Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Babraham, Cambridge, CB2 4AT, UK
* Author for correspondence (e-mail: peter.lockyer{at}bbsrc.ac.uk)
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Ras, GTPase, Raf, Reporter, Probe, Biosensor, Imaging, Signalling, Membrane, Trafficking, Plasma membrane, Golgi, Endoplasmic reticulum, Endosomes, Prenylation, Palmitoylation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In 1980, pioneering work from Scolnick and colleagues (Willingham et al., 1980) demonstrated that H-Ras and K-Ras proteins are predominantly located on the inner leaflet of the plasma membrane in Harvey and Kirsten murine sarcoma virus (MSV)-transformed cells [see Malumbres and Barbacid (Malumbres and Barbacid, 2003
) for a timeline detailing the identification of Ras oncogenes]. Immunocytochemistry indicated no specific localization of Ras in the nucleus or other intracellular sites. However, using electron microscopy, they observed that `some p21 was seen on the cytoplasmic surface of what appeared to be uncoated endocytic vesicles near the plasma membrane, and small amounts were seen on the cytoplasmic face of a few vesicles in the Golgi apparatus'. With remarkable foresight, they speculated that MSV-induced transformation by Ras involves processes associated with guanine-nucleotide-binding proteins in the plasma membrane (Willingham et al., 1980
). In hindsight, there was also evidence in many later studies that a significant amount of H-Ras is localized to intracellular structures such as the Golgi (Leevers et al., 1994
; Marais et al., 1995
; Thissen et al., 1997
), but this was not further investigated at the time.
We now know far more about how post-translational lipid modifications direct Ras proteins to associate with multiple cell membranes, including the plasma membrane (Fig. 1), so that Ras can be activated (Apolloni et al., 2000; Choy et al., 1999
; Dong et al., 2003
; Lobo et al., 2002
). Recently, significant advances have been made in understanding how post-translationally modified Ras isoforms traffic to the plasma membrane from the endoplasmic reticulum (ER) and Golgi (Apolloni et al., 2000
; Choy et al., 1999
; Dong et al., 2003
; Lobo et al., 2002
), are segregated in membrane domains (Hancock, 2003
), and are differentially internalized and signal from endosomes (Roy et al., 2002
). Perhaps one of the more provocative questions arising from this work concerns the signalling role of Ras-GTP on intracellular organelles such as the ER and Golgi (Bivona and Philips, 2003
; Chiu et al., 2002
). If these are important sites for interactions between Ras and its effectors, then their proximity to the nucleus has implications for pathways driving the control of gene transcription. In addition to endosomes, the ER and Golgi, another enigmatic location for Ras signalling are the mitochondria, which poses interesting questions about the balance between cell survival and apoptosis (and subversion by oncogenic Ras) in this compartment (Rebollo et al., 1999
).
|
In the literature, a canonical view has developed for receptor tyrosine kinase (RTK) signalling cascades in which a Ras GEF (Sos) is recruited by adaptors to the receptor in order to activate Ras at the plasma membrane (Pawson, 2004). Ras is deactivated by the recruitment of GAPs such as p120 Ras GAP through Src-homology 2 (SH2) domains to phosphotyrosine residues on the activated receptor. The delineation of this pathway was a major advance in signal transduction research more than a decade ago (Malumbres and Barbacid, 2003
; Pawson, 2004
) and was soon followed by the demonstration that Ras-GTP recruits the serine/threonine kinase Raf to the plasma membrane to facilitate Raf activation (Leevers et al., 1994
; Stokoe et al., 1994
; Traverse et al., 1993
). In addition, there are many dynamically regulated Ras GEFs and Ras GAPs that do not operate through phosphotyrosine-based recognition motifs at the RTK. For example, there is strong evidence that members of the GRP/CalDAG-GEF family and some members of the GAP1 family are specifically regulated by the second messengers Ca2+ and/or diacylglycerol (DAG) (Cullen and Lockyer, 2002
; Walker et al., 2003
). The physiological significance of such modulation is not yet clear for many of these proteins, except for Ras GRP1, which has been shown to be a major target for DAG during T-cell receptor (TCR) signalling and is needed for proper thymocyte development in the mouse (Dower et al., 2000
).
