Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS
Qingci Zhao,
Ryu Fujimiya,
Satoshi Kubo,
Christopher B. Marshall,
Mitsuhiko Ikura,
Ichio Shimada,
Noritaka Nishida
Affiliations
Qingci Zhao
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Ryu Fujimiya
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Satoshi Kubo
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Christopher B. Marshall
Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
Mitsuhiko Ikura
Princess Margaret Cancer Centre, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada
Ichio Shimada
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; Corresponding author
Noritaka Nishida
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan; Corresponding author
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.
