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Sensors 2012, 12(7), 9411-9422; doi:10.3390/s120709411
Article

Dynamics of Ras Complexes Observed in Living Cells

1,2
, 3
 and 1,2,*
Received: 12 June 2012; in revised form: 29 June 2012 / Accepted: 4 July 2012 / Published: 9 July 2012
(This article belongs to the Special Issue Live Cell-Based Sensors)
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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 K-Ras; Raf1; BiFC; membrane association; signal pathway
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.

AMA Style

Li X, Cheng Z, Jin H. Dynamics of Ras Complexes Observed in Living Cells. Sensors. 2012; 12(7):9411-9422.

Chicago/Turabian Style

Li, Xiangyong; Cheng, Zhiyong; Jin, Honglin. 2012. "Dynamics of Ras Complexes Observed in Living Cells." Sensors 12, no. 7: 9411-9422.


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