Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS

Cell Reports

卷:32

Noritaka Nishida¹  Ryu Fujimiya²  Ichio Shimada²  Qingci Zhao²  Satoshi Kubo²  Mitsuhiko Ikura³  Christopher B. Marshall³ 

[1] RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan;

[2] Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;

[3] Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada;

关键词: RAS;    small GTPase;    oncogenic mutants;    in-cell NMR;    time-resolved NMR;    molecular crowding;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: The small guanosine triphosphatase (GTPase) RAS serves as a molecular switch in signal transduction, and its mutation and aberrant activation are implicated in tumorigenesis. Here, we perform real-time, in-cell nuclear magnetic resonance (NMR) analyses of non-farnesylated RAS to measure time courses of the fraction of the active GTP-bound form (fGTP) within cytosol of live mammalian cells. The observed intracellular fGTP is significantly lower than that measured in vitro for wild-type RAS as well as oncogenic mutants, due to both decrease of the guanosine diphosphate (GDP)-GTP exchange rate (kex) and increase of GTP hydrolysis rate (khy). In vitro reconstitution experiments show that highly viscous environments promote a reduction of kex, whereas the increase of khy is stimulated by unidentified cytosolic proteins. This study demonstrates the power of in-cell NMR to directly detect the GTP-bound levels of RAS in mammalian cells, thereby revealing that the khy and kex of RAS are modulated by various intracellular factors.

【 授权许可】

Unknown