N-ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A
N-ethylmaleimide inhibits platelet-derived growth factor BB-stimulated Akt phosphorylation via activation of protein phosphatase 2A
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DOI：
10.1074/jbc.m206376200
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发表时间：
2002-10-18
影响因子：
4.8
通讯作者：
Rao, GN
中科院分区：
文献类型：
--
作者：
Yellaturu, CR;Bhanoori, M;Rao, GN
氧化还原状态在基因调控中起着重要作用。巯基维持细胞内氧化还原稳态。为了了解硫醇在氧化还原信号传导中的作用，我们研究了硫醇烷基化对血管平滑肌细胞中血小板衍生生长因子-BB（PDGF-BB）诱导的细胞存活事件的影响。PDGF-BB主要在Ser-473处刺激Akt磷酸化。N-乙基马来酰亚胺（NEM），一种硫醇烷基化剂，阻断PDGF-BB诱导的Akt磷酸化，而不影响其上游磷脂酰肌醇3-激酶（PI 3 K）。另一方面，PI 3 K的特异性抑制剂LY 294002和渥曼青霉素阻止PDGF-BB诱导的Akt及其下游效应分子p70 S6 K、核糖体蛋白S6、4 E-BP 1和eIF 4 E的磷酸化。NEM还消除了PDGF-BB诱导的p70 S6 K、核糖体蛋白S6、4 E-BP 1和eIF 4 E的磷酸化，表明巯基烷基化在Akt水平上干扰PI 3 K/Akt通路。此外，NEM阻断PDGF-BB诱导的BAD和叉头转录因子FKHR-L1的磷酸化，这些事件与细胞凋亡增加相关。NEM单独和与PDGF-BB一起增加VSMC中活性氧（ROS）的产生和蛋白磷酸酶2A（PP 2A）的活性。NEM对PDGF-BB诱导的Akt磷酸化的抑制作用可被PP 2A抑制剂fostriecin和okadaic acid、神经酰胺合成酶抑制剂fumonisin B1和ROS清除剂N-乙酰半胱氨酸（NAC）完全逆转。NAC还减弱了NEM单独或与PDGF-BB组合诱导的细胞凋亡。总之，这些发现首次证明PP 2A介导Akt和细胞存活的硫醇烷基化依赖性氧化还原调节。
The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473. N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BB-induced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger N-acetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.