Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays
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 regulate...
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doaj-358384fcdebd41f6bee0ce232b16ea822021-08-26T13:52:39ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-08-01228766876610.3390/ijms22168766Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro AssaysRaef Shams0Yoshihiro Ito1Hideyuki Miyatake2Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, RIKEN, Wako 351-0198, Saitama, JapanEmergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, RIKEN, Wako 351-0198, Saitama, JapanDepartment of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama City 338-8570, Saitama, JapanThe 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<sup>®</sup>) 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.https://www.mdpi.com/1422-0067/22/16/8766mTORC1RHEBG-Proteinallosteric activationkinase domainbinding kinetics |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Raef Shams Yoshihiro Ito Hideyuki Miyatake |
spellingShingle |
Raef Shams Yoshihiro Ito Hideyuki Miyatake Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays International Journal of Molecular Sciences mTORC1 RHEB G-Protein allosteric activation kinase domain binding kinetics |
author_facet |
Raef Shams Yoshihiro Ito Hideyuki Miyatake |
author_sort |
Raef Shams |
title |
Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays |
title_short |
Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays |
title_full |
Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays |
title_fullStr |
Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays |
title_full_unstemmed |
Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays |
title_sort |
evaluation of the binding kinetics of rheb with mtorc1 by in-cell and in vitro assays |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1661-6596 1422-0067 |
publishDate |
2021-08-01 |
description |
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<sup>®</sup>) 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. |
topic |
mTORC1 RHEB G-Protein allosteric activation kinase domain binding kinetics |
url |
https://www.mdpi.com/1422-0067/22/16/8766 |
work_keys_str_mv |
AT raefshams evaluationofthebindingkineticsofrhebwithmtorc1byincellandinvitroassays AT yoshihiroito evaluationofthebindingkineticsofrhebwithmtorc1byincellandinvitroassays AT hideyukimiyatake evaluationofthebindingkineticsofrhebwithmtorc1byincellandinvitroassays |
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1721192566886498304 |