EPIDERMAL GROWTH-FACTOR (EGF)-INDUCED GENERATION OF HYDROGEN-PEROXIDE- ROLE IN EGF RECEPTOR-MEDIATED TYROSINE PHOSPHORYLATION

Recent evidence indicates that reactive oxygen species (ROS) may funct ion as intracellular messengers in receptor signaling pathways. The po ssible role of ROS in epidermal growth factor (EGF) signaling was ther efore investigated. Stimulation of A431 human epidermoid carcinoma cel ls with EGF resulted in a transient increase in the intracellular conc entration of ROS, measured with the oxidation-sensitive fluorescent pr obe 2',7'-dichlorofluorescin diacetate and laser-scanning confocal mic roscopy. The predominant ROS produced appeared to be H2O2, because the EGF-induced increase in fluorescence was completely abolished by inco rporation of catalase into the cells by electroporation. The eliminati on of H2O2 by catalase also inhibited the EGF-induced tyrosine phospho rylation of various cellular proteins including the EGF receptor and p hospholipase C-gamma 1. The dependence of H2O2 production on the intri nsic tyrosine kinase activity of the EGF receptor and the autophosphor ylation sites located in its COOH-terminal tail was investigated. EGF failed to induce H2O2 generation in cells expressing a kinase-inactive EGF receptor. However, normal H2O2 generation was observed in cells e xpressing a mutant receptor from which the 126 COOH-terminal amino aci ds had been deleted to remove four (out of the total of five) autophos phorylation sites. These results suggest that EGF-induced H2O2 formati on requires the kinase activity but probably not the autophosphorylati on sites of the EGF receptor and that inhibition of protein tyrosine p hosphatase activity by H2O2 may be required for EGF-induced protein ty rosine phosphorylation to be manifested.