# Molecular mechanism of Rheb-dependent mTORC1 regulation

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2024 · $450,341

## Abstract

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.

## Key facts

- **NIH application ID:** 10831085
- **Project number:** 5R01GM145631-03
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Ken Inoki
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $450,341
- **Award type:** 5
- **Project period:** 2022-06-15 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10831085

## Citation

> US National Institutes of Health, RePORTER application 10831085, Molecular mechanism of Rheb-dependent mTORC1 regulation (5R01GM145631-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10831085. Licensed CC0.

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