Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays.

Raef Shams,Hideyuki Miyatake,Yoshihiro Ito

doi:10.3390/ijms22168766

Abstract

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB–GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein–protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60–167) or M-heat domain (∆M, aa 967–1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240–1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148–2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.

Highlights

The mammalian/mechanistic target of rapamycin regulates cell growth and survival through the modulation of the metabolic pathways [1,2]. mTOR assembles in two different complexes, mTOR complex 1 and mTOR complex 2, to regulate different processes [1,3]
Biochemical studies suggested that the small G-protein, Ras homolog enriched in brain (RHEB) was involved in the activation of mTOR complex 1 (mTORC1) [8,9,10]
Because mTORC1 and mTOR complex 2 (mTORC2) are often hyperactivated in cancer cells to sustain their rapid growth, its inhibition has been proposed for cancer therapy

Summary

Introduction

The mammalian/mechanistic target of rapamycin (mTOR) regulates cell growth and survival through the modulation of the metabolic pathways [1,2]. mTOR assembles in two different complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), to regulate different processes [1,3]. In the mTORC1, mTOR is a kinase complexed with other proteins, regulatory associated protein of mTOR (Raptor), the mammalian lethal with. In response to growth factors and nutrients, mTORC1 regulates a variety of life phenomena; synthesis of proteins, lipids and nucleotides, cell proliferation, and autophagy [1]. Several studies have revealed the molecular mechanisms of mTORC1 kinase activation by amino acids and growth factors [5,6,7]. Biochemical studies suggested that the small G-protein, Ras homolog enriched in brain (RHEB) was involved in the activation of mTORC1 [8,9,10]. It was found that the tuberous sclerosis complex 1/2 (TSC1/2), the upstream negative regulator of mTORC1, served as a GTPase-activating protein for RHEB [7,11,12].

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