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.

连续暴露于氧化应激会导致血管肥大，这种疾病会随着年龄的增长而影响人们。现在，由活性氧（ROS）（例如过氧化氢（H2O2）和超氧化物（O2-））引起的氧化应激现在被认为是有助于血管肥大的信号转导途径的生理激活剂。血小板衍生的生长因子（PDGF）和血管紧张素II是结合其各自受体的配体，这导致血管平滑肌细胞中H2O2的产生。对PDGF和血管紧张素II的生理反应分别增加了DNA合成和血管肥大。 ROS产生的抑制剂抑制了这两种生物反应，这表明ROS是信号转导途径的重要调节剂。 Akt信号转导途径在用血管紧张素II，PDGF以及H2O2处理的血管平滑肌细胞中由ROS调节。然而，在这些细胞中尚未表征AKT下游功能的蛋白质。使用蛋白质组学和质谱法，我们确定了丝氨酸/苏氨酸激酶MEKK3作为Akt的底物。我们假设Mekk3像AKT一样受生成ROS的配体调节。在第一个特定目的中，我们将确定MEKK3 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关联所必需的磷酸化位点将在特定目标两个中映射。研究表明，MEKK3的过表达激活了多个下游途径。例如，MEKK3可以激活JNK，ERK，P38和BMK1 MAP激酶以及NF-kappab信号通路。另外，MEKK3催化结构域的诱导表达可阻止细胞周期通过p38。但是，由内源性MEKK3调节的信号通路仍然未知。在第三个特定目的中，我们将表征MEKK3的细胞内定位，以了解激活的MEKK3位于单元格中的位置。在最后一个特定目的中，我们将表征MEKK3被AKT磷酸化后的途径。在这些研究的结论中，我们将对活性氧如何调节Akt和Mekk3的方式提供更好的机械理解，从而影响血管肥大的发展。