Small modular GBDs (GTPase-binding domains) derived from GTPase-effector proteins are useful tools for the selective detection of the active GTP-loaded GTPase conformation, be it in biochemical assays or for imaging purposes. Use of GBD probes requires careful consideration of all features of the GDB–GTPase interaction. It is innate to the strong and specific interaction with the GTP-loaded GTPase, that GBDs will protect their partner GTPases from GAP (GTPase-activating protein) action. This feature is likely to cause an increase in cellular Ras-GTP levels, in particular in leucocytes and other cells with high steady-state Ras-GDP/GTP cycling rates. By the same token, high levels of GBD expression will interrupt GTPase-initiated signalling, with implications for the activation of the very same GTPase since feedback regulatory mechanisms can impinge on this process.
- live-cell analysis
- nucleotide exchange
Small GTPases cycle between a GDP and GTP-bound conformation in a tightly regulated fashion. GEFs (guanine nucleotide-exchange factors) promote GTP loading of the GTPases, and hence GTPase activation, whereas GAPs (GTPase-activating proteins) terminate signalling by accelerating GTPase-bound GTP hydrolysis. In its GTP-bound form, Ras GTPases interact with an ever-increasing number of (potential) effector or target proteins to exert their biological function. The c-Raf kinase is the best-characterized and most firmly established effector for the three prototypical Ras proteins K, H and N-Ras (Ras, from hereon). Owing to this feature (strong and selective binding to Ras-GTP versus Ras-GDP) the GBD (GTPase-binding domain) of c-Raf has become a widely used probe for both biochemical and imaging studies of Ras activation. Indeed, a number of studies reporting on the live-cell visualization of Ras activation have drawn widespread attention in recent times [1–4]. In all cases, Ras-GTP visualization was accomplished with probes built from the c-Raf-GBD. Beyond this fundamental consensus, the experimental strategies varied (see [5,6] for explicit reviews) as did some major findings. One issue, in particular, is currently being hotly debated in the field: does Ras become activated in endomembranes in response to growth factors?
Does Ras elicit signals from the Golgi?
While all these studies agree that Ras becomes activated at the plasma membrane, available experimental data disagree on the formation of Ras-GTP on endomembranes, prominently the Golgi apparatus. It is firmly established that Ras proteins undergo a post-translational ‘maturation’ process that drives them through the Golgi on their way to the plasma membrane [7,8]. Since Ras, then, is undoubtly present at that organelle, some Ras-GTP is bound to exist at the Golgi, but the burning question really is whether endogenous endomembrane Ras is under the control of growth factor signals and whether the levels of endogenous Golgi-located Ras-GTP rise and contribute to growth factor signalling. All imaging studies referred to herein have involved the overexpression of Ras [or Ras-chimaeric FRET (fluorescence resonance energy transfer) constructs] as a way to increase signal output. Ras is rate-limiting for its own activation in most systems where this has been investigated, indicating that activation of overexpressed Ras might proceed by mechanisms that do not operate on endogenous Ras. For example, Ras exhibits saturable binding sites on the plasma membrane [5,9], meaning that overexpression could result in overflow, mislocalization and hypothetically escape from GAP action.
Ras-GTP visualization: limitations imposed by the GBD probes
Along this line of reasoning, the impact of the GBD-based probes used for imaging is a further factor to be considered. The c-Raf-GBD protects Ras-GTP from GAP action in vitro, in cell lysates and in vivo (Figure 1) [10,11] leading to accumulation of Ras-GTP. This effect on Ras-GTP may be of particular importance in cells that exhibit high steady-state Ras-GDP/GTP cycling rates. Although most studies in the Ras field have focused on the regulation of agonist-induced Ras-GTP formation, relatively little attention has been paid to the control of the basal nucleotide turnover rate on Ras. A number of studies, however, illustrate that Ras proteins exhibit dramatically different rates of resting nucleotide turnover depending on the cell type (Figure 1). Leucocytes, for example, have a very high basal nucleotide turnover on Ras, whereas in resting fibroblast and epithelial cells commonly used in Ras studies, such as COS-7 and Rat-1 cells, Ras exhibits a much slower rate of nucleotide exchange [11–14]. Intriguingly, these differences in nucleotide turnover velocities correlate with the speed at which Ras-GTP accumulates in response to growth factors (see e.g. [11,12]). These considerations make it tempting to hypothesize a causal link between the basal nucleotide turnover rate and the speed of agonist-driven Ras-GTP formation. In the context of the present discussion, GAP protection exerted by ectopic expression of c-Raf-GBD is hence expected to have a particularly strong impact on Ras-GDP/GTP levels in cells with high Ras-GDP/GTP cycling rates.
In conclusion, the better we understand Ras function the more we are forced to realize that multiple consequences of experimental manipulation can impinge on the finely tuned and balanced endogenous Ras signalling machinery. In the context of the issue of live-cell visualization of Ras-GTP formation, obtaining conclusive evidence on the identity of the subcellular platforms of Ras activation will probably have to await approaches for the visualization of endogenous Ras-GTP. However, it is not clear that conclusive evidence can be obtained at all considering that GBDs or GBD-related reporter probes for Ras-GTP as we know them today, by definition, are bound to protect Ras from GAPs and to interrupt long-established as well as recently disclosed Ras-GTP-initiated feedback loops .
Localization and Activation of Ras-like GTPases: Focused Meeting held at the Royal Agricultural College, Cirencester, U.K., 21–23 March 2005. Organized and Edited by A. Ridley (Ludwig Institute of Cancer Research, London, U.K.) and M. Seabra (Imperial College London, U.K.).
Abbreviations: GAP, GTPase-activating protein; GBD, GTPase-binding domain
- © 2005 The Biochemical Society