Phorbol Ester-induced Generation of Reactive Oxygen Species Is Protein Kinase Cβ-dependent and Required for SAPK Activation

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with protein kinase C (PKC) betaII-mediated activation of the stress-activated protein kinase (SAPK) pathway. The present studies demonstrate that the TPA response of U-937 cells includes the generation of reactive oxygen species (ROS). By contrast, the TPA-resistant U-937 cell variant (TUR), which is deficient in PKCbetaII expression, failed to respond to TPA with the induction of ROS. Moreover, we show that TPA-induced ROS production is restored in TUR cells stably transfected to express PKCbetaII. The results also demonstrate that TPA-induced ROS production is required for activation of the MEK kinase-1 (MEKK-1)--> SAPK pathway. In concert with this observation, treatment of U-937 with H(2)O(2) as a source of ROS is associated with activation of the MEKK-1-->SAPK cascade. These findings indicate that PKCbetaII is required for TPA-induced ROS production and that the MEKK-1-->SAPK pathway is activated by a ROS-mediated mechanism.