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.

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

“…Some groups, using new RBD-based specific probes, have detected Ras activation preferentially at the plasma membrane (24,25). However, studies carried out using classical fluorescent RBD probes to detect the subcellular activation of Ras in T cells have shown that TCR engagement or CD28 costimulation activate H-and N-Ras almost exclusively on the Golgi, whereas costimulation via the integrin LFA-1 activates Ras on both the Golgi and the plasma membrane (26).…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…Several advanced fluorescence based approaches [89]. Some studies have revealed endomembrane Ras activation in addition to the cell surface pool [64,[90][91][92][93], whilst others only detect plasma membranelocalised Ras activation [94][95][96]. One criticism of the evidence for endomembranous Ras activation is that it is normally seen only with over-expression of Ras.…”

“…Importantly, endogenous Ras activation has been visualised, albeit with contradictory results [91,94]. Whilst further work is clearly required to resolve these differences, Philips and colleagues used their ability to detect endogenous ER/Golgi Ras activation to investigate how this might be regulated [91].…”

TCR-Induced Activation of Ras Proceeds at the Plasma Membrane and Requires Palmitoylation of N-Ras

“…Trimeric EGFP (E3) reporter constructs were generated by adding two EGFP modules to the 59 end of previously described E1 type reporter versions (18) (Fig. 2A).…”

“…GTPases to be cloned in fusion with mCherry were excised from parental dsRed2-C1 constructs (18) by XhoI/HindIII (K-Ras, K-RasG12V), XhoI/BamHI (M-RasQ71L, TC21Q72L), or BglII/EcoRI restriction (Rap1AG12V) and subcloned into pmCherry C1 (Clontech, Mountain View, CA). pmCherry-N-Ras was subcloned accordingly by BglII/KpnI restriction using HA-NRas in pCMV (kind gift of Ian Prior, University of Liverpool, Liverpool, U.K.) as the parental plasmid.…”