Defining the Biochemical Functions of the TSC Tumor Suppressors in mTORC1 Signaling

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Project Summary/Abstract Many growth factor signaling pathways converge on the tuberous sclerosis complex (TSC) complex to activate the mechanistic target of rapamycin complex 1 (mTORC1), a key event in the promotion of anabolic processes underlying cell growth. The TSC complex is composed of the tumor suppressors, TSC1 and TSC2, and the protein TBC1D7. Within the complex, TSC2 acts as a GTPase activating protein (GAP) towards the small G- protein Rheb, which potently activates mTORC1 when it is GTP bound. The pathways that control mTORC1 are comprised of some of the most common oncogenes and tumor suppressors genetically altered in human cancers. Moreover, mutations found in TSC1 and TSC2 themselves, have been identified in sporadic cancers. In these settings, mTORC1 is aberrantly activated and results in uncontrolled cell growth. Although significant progress has been made in understanding the mTORC1 signaling network, many longstanding questions underlying the mechanistic function of the TSC complex and its regulation of Rheb remain unanswered. Our lab recently established that specific phosphorylation events on TSC2 by the growth factor signaling kinase, Akt, regulates mTORC1 activity through spatial localization of the TSC complex. However, a deeper mechanistic understanding of how phosphorylation impacts TSC2-Rheb binding and the Rheb-GDP/GTP state, and whether additional kinases regulate localization of the complex is needed. Moreover, how TSC2 binds to Rheb in different nucleotide-bound states and whether the TSC complex contains regulatory functions towards Rheb independent of GAP activity are unknown. This proposal will address these unanswered questions regarding the molecular determinants of TSC complex regulation and function through biochemical studies including in vitro TSC2-Rheb binding, Rheb nucleotide exchange and TSC2 kinase assays as well as cell based immunofluorescence microscopy and mTORC1 signaling experiments. Collectively, these mechanistic studies will provide novel molecular insights into the regulation and function of the TSC tumor suppressors and their regulation of Rheb and mTORC1 in both normal settings and cancer. To investigate these questions, I propose the following aims: 1) Map the TSC2-Rheb binding domains and characterize novel regulatory functions inherent to these regions. 2) Determine how multi-site phosphorylation of TSC2 by upstream kinases influences TSC2-Rheb binding and regulation. The research outlined in this proposal will take place at the Harvard T.H. Chan school of Public Health in the lab of Dr. Brendan Manning. Dr. Manning is an expert in the mTORC1 field and has extensive expertise in biochemical techniques and experimental design. Additionally, many of the proposed reagents and protocols have already been established in the Manning lab.