Since many RTKs are coupled to phospholipase C (PLC) signalling through PLC , perhaps it is of no surprise that there are GEFs and GAPs that are able to respond to the `products' of PLC activity - Ca2+ and DAG. What has been particularly exciting from this signalling perspective has been the recent discovery of a novel class of PLC, PLC
, as a candidate for an effector of Ras (Kelley et al., 2001
; Lopez et al., 2001
; Song et al., 2001
). The most studied target of DAG is protein kinase C (PKC), and it has long been known that PKCs integrate DAG and Ca2+ signals at the level of Ras and Raf through multiple mechanisms (Corbit et al., 2003
; Kawakami et al., 2003
; Marais et al., 1998
), including potentially inhibiting GAP activity (Downward et al., 1990
; Marais et al., 1998
; Villalonga et al., 2002
). Analysis of the spatio-temporal regulation of PKC isoforms by second messengers has offered great insight into the dynamic nature by which they are differentially recruited to membranes and scaffolds. The C2 and C1 domains of PKC are essential for these mechanisms and similar domains are built into members of the GRP and GAP1 families (Cullen and Lockyer, 2002
; Walker et al., 2003
), enabling them to respond dynamically to a given second messenger signal (Bivona et al., 2003
; Caloca et al., 2003
; Lockyer et al., 2001
; Walker et al., 2004
). Thus, it would seem that the study of Ras activity in space and real-time is of importance. Only with the development of cell-based assays to complement existing technologies will issues such as specificity, compartmentalization and effector output be truly refined.
Work in the past few years using Raf, or domains from Raf, as activity probes for Ras in live cells has highlighted unexpected mechanisms and locations for Ras signal output (Bivona and Philips, 2003; Hancock, 2003
; Hingorani and Tuveson, 2003
). Such methodologies use different domains from Raf fused to fluorescent proteins as probes, and can take advantage of additional fluorescence resonance energy transfer (FRET; Fig. 2). Here, we review the use of these biosensors, their advantages and their inherent limitations. Although we have concentrated here on Ras, activity probes for other small GTPases show equal promise (Pertz and Hahn, 2004
).
|
![]() |
Use of Ras activity probes reporting membrane localization |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Philips and co-workers have concentrated on over-expressing Ras isoforms along with a construct containing GFP fused to residues 51-131 of Raf-1 (Chiu et al., 2002). In their hands, a significant fraction of ectopic H-Ras appears to localize to the Golgi apparatus when co-expressed with GFPRBD in non-starved COS-1 cells (Chiu et al., 2002
). In the absence of serum, the GFP-RBD probe is entirely cytosolic, indicating that serum factors mediate the GTP loading of over-expressed Ras at the Golgi. In addition, constitutively active Ras co-expressed with GFP-RBD causes recruitment of the reporter to cell membranes, and no membrane-associated probe is detected by co-expression with dominant-negative Ras (Chiu et al., 2002
). H-Ras mutants that cannot be palmitoylated, and therefore are unable to traffic through the secretory pathway to the plasma membrane, induce the translocation of GFP-RBD to the ER and Golgi. This indicates that, once farnesylated, over-expressed H-Ras can be GTP loaded on endomembranes, including the ER. Although no ER-resident Ras-GTP-interacting protein has been found in mammalian cells, recent data indicate that there are novel candidates in budding yeast (Sobering et al., 2003
). Unlike H-Ras and N-Ras, K-Ras4B contains a polybasic sequence rather than sites for palmitoylation (K-Ras4A is palmitoylated but K-Ras4B is not). K-Ras4B exits the ER and bypasses the Golgi altogether en route to the plasma membrane (Fig. 1). Thus, when co-expressed with K-Ras4B, no GFP-RBD reporter localizes to the Golgi but it clearly associates with the plasma membrane (Chiu et al., 2002
).
An exciting aspect to the study became apparent when Philips and co-workers monitored the behaviour of the probe during agonist stimulation (Fig. 4). They treated serum-starved COS-1 cells over-expressing H-Ras with epidermal growth factor (EGF) or insulin and observed that the plasma membrane and the Golgi exhibit different kinetics of probe recruitment (Bivona and Philips, 2003; Chiu et al., 2002
). The translocation of the RBD to the Golgi was shown to be independent of endocytosis and was therefore due to active Ras in situ. In contrast to the recruitment of the RBD to the plasma membrane, translocation to the Golgi was dependent on Src kinases. To assay the Golgi response for endogenous Ras, Philips and co-workers used a novel `bystander' FRET technique (Chiu et al., 2002
): cyan fluorescent protein (CFP) was fused to the RBD and co-transfected with the transmembrane protein CD8 fused to yellow fluorescent protein (YFP-CD8). This labelled membranes with YFP-CD8, permitting FRET between CFP-RBD and YFP-CD8 when the RBD was bound to endogenous Ras-GTP in close proximity to over-expressed CD8. When starved cells expressing these constructs were stimulated by EGF or insulin, an increased FRET signal was detected on the plasma membrane and Golgi (Chiu et al., 2002
). Chiu et al. concluded that Src-dependent activation of a specific GEF(s) or inhibition of a specific GAP(s), or both, could be responsible for the spatial and temporal profiles of plasma membrane versus endomembrane Ras activity after mitogenic stimulation (Chiu et al., 2002
). The implication of this work was the existence of specific pathways to Ras activation on alternative membrane compartments, and therefore potentially broader Ras effector signalling output.
