Molecular mechanism of Rheb-dependent mTORC1 regulation

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Project Summary/Abstract mTORC1 is a master controller of cell growth/proliferation by regulating essential cellular anabolic processes, including protein, lipid, and nucleotide synthesis. For mTORC1 activation, while amino acids recruit mTORC1 to the lysosomal membrane through Rag small GTPases (Rags) activation, growth factors execute mTORC1 activation on the lysosome by activating Rheb small GTPase, a direct activator of mTORC1. However, the molecular mechanisms by which lysosomal mTORC1 recruited by the active Rags finds and interacts with Rheb on the lysosome have not been well understood. We recently reported that amino acid stimulation increased polyubiquitinated Rheb (Ub-Rheb) levels, which forms heteromultimers with ubiquitin-free Rheb and displays a strong binding preference for mTORC1, thereby supporting mTORC1 activation on the lysosome (Yao et al., Mol Cell, 2020). We identified that Ataxin3 acts as a key lysosomal Ub-Rheb deubiquitinase whose lysosomal localization is mitigated by amino acid-induced active Rags. We hypothesized that ubiquitin-dependent higher-order of assembly of Rheb might increase its density on the lysosome membrane and help sequester mTORC1 and its effectors for activation. However, ubiquitin ligases for Rheb, which generate Rheb polyubiquitination and support mTORC1 activation, have not been identified. Using Ub-Rheb-interacting proteomics and lysosome proteomics databases, we identified that both HUWE1 and the Cullin3 ubiquitin ligase complex interact with Rheb and positively regulate cellular mTORC1 activity without affecting Akt activity. Our preliminary data indicate that HUWE1 is required for Rheb to interact with both mTORC1 and CAD, a key enzyme for de novo pyrimidine synthesis, of which activity is stimulated by Rheb and the mTORC1-S6K1 pathway. In this proposal, we will study the molecular mechanisms by which HUWE1 and the CUL3 ubiquitin ligase complex coordinately ubiquitinate Rheb and the role of these ubiquitin ligases in the regulation of mTORC1 activity, de novo pyrimidine synthesis, and cell growth control. The specific aims of our proposed studies are to: investigate whether and how these ubiquitin ligases coordinately ubiquitinate Rheb to form catalytically competent Rheb multimers to stimulate mTORC1 activity (Aim1); to determine the roles and mechanisms of this Ub-Rheb-mTORC1-dependent signal amplification for specific downstream pathways such as de novo pyrimidine synthesis (Aim2); study the physiological relevance of this HUWE1/CUL3-Ub Rhab-mTORC1-CAD pathway in the regulation of cancer cell proliferation and tissue homeostasis such as liver and glomerular epithelial cells (Aim3). The proposed studies will provide previously unappreciated molecular mechanisms for mTORC1 activation and insight into the understanding Ub-Rheb-centered membraneless compartment for amplifying mTORC1-dependent signals for cell growth/proliferation control.