Preprint Communication Version 1 Preserved in Portico This version is not peer-reviewed

Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-cell and In vitro Assays

Version 1 : Received: 10 June 2021 / Approved: 11 June 2021 / Online: 11 June 2021 (09:14:14 CEST)

A peer-reviewed article of this Preprint also exists.

Shams, R.; Ito, Y.; Miyatake, H. Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays. Int. J. Mol. Sci. 2021, 22, 8766. Shams, R.; Ito, Y.; Miyatake, H. Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays. Int. J. Mol. Sci. 2021, 22, 8766.

Abstract

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, RHEB-GTPase. On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and FAT, 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. For this, we used the split-luciferase system (NanoBiT®) for in-cell studies, and prepared proteins for the in vitro measurements. Consequently, it was shown that RHEB binds to the whole mTOR both in the presence or absence of GTPɣS, with five-fold weaker affinity in the presence of GTPɣS. Also, RHEB bound to the truncated mTOR fragments of N-heat domain (60-167) and M-heat domain (967-1023) in a GTP independent manner. Furthermore, RHEB bound to the truncated kinase domain (2148-2300) with higher affinity also in GTP independently. In conclusion, RHEB binds two different binding sites of mTOR, which probably regulates the kinase activity of mTOR through multiple different molecular mechanisms.

Keywords

mTORC1; RHEB; G-Protein; Allosteric activation; Kinase domain; Binding kinetics

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

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