|
Bondeva et al. used a different approach, avoiding cotransfection of GFP-RBD with Ras constructs (Bondeva et al., 2002). They compared the location of the probe in normal versus H-Ras-transformed NIH 3T3 cells (Bondeva et al., 2002
). Their RBD constructs were fused at the C-terminus to GFP, unlike the Chiu et al. construct that had GFP at the N-terminus. This difference in orientation made no difference to reporter behaviour (T. Balla, personal communication). It is worth considering the physiological relevance, since Ras expression levels in virally transformed cells are likely to be substantially lower than those driven by a strong plasmid promoter in transiently transfected cells. RBD-GFP (residues 51-131) had little or no membrane localization in starved HRas-transformed NIH 3T3 cells; yet, incorporation of the Raf-1 cysteine-rich domain (CRD) into the probe (RBD-CRD residues 51-200; Fig. 2) was sufficient to induce a clear membrane translocation. This also correlated with in vitro analysis of the strength of the interactions between RBD (51-131) and Ras versus those between RBD-CRD (51-200) and Ras (Bondeva et al., 2002
). Over-expression of the RBD-CRD reporter in the studies by Bondeva et al. appeared to saturate Ras-binding sites at the membrane, suggesting that there is a limit to the Ras-binding sites available even at moderate levels of RBD-CRD expression (Bondeva et al., 2002
).
In common with Chiu et al., Bondeva et al. were able to use a GFP reporter to monitor the spatio-temporal kinetics of Ras activation, although they did not need a FRET method to detect endogenous Ras in normal NIH 3T3 cells stimulated by growth factor (Bondeva et al., 2002). However, unlike Chiu et al., they observed no localization of the reporters to the Golgi even in COS cells expressing constitutively active Ras (Bondeva et al., 2002
). Their results thus argue against the idea that Ras is significantly active on endomembranes.
![]() |
Activation of Ras on the Golgi |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FRET-based Ras activity probes |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Current FRET methods for Ras-GTP detection rely on over-expression of Ras and reporter constructs. In theory, the technique is sensitive and quantitative compared with analysis of the fluorescence intensities of GFP-RBD at the membrane versus the cytosol. Jiang and Sorkin (Jiang and Sorkin, 2002) have successfully used a corrected FRET method (Gordon et al., 1998
) to detect FRET between YFP-RBD (residues 1-153) and active CFP-Ras (Jiang and Sorkin, 2002
). In experiments with starved cells, Jiang and Sorkin detected significant levels of ectopic, active H-Ras on cell membranes - predominantly perinuclear vesicular structures that could include the Golgi (Jiang and Sorkin, 2002
). In contrast to Chiu et al., they concluded that over-expression of H-Ras led to activation of a substantial pool of small GTPase, regardless of serum starvation or even growth factor stimulation. This indicated that the method is more suitable for tracking the movement of active Ras, rather than where and how much activation is occurring per se (Jiang and Sorkin, 2002
).
The Raichu-Ras probe is an innovative FRET reporter designed to assay active Ras in live cells by reporting the sum of GEF and GAP activities on the compartment it is targeted to (Mochizuki et al., 2001). This hybrid molecule (Fig. 5A) reports the intramolecular association of an RBD (51-131) with H-Ras, although the reporter actually includes the hypervariable region of K-Ras to ensure membrane localization. The probe has produced results in marked contrast to those of Chiu et al., despite use of the same cell type and equivalent stimulation (Miyawaki, 2003
). In COS cells stimulated with EGF, the Raichu-Ras probe is clearly activated only at the plasma membrane (Mochizuki et al., 2001
) (Fig. 5B). The explanation for the discrepancy between techniques is not clear, although this could be due to the nature of the KRas4B-specific post-translational modifications on the Raichu-Ras probe, which should direct it to the plasma membrane and bypass the Golgi altogether, despite the H-Ras coding sequence. However, merely replacing the H-Ras sequence with that of Rap1 (Raichu-Rap1) generates a reporter that appears to be selectively activated at perinuclear sites, and not the plasma membrane. This supports previous observations that Rap1 is predominantly localized to intracellular compartments and not to the plasma membrane. However, the functional significance of the Mochizuki et al. study has been disputed by others, who have measured mitogenic Rap1 activation exclusively at the plasma membrane using GFP reporters in the same cell lines (Bivona et al., 2004
).
