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EMBO reports 7, 1, 46–51 (2006)
doi:10.1038/sj.embor.7400560
Published online: 11 November 2005
Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane
Martin Augsten1*, Rico Pusch2161429285, Christoph Biskup3, Knut Rennert2, Ute Wittig2, Katja Beyer4, Alfred Blume4, Reinhard Wetzker2, Karlheinz Friedrich1 & Ignacio Rubio2
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1 Institute of Biochemistry, Medical Faculty, Friedrich Schiller-University Jena, Nonnenplan 2, 07743 Jena, Germany
2 Institute of Molecular Cell Biology, Medical Faculty, Friedrich Schiller-University Jena, Drackendorfer Strasse 1, 07747 Jena, Germany
3 Institute of Physiology, Medical Faculty, Friedrich Schiller-University Jena, Teichgraben 8, 07740 Jena, Germany
4 Institute of Physical Chemistry, Martin Luther-University, Halle-Wittenberg, Mühlpforte 1, 06108 Halle, Germany
To whom correspondence should be addressed
Ignacio Rubio Tel: +49 3641 9325670; Fax: +49 3641 9325652; E-mail: b5igru@rz.uni-jena.de
* These authors contributed equally to this work
Received 21 February 2005; Accepted 21 September 2005; Published online 11 November 2005.
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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.
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