Esposito Franca, Chirico Giuseppa, Montesano Gesualdi Nicola, Posadas Inmaculada, Ammendola Rosario, Russo Tommaso, Cirino Giuseppe, Cimino Filiberto
Dipartimento di Biochimica e Biotecnologie Mediche, Universitá di Napoli Federico II, Via Pansini 5, Naples 80131, Italy.
J Biol Chem. 2003 Jun 6;278(23):20828-34. doi: 10.1074/jbc.M211841200. Epub 2003 Apr 7.
Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.
活性氧（ROS）作为第二信使参与有丝分裂信号转导。支持ROS作为信号分子作用的大多数实验数据是通过使用过氧化氢（H2O2）获得的。在没有生长因子结合的情况下，将细胞暴露于H2O2会迅速增加酪氨酸激酶受体（TKRs）的酪氨酸磷酸化，从而诱导下游信号级联反应的激活，如蛋白激酶B（AKT）的激活。另一种能够诱导细胞内ROS水平升高的分子是马来酸二乙酯（DEM），它通过消耗ROS清除剂还原型谷胱甘肽（GSH）起作用。对H2O2和DEM所产生效应的比较表明，后者诱导的氧化还原修饰比H2O2产生的修饰更温和。我们还证明，DEM诱导的氧化还原修饰不会伴随血小板衍生生长因子受体（PDGF-R）和表皮生长因子受体酪氨酸磷酸化，尽管它们能够激活细胞外调节蛋白激酶（ERKs）和AKT，但其动力学与H2O2处理后观察到的不同。这两条途径的激活不会被PDGF-R酪氨酸激酶的选择性抑制剂AG1296阻断，从而证实DEM的作用不是由TKR磷酸化介导的。相反，Src激酶抑制剂PP2（4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3,4-d]嘧啶）完全阻止DEM和H2O2诱导的AKT激活，但对ERKs途径没有影响。最后，在DEM处理的细胞中观察到PDGF-R中酪氨酸残基硝化，因此表明在生长因子受体水平可能发生不同于酪氨酸磷酸化的ROS诱导修饰，并可能参与信号通路的调节。