|
Matsuda and co-workers have taken a step further to offer a very different hypothesis for the localization of EGF-induced Ras signalling (Ohba et al., 2003). Provocatively, they have proposed that a gradient of cellular GAP activity, with the highest deactivation of Ras in the centre, radiates out to the plasma membrane. This idea was based on observing the uniform cytoplasmic expression of an artificial cAMP-responsive Ras GEF (e-GRF) but functional activation of Ras only at the peripheral plasma membrane (Ohba et al., 2003
). In addition, Ras appeared to be uniformly activated in EGF-stimulated cells when Raichu-Ras probes with reduced sensitivity to GAPs were used (Ohba et al., 2003
). Kinetic simulations of GEF and GAP activity were integrated into a virtual cell model to support the theory of a GAP gradient. At first glance, this is an eccentric proposition given that several potent Ras GAPs, including GAP1m, which was used in the Raichu-Ras studies, are specific sensors of signalling events at the plasma membrane. This is because they dynamically translocate from the cytosol to this compartment in response to an agonist-evoked stimulus, such as the localized generation of a second messenger. For example, p120 Ras GAP is believed to terminate Ras signalling by being recruited to RTKs (Pawson, 2004
), GAP1m is a high-affinity receptor for phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane (Lockyer et al., 1999
) and CAPRI is a Ca2+-activated Ras GAP when it translocates to the cell periphery (Bivona et al., 2003
; Lockyer et al., 2001
). It is of course possible that there are specific Ras GAPs that are active in the perinuclear region of the cell. Neurofibromin (NF-1) is just such a candidate and has been localized to both mitochondria (Roudebush et al., 1997
) and microtubules (Gregory et al., 1993
). However, the exceptional difficulty of working with NF-1 has precluded detailed study of its molecular regulation. Further experimental testing of the virtual cell theory of a Ras GAP gradient is required for it to gain credence (Ohba et al., 2003
).
Full-length Raf-1 has also been tried as a FRET or membrane-localization reporter for active Ras with varying degrees of success (Bondeva et al., 2002; Hibino et al., 2003
; Jiang and Sorkin, 2002
). This might reflect the complex regulation of Raf-1 membrane recruitment (Bondeva et al., 2002
) or the unfavourable positioning of fluorescent protein partners in Ras and Raf for FRET analysis (Jiang and Sorkin, 2002
). For detectable translocation of GFP-Raf-1 in EGF-stimulated cells, Hibino et al. had to over-express Ras (Hibino et al., 2003
). They observed sustained recruitment of a small proportion of Raf to membrane ruffles for more than 60 minutes (Hibino et al., 2003
). The physiological significance of this result is unclear and requires further investigation given that Ras activity returns to low levels within 30-60 minutes of mitogenic stimulation (Hibino et al., 2003
). Moreover, they also observed perinuclear accumulation of Raf-1, suggesting active Ras is present on endomembranes (Hibino et al., 2003
).
![]() |
Perspectives |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When considering the spatio-temporal analysis of Ras activity, it is worth noting some specific effects that over-expression might influence. There are interesting differences in palmitoylation between Ras isoforms - for example, H-Ras is probably twice as palmitoylated as N-Ras (Hancock, 2003). The degree of palmitoylation could vary between membrane compartments and might even depend on the activation status of the small GTPase (Baker et al., 2003
). If there are differences in palmitoylation between proteins and between cellular compartments, then this could be a concern when one compares over-expression results obtained with different Ras isoforms. The post-translational enzymatic machinery might have to work much harder to modify H-Ras, and this could impact on differences in localization and activation status. Thus, validation of endogenous Ras activity is particularly important - for example, by the bystander FRET method (Chiu et al., 2002
). Ras might also influence the dynamics of the very organelles that it traffics through in the secretory pathway; for example, inducible expression of oncogenic N-Ras causes the collapse of the Golgi complex and an increase in constitutive protein transport in NRK cells (Babia et al., 1999
).
Experiments to determine the spatio-temporal pattern of activation of Ras by EGF stimulation have led to different conclusions: co-expression of H-Ras and GFP-RBD indicated a rapid activation at the plasma membrane followed by later activation on the Golgi (Chiu et al., 2002), whereas analysis with Raichu-Ras indicated activation at the plasma membrane but not at perinuclear sites, where Rap1-GTP loading was clearly enhanced (Mochizuki et al., 2001
). The Raichu-Ras probe is an odd mixture of H-Ras and the RBD, membrane localization being provided by the hypervariable region of KRas4B (Mochizuki et al., 2001
). It is becoming increasingly apparent that the nature of the hypervariable group on Ras proteins has significant influence on signalling specificity by determining membrane microlocalization and the efficiency of interaction with GEFs, effectors and even GAPs (Jaumot et al., 2002
). Differences in post-translational modification between Ras isoforms and the possible influence on GEF specificity is a factor that should be considered when using chimeric Ras molecules such as Raichu-Ras. There is evidence that GEFs are sensitive to the prenylation status of Ras-family GTPases (Gotoh et al., 2001
) and Sos1 requires prenylation of Ras proteins for efficient nucleotide exchange (Porfiri et al., 1994
). If the Raichu-Ras probe is further developed, it would be interesting to couple H-, N- and K-Ras GTPases with their respective hypervariable domains and then determine the spatio-temporal profile of activation to see whether there are any differences.
