Epidermal growth factor (EGF)-induced generation of hydrogen peroxide - Role in EGF receptor-mediated tyrosine phosphorylation
Epidermal growth factor (EGF)-induced generation of hydrogen peroxide - Role in EGF receptor-mediated tyrosine phosphorylation
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DOI：
10.1074/jbc.272.1.217
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发表时间：
1997-01-03
影响因子：
4.8
通讯作者：
Rhee, SG
中科院分区：
文献类型：
--
作者：
Bae, YS;Kang, SW;Rhee, SG
最近的证据表明活性氧（ROS）可能在受体信号通路中起细胞内信使的作用。因此研究了ROS在表皮生长因子（EGF）信号传导中的可能作用。用氧化敏感荧光探针2′，7′-双醋酸二氯荧光素和激光扫描共聚焦显微镜检测到，EGF刺激A431人表皮样癌细胞导致细胞内ROS浓度瞬间升高。产生的主要ROS似乎是H2O2，因为通过电穿孔将过氧化氢酶掺入细胞，完全消除了egf诱导的荧光增加。过氧化氢酶对H2O2的消除也抑制了EGF诱导的各种细胞蛋白的酪氨酸磷酸化，包括EGF受体和磷脂酶c - γ 1。研究了H2O2的产生对EGF受体内在酪氨酸激酶活性及其cooh末端末端的自磷酸化位点的依赖。在表达激酶失活的EGF受体的细胞中，EGF不能诱导H2O2生成。然而，在表达突变受体的细胞中观察到正常的H2O2生成，其中126个cooh末端氨基酸被删除，以去除4个（总共5个）自磷酸化位点。这些结果表明，EGF诱导的H2O2形成需要激酶活性，但可能不需要EGF受体的自磷酸化位点，并且H2O2可能需要抑制蛋白酪氨酸磷酸酶活性才能表现出EGF诱导的蛋白酪氨酸磷酸化。
Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers in receptor signaling pathways. The possible role of ROS in epidermal growth factor (EGF) signaling was therefore investigated. Stimulation of A431 human epidermoid carcinoma cells with EGF resulted in a transient increase in the intracellular concentration of ROS, measured with the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin diacetate and laser-scanning confocal microscopy. The predominant ROS produced appeared to be H2O2, because the EGF-induced increase in fluorescence was completely abolished by incorporation of catalase into the cells by electroporation. The elimination of H2O2 by catalase also inhibited the EGF-induced tyrosine phosphorylation of various cellular proteins including the EGF receptor and phospholipase C-gamma 1. The dependence of H2O2 production on the intrinsic tyrosine kinase activity of the EGF receptor and the autophosphorylation 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 expressing a mutant receptor from which the 126 COOH-terminal amino acids had been deleted to remove four (out of the total of five) autophosphorylation sites. These results suggest that EGF-induced H2O2 formation requires the kinase activity but probably not the autophosphorylation sites of the EGF receptor and that inhibition of protein tyrosine phosphatase activity by H2O2 may be required for EGF-induced protein tyrosine phosphorylation to be manifested.