Sensors | |
Dynamics of Ras Complexes Observed in Living Cells | |
Xiangyong Li2  Zhiyong Cheng1  | |
[1] Wuhan Mechanical Technology College, Wuhan 430075, China; E-Mail:;Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China; E-Mail: | |
关键词: K-Ras; Raf1; BiFC; membrane association; signal pathway; | |
DOI : 10.3390/s120709411 | |
来源: mdpi | |
【 摘 要 】
K-Ras works as a switch in many important intracellular signaling pathways and plays important roles in cell growth, proliferation, differentiation and carcinogenesis. For signal transduction from K-Ras to Raf1, the best-characterized effector of K-Ras, the general view is that Ras recruits Raf1 from the cytoplasm to the cell membrane. To elucidate this process, we constructed a series of fusion proteins (including Raf1 and K-Ras fused with either fluorescent proteins or fluorescent protein fragments) to compare subcellular localizations of these proteins. Bimolecular fluorescence complementation (BiFC) and a co-transfection system were used. In the BiFC system, the K-Ras/Raf1 complexes were mainly located in the cell membrane, while the Raf1 control was uniformly distributed in the cytoplasm. However, the complexes of Raf1 and K-RasC185S, a K-Ras mutant which loses membrane-localization, were also able to accumulate in the cell membrane. In contrast, an apparent cytosolic distribution pattern was observed in cells co-transfected with mcerulean-Raf1 and EGFP-K-RasC185S, suggesting that the membrane localization of K-Ras/Raf1 complexes is not entirely dependent on K-Ras, and that other factors, such as the irreversible conformation formed between K-Ras and Raf1 may play a role. This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane.
【 授权许可】
CC BY
© 2012 by the authors; licensee MDPI, Basel, Switzerland.
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
RO202003190043043ZK.pdf | 710KB | download |