The study investigates the role of reactive oxygen species (ROS), particularly hydrogen peroxide (H₂O₂), in epidermal growth factor (EGF) signaling. Using A431 human epidermoid carcinoma cells, the researchers found that EGF stimulation led to a transient increase in intracellular ROS levels, primarily H₂O₂, as measured by the oxidation-sensitive fluorescent probe 2,7-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The addition of catalase, which specifically dismutates H₂O₂, completely abolished the EGF-induced increase in fluorescence, suggesting that H₂O₂ is the predominant ROS produced. Further experiments showed that the elimination of H₂O₂ by catalase inhibited EGF-induced tyrosine phosphorylation of various cellular proteins, including the EGF receptor and phospholipase C-γ1 (PLC-γ1). The study also explored the dependence of H₂O₂ production on the intrinsic tyrosine kinase activity of the EGF receptor and its autophosphorylation sites. EGF failed to induce H₂O₂ generation in cells expressing a kinase-inactive EGF receptor, but normal H₂O₂ generation was observed in cells expressing a mutant receptor lacking four out of five autophosphorylation sites. These results suggest that EGF-induced H₂O₂ formation requires kinase activity but not autophosphorylation sites, and that inhibition of protein tyrosine phosphatase (PTPase) activity by H₂O₂ may be necessary for EGF-induced protein tyrosine phosphorylation.The study investigates the role of reactive oxygen species (ROS), particularly hydrogen peroxide (H₂O₂), in epidermal growth factor (EGF) signaling. Using A431 human epidermoid carcinoma cells, the researchers found that EGF stimulation led to a transient increase in intracellular ROS levels, primarily H₂O₂, as measured by the oxidation-sensitive fluorescent probe 2,7-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The addition of catalase, which specifically dismutates H₂O₂, completely abolished the EGF-induced increase in fluorescence, suggesting that H₂O₂ is the predominant ROS produced. Further experiments showed that the elimination of H₂O₂ by catalase inhibited EGF-induced tyrosine phosphorylation of various cellular proteins, including the EGF receptor and phospholipase C-γ1 (PLC-γ1). The study also explored the dependence of H₂O₂ production on the intrinsic tyrosine kinase activity of the EGF receptor and its autophosphorylation sites. EGF failed to induce H₂O₂ generation in cells expressing a kinase-inactive EGF receptor, but normal H₂O₂ generation was observed in cells expressing a mutant receptor lacking four out of five autophosphorylation sites. These results suggest that EGF-induced H₂O₂ formation requires kinase activity but not autophosphorylation sites, and that inhibition of protein tyrosine phosphatase (PTPase) activity by H₂O₂ may be necessary for EGF-induced protein tyrosine phosphorylation.