Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B
Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B
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DOI：
10.1074/jbc.m201070200
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发表时间：
2002-11-08
影响因子：
4.8
通讯作者：
Cuadrado, A
中科院分区：
文献类型：
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作者：
Martín, D;Salinas, M;Cuadrado, A
本研究旨在阐明过氧化氢诱导细胞死亡过程中Akt/蛋白激酶B（PKB）存活通路下调的机制。H2 O2产生Akt/PKB的早期激活和DNA损伤，随后是p53水平的稳定，活性氧（ROS）的形成，并通过谷胱甘肽敏感的中性鞘磷脂酶的激活产生神经酰胺。这些事件与Akt的长期去磷酸化和随后的降解相关。一个膜靶向的活性Akt版本衰减凋亡，但不坏死诱导过氧化氢和更耐去磷酸化和蛋白水解诱导的过氧化氢的凋亡浓度。外源性谷胱甘肽的加入阻止了Akt的蛋白水解，表明ROS和神经酰胺在Akt降解中的作用。然而，Akt在转染野生型和显性负性p53突变体的细胞中类似地降解，表明Akt在氧化损伤下的降解可能是p53非依赖性的。caspase组I和III的特异性抑制剂阻止Akt/PKB和聚（ADP-核糖）聚合酶在细胞凋亡，但不坏死H2 O2浓度的蛋白水解。令人惊讶的是，在caspase-3缺陷的MCF-7细胞中，Akt对H2 O2诱导的降解比caspase-3底物聚（ADP-核糖）聚合酶更敏感。此外，Akt/PKB双突变体Akt（D108 A，D119 A），这是不被半胱天冬酶-3切割，和三重突变体（D453 A，D455 A，D456 A），这缺乏半胱天冬酶-3切割的共有序列，也降解过氧化氢处理的细胞。我们的研究结果表明，强氧化剂产生细胞内ROS和神经酰胺，这在长期导致Akt的去磷酸化和caspase-3的非依赖性蛋白水解下调。
This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis.