A bimolecular fluorescent complementation screen reveals complex roles of endosomes in Ras-mediated signaling

• Published in: Methods in enzymology Vol. 535; p. 25
• Main Authors: Zheng, Ze-Yi, Chang, Eric C
• Format: Journal Article
• Language: English
• Published: United States 2014
• Subjects: Animals; Bacterial Proteins - biosynthesis; Cell Line, Tumor; Endosomes - metabolism; Gene Library; HEK293 Cells; Humans; Luminescent Proteins - biosynthesis; Mice; Microscopy, Fluorescence; NIH 3T3 Cells; Protein Binding; Protein Interaction Mapping - methods; Protein Transport; ras Proteins - metabolism; Recombinant Fusion Proteins - biosynthesis; Signal Transduction; Signal transduction; Transformation; ESCRT; Oncogene; VPS4A; Ras GTPases; BiFC; CHMP6; Endosome
• ISSN: 1557-7988
• DOI: 10.1016/B978-0-12-397925-4.00002-X

While Ras GTPases are best known for mediating growth factor signaling on the plasma membrane, these proteins also have surprisingly complex activities in the endosome. Assisted by a method called bimolecular fluorescent complementation (BiFC), which can detect weak and transient protein-protein interactions and reveal where the binding takes place in live cells, we have identified three effectors, Cdc42, CHMP6, and VPS4A that interact with Ras proteins in endosomes. These effectors are all necessary for Ras-induced transformation, suggesting that for Ras proteins to efficiently induce tumor formation, they must also activate effectors in cytoplasm, such as those in endosomes. Here, we describe how BiFC can be used to detect and screen for Ras effectors and for readily revealing where in the cell the binding occurs.