title: Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane.
abstract: Ras-GTP imaging studies using the Ras-binding domain (RBD) of the Ras effector c-Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras-GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras-GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist-induced endogenous Ras activation at the plasma membrane (PM) of COS-7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant-negative RasS17N and pharmacological manipulations matches Ras-GTP formation assessed biochemically. Our data illustrate that endogenous Golgi-located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist-induced Ras activation.
authors.affiliation: Institute of Biochemistry, Medical Faculty, Friedrich Schiller-University Jena, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Augsten
authors.forename: Rico
authors.initials: R
authors.lastname: Pusch
authors.forename: Christoph
authors.initials: C
authors.lastname: Biskup
authors.forename: Knut
authors.initials: K
authors.lastname: Rennert
authors.forename: Ute
authors.initials: U
authors.lastname: Wittig
authors.forename: Katja
authors.initials: K
authors.lastname: Beyer
authors.forename: Alfred
authors.initials: A
authors.lastname: Blume
authors.forename: Reinhard
authors.initials: R
authors.lastname: Wetzker
authors.forename: Karlheinz
authors.initials: K
authors.lastname: Friedrich
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rubio
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence / Q000379:methods*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D018631:ras Proteins