The Tuberous Sclerosis Complex 2 (TSC2) tumor suppressor functions as a gatekeeper for mTOR activity in the PI3K signaling pathway. The mTOR kinase regulates several cellular functions, including protein synthesis, cell growth and autophagy, with TSC2 functioning as a rheostat to regulate mTOR by integrating mitogenic signals with energy availability. Interestingly, TSC2-deficent cells have aberrant redox homeostasis, and our preliminary data indicate TSC2 is required for mTOR repression by reactive oxygen species (ROS), leading us to hypothesize that TSC2 represses mTOR in response to oxidative stress. In addition, we found that the cellular damage sensor ATM is activated by ROS, and that in the absence of either ATM or TSC2, cells are unable to appropriately repress mTOR in response to ROS. It is known that TSC2 localizes to cellular endomembranes, and that membrane localization regulates TSC2 function. However, the specific endomembrane(s) to which this tumor suppressor localizes has not been determined. Importantly, we found that both ATM and TSC2 localize to the peroxisome, a major site for 2-oxidation of fatty acids and generation of ROS in the cell. These data have led us to propose the hypothesis that ATM and TSC2 participate in a stress response pathway at the peroxisome that responds to ROS to repress mTOR signaling. As mTOR is a negative regulator of autophagy, we hypothesize that the physiological role for TSC2-mediated mTOR repression in response to ROS is to regulate autophagy and peroxisome turnover. The overarching hypothesis of this proposal is that the TSC2 tumor suppressor participates downstream of ATM in a novel stress response pathway at the peroxisome to repress mTOR signaling and regulate autophagy. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1: Test the hypothesis that TSC2 and components of this mTOR regulatory node (the TSC2 activation partner TSC1, its GAP target Rheb and mTOR) localize to the peroxisome. Specific Aim 2: Identify the signaling pathway(s) that regulate TSC2 function at the peroxisome. Specific Aim 3: Test the hypothesis that peroxisomal TSC2 represses mTOR to regulate autophagy and/or peroxisomal turnover by pexophagy. The experiments proposed in this application will allow us to test our hypothesis that TSC2 participates downstream of ATM in a novel stress response pathway at the peroxisome to repress mTOR and regulate cellular autophagy. If this hypothesis is correct, and we are successful at demonstrating that this peroxisome- localized pathway has physiological significance, these studies will for the first time focus attention on the peroxisome as a signaling organelle. In addition, they will lay the groundwork for future studies to understand how cell signaling at the peroxisome participates in carcinogensis and open the possibility for the development of strategies targeting the peroxisome to regulate cell signaling and autophagy in tumor cells. PUBLIC HEALTH RELEVANCE: The overarching hypothesis of this proposal is that TSC2 participates in a novel stress response pathway at the peroxisome to repress mTOR signaling and regulate cellular autophagy. Understanding the mechanisms that regulate the process of autophagy or influence the cell's decision to initiate this process has important implications for carcinogenesis and cancer therapy. A better understanding of the mechanisms that control this process has the potential to aid in the design of prevention strategies targeting the peroxisome and signaling pathways that localize to this organelle. In addition, in the future they may aid in the design of therapeutics that limit the ability of tumor cells to utilize autophagy as a survival pathway and/or promote autophagy-induced programmed cell death in response to therapy.