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Dynamics of Ras Complexes Observed in Living Cells
AbstractK-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.
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Li, X.; Cheng, Z.; Jin, H. Dynamics of Ras Complexes Observed in Living Cells. Sensors 2012, 12, 9411-9422.View more citation formats
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.