Regulation of MEKK3 by Oxidative Stress

持续暴露在氧化应激下会导致血管肥大，随着年龄的增长，这种情况会影响到人们。过氧化氢(H_2O_2)、超氧阴离子(O_2~-)等活性氧物种(ROS)引起的氧化应激是导致血管肥大的信号转导通路的生理性激活物。血小板衍生生长因子(PDGF)和血管紧张素II是与其各自的受体结合的配体，导致血管平滑肌细胞产生过氧化氢。对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。血管紧张素II、血小板衍生生长因子和过氧化氢对血管平滑肌细胞Akt信号转导通路的调节受ROS的影响。然而，Akt下游发挥作用的蛋白质在这些细胞中还没有被鉴定。利用蛋白质组学和质谱仪，我们已经确定MEKK3，一个丝氨酸/苏氨酸激酶，是Akt的底物。我们假设MEKK3和Akt一样，是由产生ROS的配体调控的。在第一个特定目标中，我们将确定MEKK3的Akt磷酸化激活该激酶的机制。许多Akt底物，如Bad和Forkhead转录因子，与14-3-3蛋白相关，MEKK3也不例外。14-3-3结合所需的磷酸化位点将在特定目标2中绘制。研究表明，MEKK3的过度表达激活了多条下游通路。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催化区的可诱导表达可通过p38阻止细胞周期进程。然而，受内源性MEKK3调控的信号通路仍不清楚。在第三个特定目标中，我们将表征MEKK3在细胞内的定位，目的是了解激活的MEKK3在细胞中的位置。在最后一个特定目标中，我们将描述被Akt磷酸化后受MEKK3调控的途径。在这些研究的结论中，我们将更好地从机制上理解活性氧如何调节Akt和MEKK3，从而影响血管肥厚的发展。

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.