RRC ID 33949
Author Hibino K, Shibata T, Yanagida T, Sako Y.
Title A RasGTP-induced conformational change in C-RAF is essential for accurate molecular recognition.
Journal Biophys J
Abstract The dysregulation of Ras-RAF signaling is associated with many types of human cancer. However, the kinetic and dynamic features of the mutual molecular recognition of Ras and RAF remain unknown. Here, we developed a technique for imaging single-pair fluorescence resonance energy transfer in living cells, and coupled this technique to single-molecule kinetic analysis to investigate how C-RAF (a subtype of RAF) molecules distinguish the active form of Ras (RasGTP) from the inactive form (RasGDP). Functional fragments of C-RAF containing the Ras-binding domains did not detect the switch in Ras activity in living cells as efficiently as did C-RAF. Single-molecule analysis showed that RasGDP associates with closed-conformation C-RAF, whereas the association of C-RAF with RasGTP immediately triggers the open RAF conformation, which induces an effective interaction between C-RAF and RasGTP. Spontaneous conformational changes from closed C-RAF to the open form rarely occur in quiescent cells. The conformational change in C-RAF is so important to Ras-RAF molecular recognition that C-RAF mutants lacking the conformational change cannot distinguish between RasGDP and RasGTP. The manipulation of the conformation of an effector molecule is a newly identified function of RasGTP.
Volume 97(5)
Pages 1277-87
Published 2009-9-2
DOI 10.1016/j.bpj.2009.05.048
PII S0006-3495(09)01059-5
PMID 19720015
PMC PMC3325157
MeSH Binding Sites Cell Membrane / metabolism Epidermal Growth Factor / metabolism Fluorescence Resonance Energy Transfer / methods Guanosine Diphosphate / metabolism Guanosine Triphosphate / metabolism HeLa Cells Humans Intracellular Space / metabolism Kinetics Microscopy, Confocal Models, Biological Mutation Protein Conformation Proto-Oncogene Proteins c-raf / chemistry* Proto-Oncogene Proteins c-raf / genetics Proto-Oncogene Proteins c-raf / metabolism* Transfection Video Recording ras Proteins / metabolism*
IF 3.854
Times Cited 23
DNA material GL023 (RDB13254)