The mammalian target of rapamycin (mTOR) has a central role in the regulation of cell growth and proliferation. By integrating signals derived from nutrients, energy status, and growth factors, the mTOR kinase controls protein translation by phosphorylating downstream effectors of growth. Deregulation of the mTOR pathway is implicated in a number of human diseases such as cancer and diabetes. In particular, the mTOR pathway is hyperactive in tuberous sclerosis complex (TSC), a mental retardation and cancer-prone syndrome affecting 1 in 6,000 people in the United States. This particular cancer results from mutations in either TSC1 or TSC2 tumor suppressor genes, which negatively regulate the mTOR activity via a small GTPase protein, Rheb. We do not yet know how loss of TSC1/2 function leads to tumor formation.

Our understanding of the mTOR pathway in many organisms has expanded considerably as several novel proteins essential for the function of the mTOR kinase have been identified and characterized. Our previous studies show that mTOR participates in two distinct multiprotein complexes, one of which, mTORC1, is acutely sensitive to the small molecule rapamycin and is regulated by TSC1/2. Rapamycin, a Food and Drug Administration-approved immunosuppressant, is currently in clinical trials for the treatment of certain human cancers, including TSC. Early work in animal models suggests the possibility that suppression of the mTORC1 pathway with rapamycin may be beneficial to TSC. Given that the mTORC1 pathway is an important target in TSC treatment, understanding how impairment of TSC1/2 function results in the activation of mTORC1 is critical. With the long-term goal of developing therapies based on the mTORC1 regulatory mechanisms, our structural and biochemical studies aim to find efficient means of regulating this signaling network. Therefore, we will carry out structural analysis of the key components of mTORC1 as well as mechanistic analysis of mTORC1 activation via Rheb.

The first specific aim will elucidate the molecular mechanism through which Rheb activates the mTORC1 pathway, downstream of TSC1/2. We will identify Rheb-induced Raptor phosphorylation sites by mass spectrometry and determine their functions and mechanisms of action in the activation of mTORC1 using functional assays. Our results will decipher the unknown link between Rheb and mTORC1 activation and provide important data regarding dysfunctions in TSC at the molecular level.

In addition to biochemical analysis of the Rheb-mediated mTORC1 activation, we aim to characterize the structures of the catalytic domain of mTORC1 and its interacting proteins to further our understanding of key protein-protein interactions and their functions. To accomplish this, we will design and apply a multidisciplinary approach, combining tools of structural biology, cell biology, and biochemistry. In particular, we will determine structures of the mTOR kinase domain with high-resolution X-ray diffraction, and we will utilize small-angle X-ray scattering and cryo-electron microscopy to construct an intermediate-resolution envelope of the high molecular weight complex. With the results obtained through successful experiments, we will create an adequate framework in which the high-resolution models that are generated by protein crystallography can be meaningfully fitted. This study will present new insights into understanding the protein-protein interactions, docking geometries, and stoichiometry of binding within the mTORC1 complex, as well as providing important information about molecular mechanisms of the mTORC1 regulation and their implications in TSC. Moreover, since inhibition of mTORC1 signaling is thought to be a promising strategy for anticancer therapeutic developments, the proposed work will provide critical information for effective drug designs.