Regulation of MEKK3 by Oxidative Stress

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。 由活性氧（reactive oxygen species，ROS）如过氧化氢（hydrogen peroxide，H2 O2）和超氧阴离子（superoxide，O2-）引起的氧化应激被认为是导致血管肥大的信号转导通路的生理激活剂。 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，其导致血管平滑肌细胞中产生H2 O2。 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，表明ROS是信号转导途径的重要调节剂。Akt信号转导通路在血管紧张素II、PDGF以及H2 O2处理的血管平滑肌细胞中受ROS调节。 然而，在这些细胞中，Akt下游起作用的蛋白质尚未被表征。 使用蛋白质组学和质谱，我们已经确定MEKK 3，丝氨酸/苏氨酸激酶，作为Akt的底物。 我们假设MEKK 3，像Akt一样，受产生ROS的配体调节。在第一个具体目标中，我们将确定MEKK 3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，MEKK 3也不例外。 14-3-3结合所必需的磷酸化位点将在特定目标2中定位。研究表明，MEKK 3的过表达激活了多个下游通路。 例如，MEKK 3可以激活JNK、ERK、p38和BMK 1 MAP激酶，以及NF-κ B信号通路。 此外，MEKK 3的催化结构域的诱导型表达通过p38阻止细胞周期进程。 然而，由内源性MEKK 3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MEKK 3的细胞内定位，目的是了解活化的MEKK 3位于细胞中的何处。 在最后一个具体的目标中，我们将表征MEKK 3被Akt磷酸化后调节的途径。 在这些研究的结论，我们将提供一个更好的机制了解活性氧如何调节Akt和MEKK 3，从而影响血管肥大的发展。

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.