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Live‐cell imaging of endogenous Ras‐GTP illustrates predominant Ras activation at the plasma membrane
Author(s) -
Augsten Martin,
Pusch Rico,
Biskup Christoph,
Rennert Knut,
Wittig Ute,
Beyer Katja,
Blume Alfred,
Wetzker Reinhard,
Friedrich Karlheinz,
Rubio Ignacio
Publication year - 2006
Publication title -
embo reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.584
H-Index - 184
eISSN - 1469-3178
pISSN - 1469-221X
DOI - 10.1038/sj.embor.7400560
Subject(s) - endogeny , golgi apparatus , gtp' , gtpase , microbiology and biotechnology , anti apoptotic ras signalling cascade , jurkat cells , g protein , effector , gtpase activating protein , chemistry , biology , signal transduction , enzyme , biochemistry , endoplasmic reticulum , t cell , mapk/erk pathway , immunology , immune system
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.