This A2 proposal is focused on renal disease in Tuberous Sclerosis Complex (TSC). TSC is an autosomal dominant disorder in which the renal manifestations include angiomyolipomas, polycystic kidney disease, and carcinoma. We and others have shown that loss of tuberin (TSC2) upregulates Rheb activity. Rheb activates the mammalian target of rapamycin (mTOR). In published work, we demonstrated mitotic regulation of hamartin (TSC1) by the cyclin-dependent kinase CDK1. In unpublished work, we have found that hamartin is centrosome-localized and interacts with the mitotic kinase Plk1. Consistent with a central role of hamartin (TSC1) in mitosis, we also found that Tsc1''' mouse embryonic fibroblasts (MEFs) have mitotic and centrosome defects. This proposal centers on two inter-related central hypotheses: 1) mitotic regulation of hamartin plays a key role in the function of the hamartin/tuberin complex, and 2) Rheb is the critical downstream target through which hamartin (TSC1) and tuberin (TSC2) function as tumor suppressors. To address these hypotheses, we propose the following Specific Aims. Aim 1: To define the impact of mitotic phosphorylation of hamartin (TSC1) on Rheb activity and the hamartin-Plkl interaction. Aim 2: To test the hypothesis that loss of hamartin (TSC1) results in centrosomal and mitotic defects. Aim 3: To determine whether Rheb-dependent pathways are activated in sporadic human renal tumors. Aim 4: To determine whether Rheb expression in the kidney induces tumors. Our published and preliminary data point toward a critical role of hamartin in regulation of mitosis (Aims 1 and 2), with broad potential cancer relevance. In Aims 3 and 4 we will examine the role of Rheb in renal tumorigenesis in vivo, using both human tumors and transgenic mice. We anticipate that this project will elucidate the central pathways leading to tumorigenesis in TSC. Lay summary. TSC is a genetic disease in which patients can develop kidney tumors, kidney cancer, and kidney cysts. This project will use biochemistry, cell culture, human specimens, and animal models to elucidate the cause of kidney disease in TSC. The cellular and biochemical pathways that cause kidney disease in TSC are likely to be closely related to the pathways that cause kidney tumors and cysts in other individuals.