Molecular Basis of the Acceleration of the GDP-GTP Exchange of Human Ras Homolog Enriched in Brain by Human Translationally Controlled Tumor Protein

Xianchi Dong,Bei Yang,Yingjie Li,Chen Zhong,Jianping Ding

doi:10.1074/jbc.m109.012823

Abstract

Ras homolog enriched in brain (Rheb), a small GTPase, positively regulates the mTORC1 pathway. The GDP-GTP exchange of Rheb has been suggested to be facilitated by translationally controlled tumor protein (TCTP). Here we demonstrate that human TCTP (hTCTP) interacts with human Rheb (hRheb) and accelerates its GDP release in vitro and that hTCTP activates the mTORC1 pathway in vivo. To investigate the underlying mechanism, we built structure models of GDP- and GTP-bound hRheb in complexes with hTCTP and performed molecular dynamics simulations of the models, which predict key residues involved in the interactions and region of hRheb undergoing conformational change during the GDP-GTP exchange. These results are verified with site-directed mutagenesis and in vitro biochemical and in vivo cell biological analyses. Furthermore, a crystal structure of the E12V mutant hTCTP, which lacks the guanine nucleotide exchange factor activity, shows that the deficiency appears to be caused by loss of a salt-bridging interaction with Lys-45 of hRheb. These data collectively provide insights into the molecular mechanisms of how hTCTP interacts with hRheb and activates the mTORC1 pathway.

Highlights

A central regulator of cell growth and cell proliferation by integrating signals, including growth factors, nutrients, and energy status, and has been implicated to play important roles in cancer cell metabolism [1]. mammalian target of rapamycin (mTOR) functions in the context of two distinct multiprotein complexes
2 To whom correspondence may be addressed: Tel.: 86-21-5492-1626; Fax: 86-21-5492-1116; E-mail: czhong@sibs.ac.cn. 3 To whom correspondence may be addressed: Tel.: 86-21-5492-1619; Fax: 86-21-5492-1116; E-mail: jpding@sibs.ac.cn. 4 The abbreviations used are: mTOR, the mammalian target of rapamycin; Rheb, Ras homolog enriched in brain; hRheb, human Rheb; dRheb, Drosophila Rheb; TCTP, translationally controlled tumor protein; hTCTP, human TCTP; dTCTP, Drosophila TCTP; GEF, guanine nucleotide exchange factor; MD, molecular dynamics; TSC, tuberous sclerosis complex; r.m.s.d., root mean square deviation; S6K1, S6 kinase 1; 4EBP1, eukaryote initiation factor 4E-binding protein 1; SGK1, serum- and glucocorticoid-induced protein kinase 1; GST, glutathione S-transferase; siRNA, small interference RNA
Two research groups recently reported that hTCTP has no GEF activity toward hRheb and suggested that hTCTP is not involved in the mTORC1 pathway [11, 17]

Summary

Introduction

A central regulator of cell growth and cell proliferation by integrating signals, including growth factors, nutrients, and energy status, and has been implicated to play important roles in cancer cell metabolism [1]. mTOR functions in the context of two distinct multiprotein complexes. Our modeling and simulation results predicted the key residues that are involved in the hTCTP-hRheb interaction and the important region that undergoes conformational change during the GDP-GTP exchange of hRheb and reached a different conclusion than that by Rehmann et al [17].

Results

Conclusion