Is the RBD sufficiently specific to report Ras activation over Rap? The answer so far is yes but only because so many studies have relied on the co-expression of a Ras isoform, and little detectable membrane localization has been seen in untransfected cells. The probe simply does not appear sensitive enough to report endogenous Ras-GTP with a great dynamic range - this conclusion is based purely on quantifying the amount of fluorescence at a given membrane - so interference from endogenous Rap has not been so much of an issue. There has been no evidence that the RBD is efficiently recruited to Rap1 in live cells even following over-expression of Rap-GTP (Chiu et al., 2002), although the RBD does work well in the Raichu-Rap1 intramolecular FRET reporter (Miyawaki, 2003
).
Is the RBD useful if it inhibits effector and GAP interactions? In the case of Raichu-Ras, this does not appear to be a problem (Mochizuki et al., 2001); however, it might be when GFP-RBD is expressed alone. For example, Chiu et al. reported that insulin activates Ras on the plasma membrane and Golgi, using over-expressed H-Ras and GFP-RBD (Chiu et al., 2002
). If the RBD inhibits coupling to Raf then this could have major consequences for GEF stimulation of Ras-GTP, since the mitogen-activated protein kinase kinase (MEK) can feedback on the Grb2-Sos complex to limit insulin-dependent Ras activation (Waters et al., 1995
). However, such potential problems might not be as serious as they first appear. Although the RBD inhibits stimulation of the GTPase activity of Ras by p120 Ras GAP in vitro and blocks oncogenic-Ras-mediated germinal vesicle breakdown (GVBD) in Xenopus oocytes, it has no effect on progesterone or insulin stimulation of GVBD through the endogenous Ras pathway (Scheffler et al., 1994
). Similarly, RBD-GFP (51-131) inhibits ERK2 responses in cells in which RasG12V is over-expressed but has no effect on ERK2 activity in phorbol ester- or EGF-treated cells or in Ras-transformed fibroblasts (Bondeva et al., 2002
). This strongly suggests that expression of the minimal RBD has minor effects on downstream Ras signalling and should not be viewed as a significant limitation of the method.
What is the significance and purpose of Ras signalling on the Golgi? This is a difficult question to answer given the difficulty of analysing endogenous Ras signalling on this compartment. H-Ras and N-Ras are on the Golgi, and they must traffic through the compartment on the way to the plasma membrane. So how much endogenous Ras is on the Golgi in a specific cellular context? If this pool is active then what Golgi-localized Ras effectors are engaged? And what are the consequences of compartmentalized Ras signalling? In support of the findings of Philips and co-workers, Perez de Castro et al. have recently shown that low-grade TCR stimulation of Jurkat T cells is specific to endogenous N-Ras and significant endogenous N-Ras resides on the Golgi as judged by immunocytochemistry (Perez de Castro et al., 2004). They detected active N-Ras only on the Golgi of Jurkat cells, using over-expression of N-Ras and YFP-RBD (Perez de Castro et al., 2004
). Furthermore, Mitin et al. have evidence of the first Golgi-localized Ras effector, Rain (Mitin et al., 2004
). Ectopic Rain is present at a perinuclear, juxta-Golgi region and is recruited to the trans-Golgi region following expression of activated Ras. This suggests that Rain can serve as an effector of Golgi-localized Ras because its localization is influenced by Ras-GTP. A caveat to these studies is that antibodies are not yet available to determine the endogenous location of Rain. Mitin et al. also discovered that Rain co-operates with Raf to cause synergistic transformation of NIH 3T3 cells; thus, Rain is a candidate for a Ras effector on the Golgi. These two studies have provided further evidence that compartmentalized Ras is likely to offer spatially and temporally restricted signalling output (Bivona and Philips, 2003
), which should modify the view that Ras operates exclusively at the plasma membrane.
In summary, the use of real-time analysis of Ras signalling events has offered up some surprises and a few new controversies. There are limitations to the techniques, which should be considered when interpreting data, and the requirement for over-expression of Ras is currently an unwanted necessity. Despite these criticisms, many of the studies that have used real-time imaging have provided new insight into the kinetics of Ras activation and deactivation, and have raised the issue of compartmentalized Ras signalling. It will be fascinating to see how the study of the spatio-temporal regulation of Ras signalling on multiple cellular compartments inevitably develops.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apolloni, A., Prior, I. A., Lindsay, M., Parton, R. G. and Hancock, J. F. (2000). H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol. Cell. Biol. 20, 2475-2487.
