title: A bimolecular fluorescent complementation screen reveals complex roles of endosomes in Ras-mediated signaling.
abstract: 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.
authors.affiliation: Lester and Sue Smith Breast Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Ze-Yi
authors.initials: ZY
authors.lastname: Zheng
authors.affiliation: Lester and Sue Smith Breast Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA. Electronic address: echang1@bcm.tmc.edu.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Chang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism*; D015723:Gene Library; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D018631:ras Proteins
keywords: BiFC; CHMP6; ESCRT; Endosome; Oncogene; Ras GTPases; Signal transduction; Transformation; VPS4A