorylation of p70 at Thr and Thr/Ser Involves Hydrogen Peroxide and Mammalian Target of Rapamycin but not Akt and Atypical Protein Kinase C

The p70 S6 kinase (p70) is a Ser/Thr kinase that plays an important role in cell growth, transformation, and the transition of the cell cycle in mammalian cells. Because UV radiation has been reported to induce activation of p70, which is believed to play some role in the carcinogenic effects of sun exposure, the present study investigated the signaling pathways involved in this activation induced by UV radiation in mouse epidermal JB6 Cl41 cells. Exposure of cells to UV radiation led to marked increases in p70 activity and phosphorylation at Thr and Thr/ Ser. UV radiation also generated reactive oxygen species as measured by electron spin resonance and by H2O2 and O2 . fluorescence staining assays in JB6 Cl 41 cells. The scavenging of UV-generated H2O2 by N-acety-L-cyteine (a general antioxidant) or catalase (a specific H2O2 scavenger) inhibited p70 phosphorylation at Thr and Thr/Ser, whereas pretreatment of cells with sodium formate (an OH radical scavenger) or superoxide dismutase (an O2 . radical scavenger) did not show any inhibitory effects. Importantly, UV-induced increases in p70 phosphorylation at Thr and Thr/Ser were dramatically inhibited by pretreatment of cells with rapamycin, LY294002, or PD98059, whereas overexpression of dominant-negative mutants of PKC / and Akt1 did not inhibit p70 phosphorylation at Thr and Thr/Ser. These results demonstrated that H2O2, phosphatidylinositol 3-kinase, and mammalian target of rapamycin were important players for UV-induced p70 phosphorylation at Thr and Thr/Ser, whereas Akt and atypical protein kinase C were not involved in this activation. The role of H2O2 in p70 S6k phosphorylation at Thr and Thr/Ser was further supported by the findings that treatment of cells with H2O2 also caused p70 S6k phosphorylation at Thr and Thr/Ser.