REACTIVE OXYGEN SPECIES MEDIATE PHORBOL ESTER-REGULATED TYROSINE PHOSPHORYLATION AND PHOSPHOLIPASE-A(2) ACTIVATION - POTENTIATION BY VANADATE

We have previously shown that vanadate potentiates the activating effe ct of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pat hway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) t hat participate in this process. Treatment of macrophages with vanadat e plus TPA led to activation of protein kinase C (PKC) and NADPH oxida se (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) ac tivity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activit ies were assessed in cell lysates). Inhibition of ROS formation by dip henyleneiodonium (DPI) prevented PTP inhibition, PTK activation and pr otein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O 2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selec tive inhibitors) prevented the above-mentioned effects of vanadate plu s TPA, but not of vanadate plus H2O2. Collectively, the results show t hat ROS formation induced by TPA in association with vanadate is essen tial in the modulation of protein tyrosine phosphorylation and PLA2 ac tivity.