Babia, T., Ayala, I., Valderrama, F., Mato, E., Bosch, M., Santaren, J. F., Renau-Piqueras, J., Kok, J. W., Thomson, T. M. and Egea, G. (1999). N-Ras induces alterations in Golgi complex architecture and in constitutive protein transport. J. Cell Sci. 112, 477-489.
Baker, T. L., Zheng, H., Walker, J., Coloff, J. L. and Buss, J. E. (2003). Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-Ras. J. Biol. Chem. 278, 19292-19300.
Bivona, T. G., Perez de Castro, I., Ahearn, I. M., Grana, T. M., Chiu, V. K., Lockyer, P. J., Cullen, P. J., Pellicer, A., Cox, A. D. and Philips, M. R. (2003). Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature 424, 694-698.[CrossRef][Medline]
Bivona, T. G. and Philips, M. R. (2003). Ras pathway signaling on endomembranes. Curr. Opin. Cell Biol. 15, 136-142.[CrossRef][Medline]
Bivona, T. G., Wiener, H. H., Ahearn, I. M., Silletti, J., Chiu, V. K. and Philips, M. R. (2004). Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion. J. Cell Biol. 164, 461-470.
Bondeva, T., Balla, A., Varnai, P. and Balla, T. (2002). Structural determinants of Ras-Raf interaction analyzed in live cells. Mol. Biol. Cell 13, 2323-2333.
Bourne, H. R., Sanders, D. A. and McCormick, F. (1990). The GTPase superfamily - a conserved switch for diverse cell functions. Nature 348, 125-132.[CrossRef][Medline]
Caloca, M. J., Zugaza, J. L. and Bustelo, X. R. (2003). Exchange factors of the RasGRP family mediate Ras activation in the Golgi. J. Biol. Chem. 278, 33465-33473.
Chamberlain, C. E., Kraynov, V. S. and Hahn, K. M. (2000). Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. Methods Enzymol. 325, 389-400.[CrossRef][Medline]
Chiu, V. K., Bivona, T., Hach, A., Sajous, J. B., Silletti, J., Wiener, H., Johnson, R. L., Cox, A. D. and Philips, M. R. (2002). Ras signalling on the endoplasmic reticulum and the Golgi. Nat. Cell Biol. 4, 343-350.[CrossRef][Medline]
Choy, E., Chiu, V. K., Silletti, J., Feoktistov, M., Morimoto, T., Michaelson, D., Ivanov, I. E. and Philips, M. R. (1999). Endomembrane trafficking of Ras: The CAAX motif targets proteins to the ER and Golgi. Cell 98, 69-80.[Medline]
Corbit, K. C., Trakul, N., Eves, E. M., Diaz, B., Marshall, M. and Rosner, M. R. (2003). Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein. J. Biol. Chem. 278, 13061-13068.
Cullen, P. J. and Lockyer, P. J. (2002). Integration of calcium and Ras signalling. Nat. Rev. Mol. Cell Biol. 3, 339-348.[CrossRef][Medline]
Dong, X., Mitchell, D. A., Lobo, S., Zhao, L., Bartels, D. J. and Deschenes, R. J. (2003). Palmitoylation and plasma membrane localization of Ras2p by a nonclassical trafficking pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 23, 6574-6584.
Dower, N. A., Stang, S. L., Bottorff, D. A., Ebinu, J. O., Dickie, P., Ostergaard, H. L. and Stone, J. C. (2000). RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat. Immunol. 1, 317-321.[CrossRef][Medline]
Downward, J. (2003). Targeting ras signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11-22.[CrossRef][Medline]
Downward, J., Graves, J. D., Warne, P. H., Rayter, S. and Cantrell, D. A. (1990). Stimulation of p21ras upon T-cell activation. Nature 346, 719-723.[CrossRef][Medline]
Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. and Stone, J. C. (1998). RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Science 280, 1082-1086.
Ebinu, J. O., Stang, S. L., Teixeira, C., Bottorff, D. A., Hooton, J., Blumberg, P. M., Barry, M., Bleakley, R. C., Ostergaard, H. L. and Stone, J. C. (2000). RasGRP links T-cell receptor signaling to Ras. Blood 95, 3199-3203.
Gibbs, J. B. (1995). Determination of guanine nucleotides bound to Ras in mammalian cells. Methods Enzymol. 255, 118-125.[Medline]
Gordon, G. W., Berry, G., Liang, X. H., Levine, B. and Herman, B. (1998). Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702-2713.
Gotoh, T., Tian, X. and Feig, L. A. (2001). Prenylation of target GTPases contributes to signaling specificity of Ras-guanine nucleotide exchange factors. J. Biol. Chem. 276, 38029-38035.
Gregory, P. E., Gutmann, D. H., Mitchell, A., Park, S., Boguski, M., Jacks, T., Wood, D. L., Jove, R. and Collins, F. S. (1993). Neurofibromatosis type-1 gene-product (neurofibromin) associates with microtubules. Somatic Cell Mol. Genet. 19, 265-274.[Medline]
Hancock, J. F. (2003). Ras proteins: different signals from different locations. Nature Rev. Mol. Cell. Biol. 4, 373-384.[CrossRef][Medline]
Hibino, K., Watanabe, T. M., Kozuka, J., Iwane, A. H., Okada, T., Kataoka, T., Yanagida, T. and Sako, Y. (2003). Single- and multiple-molecule dynamics of the signaling from H-Ras to cRaf-1 visualized on the plasma membrane of living cells. Chemphyschem. 4, 748-753.[CrossRef][Medline]
Hingorani, S. R. and Tuveson, D. A. (2003). Ras redux: rethinking how and where Ras acts. Curr. Opin. Genet. Dev. 13, 6-13.[CrossRef][Medline]
Jaumot, M., Yan, J., Clyde-Smith, J., Sluimer, J. and Hancock, J. F. (2002). The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase. J. Biol. Chem. 277, 272-278.
Jiang, X. and Sorkin, A. (2002). Coordinated traffic of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells. Mol. Biol. Cell 13, 1522-1535.
Kawakami, Y., Kitaura, J., Yao, L., McHenry, R. W., Newton, A. C., Kang, S., Kato, R. M., Leitges, M., Rawlings, D. J. and Kawakami, T. (2003). A Ras activation pathway dependent on Syk phosphorylation of protein kinase C. Proc. Natl. Acad. Sci. USA 100, 9470-9475.
Kelley, G. G., Reks, S. E., Ondrako, J. M. and Smrcka, A. V. (2001). Phospholipase C epsilon: a novel Ras effector. EMBO J. 20, 743-754.
Leevers, S. J., Paterson, H. F. and Marshall, C. J. (1994). Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369, 411-414.[CrossRef][Medline]
Lobo, S., Greentree, W. K., Linder, M. E. and Deschenes, R. J. (2002). Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J. Biol. Chem. 277, 41268-41273.
Lockyer, P. J., Wennstrom, S., Kupzig, S., Venkateswarlu, K., Downward, J. and Cullen, P. J. (1999). Identification of the Ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate protein in vivo. Curr. Biol. 9, 265-268.[CrossRef][Medline]
Lockyer, P. J., Kupzig, S. and Cullen, P. J. (2001). CAPRI regulates Ca2+-dependent inactivation of the Ras-MAPK pathway. Curr. Biol. 11, 981-986.[CrossRef][Medline]
Lopez, I., Mak, E. C., Ding, J. R., Hamm, H. E. and Lomasney, J. W. (2001). A novel bifunctional phospholipase C that is regulated by G alpha(12) and stimulates the Ras/mitogen-activated protein kinase pathway. J. Biol. Chem. 276, 2758-2765.
Lorenzo, P. S., Kung, J. W., Bottorff, D. A., Garfield, S. H., Stone, J. C. and Blumberg, P. M. (2001). Phorbol esters modulate the ras exchange factor RasGRP3. Cancer Res. 61, 943-949.
Malumbres, M. and Barbacid, M. (2003). RAS oncogenes: the first 30 years. Nat. Rev. Cancer 3, 459-465.[CrossRef][Medline]
Marais, R., Light, Y., Paterson, H. F. and Marshall, C. J. (1995). Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. EMBO J. 14, 3136-3145.[Abstract]
Marais, R., Light, Y., Mason, C., Paterson, H., Olson, M. F. and Marshall, C. J. (1998). Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280, 109-112.
Mitin, N. Y., Ramocki, M. B., Zullo, A. J., Der, C. J., Konieczny, S. F. and Taparowsky, E. J. (2004). Identification and characterization of rain, a novel Ras-interacting protein with a unique subcellular localization. J. Biol. Chem. 279, 22353-22361.
Miyawaki, A. (2003). Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295-305.[Medline]
Mochizuki, N., Yamashita, S., Kurokawa, K., Ohba, Y., Nagai, T., Miyawaki, A. and Matsuda, M. (2001). Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411, 1065-1068.[CrossRef][Medline]
Ohba, Y., Kurokawa, K. and Matsuda, M. (2003). Mechanism of the spatio-temporal regulation of Ras and Rap1. EMBO J. 22, 859-869.
Pawson, T. (2004). Specificity in signal transduction. From phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116, 191-203.[CrossRef][Medline]
Perez de Castro, I., Bivona, T. G., Philips, M. R. and Pellicer, A. (2004). Ras Activation in Jurkat T cells following Low-Grade Stimulation of the T-Cell Receptor Is Specific to N-Ras and Occurs Only on the Golgi Apparatus. Mol. Cell. Biol. 24, 3485-3496.
