| 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
|
|
WOS Category
|
BIOPHYSICS
|
| Resource |
| DNA material |
GL023 (RDB13254) |
