Regulation of MEKK3 by Oxidative Stress

氧化应激对 MEKK3 的调节

• 批准号: 6533931
• 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
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6533931
• 关键词: aging; angiotensin II; catalase; enzyme activity; enzyme induction /repression; fluorescence microscopy; hydrogen peroxide; laboratory rabbit; liquid chromatography mass spectrometry; microtubule associated protein; mitogen activated protein kinase; nuclear factor kappa beta; oncoproteins; oxidative stress; phosphorylation; platelet derived growth factor; protein kinase; protein protein interaction; proteomics; superoxides; vascular smooth muscle

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，这是一种随着年龄增长而影响人们的疾病。过氧化氢（H2O2）和超氧化物（O2-）等活性氧（ROS）引起的氧化应激现已被认为是导致血管肥大的信号转导途径的生理激活因子。血小板衍生生长因子（PDGF）和血管紧张素II是与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因子。在血管紧张素II、PDGF和H2O2处理的血管平滑肌细胞中，Akt信号转导通路受ROS调控。然而，Akt下游的功能蛋白在这些细胞中尚未被表征。通过蛋白质组学和质谱分析，我们确定了丝氨酸/苏氨酸激酶MEKK3是Akt的底物。我们假设MEKK3和Akt一样，受产生ROS的配体调控。在第一个具体目标中，我们将确定Akt磷酸化MEKK3激活该激酶的机制。许多Akt底物，如Bad和Forkhead转录因子，都与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具体目标二中绘制。研究表明，MEKK3的过表达激活了多种下游通路。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调控的信号通路尚不清楚。在第三个特定目标中，我们将描述MEKK3的细胞内定位，目的是了解活化的MEKK3在细胞中的位置。在最后一个具体目标中，我们将描述MEKK3被Akt磷酸化后的调控途径。在这些研究的结论中，我们将更好地了解活性氧如何调节Akt和MEKK3，从而影响血管肥大的发生。