Department of Human Sciences, Society, and Health, University of Cassino and Southern Lazio, 03043 Cassino, Italy.
Oxid Med Cell Longev. 2012;2012:792705. doi: 10.1155/2012/792705. Epub 2012 Oct 31.
Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras.
过氧化氢加入培养的星形胶质细胞后，Ha-Ras 和 Ki-Ras 的表达迅速而短暂地增加。用 Raf-1 的 GTP-Ras 结合域进行的下拉实验表明，氧化应激显著增加了 Ha-Ras 的激活，而假定的法尼基化激活形式的 Ki-Ras 仅略有增加。Ha-Ras 和 Ki-Ras 的增加都对蛋白质合成抑制剂环己酰亚胺不敏感，并且被蛋白酶体抑制剂 MG-132 阻断。此外，暴露于过氧化氢会降低泛素化 Ras 蛋白的水平，表明氧化应激通过泛素/蛋白酶体途径导致两种同工型的降解减少。事实上，Ha-Ras 和 Ki-Ras 的后期减少是由于蛋白酶体降解的恢复，因为它对 MG-132 敏感。PD98059 可抑制 MAP 激酶途径，可消除 Ha-Ras 水平的后期减少，而 PD98059 对 Ki-Ras 的后期减少没有影响。我们得出结论，氧化应激在培养的星形胶质细胞中差异调节 Ha-Ras 和 Ki-Ras 的表达，并且氧化应激本身或通过其他因素激活 MAP 激酶途径可能作为一种失效安全机制，限制潜在有害的 Ha-Ras 的持续表达。
