Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane

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 Ra...

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Bibliographic Details
Published in:EMBO reports 2006-01, Vol.7 (1), p.46-51
Main Authors: Augsten, Martin, Pusch, Rico, Biskup, Christoph, Rennert, Knut, Wittig, Ute, Beyer, Katja, Blume, Alfred, Wetzker, Reinhard, Friedrich, Karlheinz, Rubio, Ignacio
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cercopithecus aethiops
COS Cells
Genes, Reporter
Golgi
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Growth factors
Guanosine Triphosphate - metabolism
Humans
imaging
Jurkat Cells
localization
Microscopy, Fluorescence - methods
PC12 Cells
plasma membrane
Probes
Protein Structure, Tertiary
Ras
ras Proteins - genetics
ras Proteins - metabolism
Rats
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Scientific Report
Side effects
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Summary: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.
ISSN:1469-221X
1469-3178
1469-221X
DOI:10.1038/sj.embor.7400560