A RasGTP-induced conformational change in C-RAF is essential for accurate molecular recognition.
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.
Hibino
Kayo
K
Cellular Informatics Laboratory, RIKEN, Wako, Japan.
Shibata
Tatsuo
T
Yanagida
Toshio
T
Sako
Yasushi
Y
D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D008954:Models, Biological; D009154:Mutation; D011487:Protein Conformation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014741:Video Recording; D018631:ras Proteins / Q000378:metabolism*
D006153:Guanosine Diphosphate; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins