Regulation of MEKK3 by Oxidative Stress

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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.