Pertz, O. and Hahn, K. M. (2004). Designing biosensors for Rho family proteins - deciphering the dynamics of Rho family GTPase activation in living cells. J. Cell Sci. 117, 1313-1318.
Porfiri, E., Evans, T., Chardin, P. and Hancock, J. (1994). Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange. J. Biol. Chem. 269, 22672-22677.
Rebollo, A., Perez-Sala, D. and Martinez, A. C. (1999). Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis. Oncogene 18, 4930-4939.[CrossRef][Medline]
Roudebush, M., Slabe, T., Sundaram, V., Hoppel, C. L., Golubic, M. and Stacey, D. W. (1997). Neurofibromin colocalizes with mitochondria in cultured cells. Exp. Cell Res. 236, 161-172.[CrossRef][Medline]
Roy, S., Wyse, B. and Hancock, J. F. (2002). H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis. Mol. Cell. Biol. 22, 5128-5140.
Satoh, T. and Kaziro, Y. (1995). Measurement of Ras-bound guanine nucleotide in stimulated hematopoietic cells. Methods Enzymol. 255, 149-155.[Medline]
Scheffler, J. E., Waugh, D. S., Bekesi, E., Kiefer, S. E., LoSardo, J. E., Neri, A., Prinzo, K. M., Tsao, K. L., Wegrzynski, B., Emerson, S. D. et al. (1994). Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP. J. Biol. Chem. 269, 22340-22346.
Sobering, A. K., Romeo, M. J., Vay, H. A. and Levin, D. E. (2003). A novel Ras inhibitor, Eri1, engages yeast Ras at the endoplasmic reticulum. Mol. Cell. Biol. 23, 4983-4990.
Song, C., Hu, C. D., Masago, M., Kariya, K., Yamawaki-Kataoka, Y., Shibatohge, M., Wu, D. M., Satoh, T. and Kataoka, T. (2001). Regulation of a novel human phospholipase C, PLC epsilon, through membrane targeting by Ras. J. Biol. Chem. 276, 2752-2757.
Stokoe, D., Macdonald, S. G., Cadwallader, K., Symons, M. and Hancock, J. F. (1994). Activation of Raf as a result of recruitment to the plasma membrane. Science 264, 1463-1467.[Medline]
Taylor, S. J., Resnick, R. J. and Shalloway, D. (2001). Nonradioactive determination of Ras-GTP levels using activated ras interaction assay. Methods Enzymol. 333, 333-342.[CrossRef][Medline]
Thissen, J. A., Gross, J. M., Subramanian, K., Meyer, T. and Casey, P. J. (1997). Prenylation-dependent association of Ki-Ras with microtubules. Evidence for a role in subcellular trafficking. J. Biol. Chem. 272, 30362-30370.
Traverse, S., Cohen, P., Paterson, H., Marshall, C., Rapp, U. and Grand, R. J. (1993). Specific association of activated MAP kinase kinase kinase (Raf) with the plasma membranes of ras-transformed retinal cells. Oncogene 8, 3175-3181.[Medline]
van Triest, M., de Rooij, J. and Bos, J. L. (2001). Measurement of GTP-bound Ras-like GTPases by activation-specific probes. Methods Enzymol. 333, 343-348.[Medline]
Villalonga, P., Lopez-Alcala, C., Chiloeches, A., Gil, J., Marais, R., Bachs, O. and Agell, N. (2002). Calmodulin prevents activation of Ras by PKC in 3T3 fibroblasts. J. Biol. Chem. 277, 37929-37935.
Walker, S. A., Cullen, P. J., Taylor, J. A. and Lockyer, P. J. (2003). Control of Ras cycling by Ca2+. FEBS Lett. 546, 6-10.[CrossRef][Medline]
Walker, S. A., Kupzig, S., Bouyoucef, D., Davies, L. C., Tsuboi, T., Bivona, T. G., Cozier, G. E., Lockyer, P. J., Buckler, A., Rutter, G. A. et al. (2004). Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca(2+) oscillations. EMBO J. 23, 1749-1760.
Waters, S. B., Holt, K. H., Ross, S. E., Syu, L. J., Guan, K. L., Saltiel, A. R., Koretzky, G. A. and Pessin, J. E. (1995). Desensitization of Ras activation by a feedback disassociation of the SOS-Grb2 complex. J. Biol. Chem. 270, 20883-20886.
Willingham, M. C., Pastan, I., Shih, T. Y. and Scolnick, E. M. (1980). Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell 19, 1005-1014.[Medline]
Zimmermann, T., Rietdorf, J., Girod, A., Georget, V. and Pepperkok, R. (2002). Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett. 531, 245-249.[CrossRef][Medline]