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) beta II-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 PKC beta II 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 PKC beta II, The results also demonstrate that TPA-induced ROS production is required for activation of the MEK kinase-1 (MEKI-1)--> SAPK pathway. In concert with this observation, treatment of U-937 with H2O2 as a source of ROS is associated with activation of the MEKK-1-->SAPK cascade. These findings indicate that PKC beta II is required for TPA-induced ROS production and that the MEKK-1-->SAPK pathway is activated by a ROS-mediated mechanism.