title: Dynamics of Ras complexes observed in living cells.
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.
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. xlxyongli@gmail.com
authors.forename: Xiangyong
authors.initials: X
authors.lastname: Li
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Cheng
authors.forename: Honglin
authors.initials: H
authors.lastname: Jin
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins