Hydrogen Peroxide-Induced Activation of SAPK/JNK Regulated by Phosphatidylinositol 3-Kinase in Chinese Hamster V79 Cells

To clarify activation mechanisms of stress-activated protein kinase/C-Jun N-terminal kinase (SAPK/JNK) during oxidative stress, the roles of phosphatidylinositol 3-kinase (PI 3-kinase), concentration of intracellular calcium ([Ca2+](i)), and cyclic AMP-dependent kinase (PKA) in hydrogen peroxide (H2O2)-induced SAPK/JNK activation were examined in Chinese hamster V79 cells. SAPK/JNK was dose-dependently activated after H2O2 treatment ( from 10 mu M to 1 m M), and a PI 3-kinase inhibitor (wortmaninn), intracellular calcium chelator (BAPTA-AM), and P K A activator ( dibutyl cyclic A M P and forskolin) inhibited this activation. An increase in [Ca2+](i) was observed after treatment with H2O2. Immunoprecipitation revealed that a PI 3-kinase regulatory subunit, p85 alpha, was associated with insulin receptor substance 1 (IRS-1) phosphorylated by H2O2 treatment. Furthermore, the formation of this complex of p85 alpha and phospho-IRS-1 was abolished by the presence of BAPTA-AM but not forskolin. These results indicated that the PI 3-kinase activated through phosphorylation of IRS-1 upstream of SAPK/JNK after H2O2 treatment of V79 cells and that [Ca2+](i) was a regulation factor for phosphorylation of IRS-1. Antiox. Redox Signal. 1, 113-121.