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Dynamics of Ras Complexes Observed in Living Cells
Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China
MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
Wuhan Mechanical Technology College, Wuhan 430075, China
* Author to whom correspondence should be addressed.
Received: 12 June 2012; in revised form: 29 June 2012 / Accepted: 4 July 2012 / Published: 9 July 2012
Abstract: 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.
Keywords: K-Ras; Raf1; BiFC; membrane association; signal pathway
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MDPI and ACS Style
Li, X.; Cheng, Z.; Jin, H. Dynamics of Ras Complexes Observed in Living Cells. Sensors 2012, 12, 9411-9422.
Li X, Cheng Z, Jin H. Dynamics of Ras Complexes Observed in Living Cells. Sensors. 2012; 12(7):9411-9422.
Li, Xiangyong; Cheng, Zhiyong; Jin, Honglin. 2012. "Dynamics of Ras Complexes Observed in Living Cells." Sensors 12, no. 7: 9411-9422.