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 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.
Augsten
Martin
M
Institute of Biochemistry, Medical Faculty, Friedrich Schiller-University Jena, Germany.
Pusch
Rico
R
Biskup
Christoph
C
Rennert
Knut
K
Wittig
Ute
U
Beyer
Katja
K
Blume
Alfred
A
Wetzker
Reinhard
R
Friedrich
Karlheinz
K
Rubio
Ignacio
I
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*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D018631:ras Proteins