Regulation of MEKK3 by Oxidative Stress

氧化应激对 MEKK3 的调节

基本信息

批准号：
6934496
负责人：
RICHARD R VAILLANCOURT
金额：
$ 22.73万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-15 至 2008-08-31
项目状态：
已结题

项目摘要

Continuous exposure to oxidative stress contributes to vascular hypertrophy, a condition that affects people as they age. Oxidative stress caused by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2-) are now recognized as physiological activators of signal transduction pathways that contribute to vascular hypertrophy. Platelet- derived growth factor (PDGF) and angiotensin II are ligands that bind to their respective receptors, which results in the production of H2O2 in vascular smooth muscle cells. The physiological response to PDGF and angiotensin II is increased DNA synthesis and vascular hypertrophy, respectively. Both biological responses are attenuated by inhibitors of ROS production, suggesting that ROS are important regulators of signal transduction pathways. The Akt signal transduction pathway is regulated by ROS in vascular smooth muscle cells treated with angiotensin II, PDGF, as well as H2O2. However, the proteins that function downstream of Akt have not been characterized in these cells. Using proteomics and mass spectrometry, we have identified MEKK3, a serine/threonine kinase, as a substrate of Akt. We hypothesize that MEKK3, like Akt, is regulated by ligands that generate ROS. In the first specific aim, we will determine the mechanism by which Akt phosphorylation of MEKK3 activates this kinase. Many Akt substrates, like Bad and the Forkhead transcription factor, associate with 14-3-3 proteins and MEKK3 is no exception. The phosphorylation sites that are necessary for 14-3-3 association will be mapped in specific aim two. Studies have shown that over-expression of MEKK3 activates multiple downstream pathways. For example, MEKK3 can activate the JNK, ERK, p38, and BMK1 MAP kinases, as well as the NF-kappaB signaling pathway. In addition, inducible expression of the catalytic domain of MEKK3 arrests cell cycle progression through p38. However, the signaling pathways that are regulated by endogenous MEKK3 remain unknown. In the third specific aim, we will characterize the intracellular localization of MEKK3 with the goal of understanding where activated MEKK3 is located in the cell. In the last specific aim, we will characterize the pathway that is regulated by MEKK3 after it is phosphorylated by Akt. At the conclusion of these studies, we will provide a better mechanistic understanding of how reactive oxygen species regulate Akt and MEKK3, and therefore affect the development of vascular hypertrophy.

持续暴露于氧化应激会导致血管肥大，这种情况会随着年龄的增长而影响人们。由过氧化氢 (H2O2) 和超氧化物 (O2-) 等活性氧 (ROS) 引起的氧化应激现在被认为是导致血管肥大的信号转导途径的生理激活剂。血小板衍生生长因子 (PDGF) 和血管紧张素 II 是与其各自受体结合的配体，导致血管平滑肌细胞产生 H2O2。 PDGF 和血管紧张素 II 的生理反应分别是 DNA 合成增加和血管肥大。这两种生物反应都会被 ROS 产生的抑制剂减弱，这表明 ROS 是信号转导途径的重要调节剂。在用血管紧张素 II、PDGF 和 H2O2 处理的血管平滑肌细胞中，Akt 信号转导途径受到 ROS 的调节。然而，这些细胞中在 Akt 下游起作用的蛋白质尚未得到表征。使用蛋白质组学和质谱分析，我们确定 MEKK3（一种丝氨酸/苏氨酸激酶）是 Akt 的底物。我们假设 MEKK3 与 Akt 一样，受到产生 ROS 的配体的调节。在第一个具体目标中，我们将确定 MEKK3 的 Akt 磷酸化激活该激酶的机制。许多 Akt 底物（例如 Bad 和 Forkhead 转录因子）与 14-3-3 蛋白相关，MEKK3 也不例外。 14-3-3 关联所需的磷酸化位点将在特定目标二中进行定位。研究表明，MEKK3 的过度表达会激活多个下游途径。例如，MEKK3 可以激活 JNK、ERK、p38 和 BMK1 MAP 激酶以及 NF-kappaB 信号通路。此外，MEKK3 催化结构域的诱导表达通过 p38 阻止细胞周期进程。然而，内源性 MEKK3 调节的信号通路仍然未知。在第三个具体目标中，我们将表征 MEKK3 的细胞内定位，旨在了解激活的 MEKK3 在细胞中的位置。在最后一个具体目标中，我们将表征 MEKK3 被 Akt 磷酸化后受其调节的途径。在这些研究结束时，我们将对活性氧如何调节 Akt 和 MEKK3，从而影响血管肥大的发展提供更好的机制理解。