Activation of PI 3-Kinase Is Required For PDGF-induced H2O2 Production

生长因子对多种细胞的刺激作用 导致细胞内浓度的一过性增加 生长因子诱导蛋白酪氨酸所需的过氧化氢 磷酸化。应激时产生的过氧化氢的影响 表皮生长因子对蛋白质酪氨酸活性的影响 磷酸酶1B(PTP1B)在A431人中的研究 表皮样癌细胞。过氧化氢灭活重组PTP1B 在体外通过氧化其催化部位的半胱氨酸，最有可能生成亚磺酸 酸。被氧化的酶被更有效地激活 硫氧还蛋白比谷氧还蛋白或谷胱甘肽 生理浓度。防止过氧化氢氧化 碘乙酸对PTP1B催化半胱氨酸的修饰 这表明应该有可能监测氧化状态 PTP1B在细胞内的放射性掺入 在细胞存在的情况下裂解后的酶 放射性标记的碘乙酸。这种放射性的量 与A431细胞的PTP1B免疫沉淀相关 用EGF刺激10分钟，比之前减少了27% 与未受刺激的细胞的PTP1B相关。金额的多少 与PTP1B相关的碘乙酸源性放射性 在用EGF刺激细胞后达到最小10分钟 在40分钟后恢复到基准值，这表明氧化 PTP1B的表达在细胞内是可逆的。这些结果表明， 受体酪氨酸激酶的结合激活 相应的增长因素可能不足以增加 细胞内蛋白质酪氨酸磷酸化的稳定水平，以及 过氧化氢对蛋白酪氨酸磷酸酶的同时抑制作用 也可能是必需的。

Stimulation of various cells with growth factors results in a transient increase in the intracellular concentration of H2O2 that is required for growth factor-induced protein tyrosine phosphorylation. The effect of H2O2 produced in response to epidermal growth factor (EGF) on the activity of protein tyrosine phosphatase 1B (PTP1B) was investigated in A431 human epidermoid carcinoma cells. H2O2 inactivated recombinant PTP1B in vitro by oxidizing its catalytic site cysteine, most likely to sulfenic acid. The oxidized enzyme was reactivated more effectively by thioredoxin than by glutaredoxin or glutathione at their physiological concentrations. Oxidation by H2O2 prevented modification of the catalytic cysteine of PTP1B by iodoacetic acid, suggesting that it should be possible to monitor the oxidation state of PTP1B in cells by measuring the incorporation of radioactivity into the enzyme after lysis of the cells in the presence of radiolabeled iodoacetic acid. The amount of such radioactivity associated with PTP1B immunoprecipitated from A431 cells that had been stimulated with EGF for 10 min was 27% less than that associated with PTP1B from unstimulated cells. The amount of iodoacetic acid-derived radioactivity associated with PTP1B reached a minimum 10 min after stimulation of cells with EGF and returned to baseline values by 40 min, suggesting that the oxidation of PTP1B is reversible in cells. These results indicate that the activation of a receptor tyrosine kinase by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells, and that concurrent inhibition of protein tyrosine phosphatases by H2O2 might also be required.