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MINIREVIEW Compartmentalized signalling: Ras proteins and signalling nanoclusters Jasminka Omerovic and Ian A. Prior Physiological Laboratory, University of Liverpool, UK Ras proteins are small GTPases that operate as molec- ular switches controlling the relay of signals from cell-surface receptors to a diverse array of intracellular effector cascades responsible for regulating cell prolif- eration, differentiation and apoptosis [1]. Hyperactivat- ing mutations of Ras promote cell transformation and contribute to oncogenesis in 15% of human cancer patients (data obtained from the Wellcome Trust Sanger Institute Cancer Genome Project http://www. sanger.ac.uk/genetics/CGP). Although clearly impor- tant from a human health perspective, Ras has also recently emerged as a key model system for investigat- ing how the outputs of signalling networks are spatially regulated. This is because the three Ras proto-oncogenes encoding four isoforms (H-Ras, K-Ras4A, K-Ras4B and N-Ras) are almost identical and all (except K-Ras4A) are ubiquitously expressed; yet do not exhibit complete functional redundancy [2]. It is proposed that this is because of their differential localization to the plasma membrane and intracellular organelles which potentially allows each isoform to come into contact with different pools of activators and effectors. Guanine nucleotide-exchange factors (GEFs) are Ras activators that promote the exchange of GDP for GTP; this results in a conformational switch in the Ras tertiary structure revealing an effector interaction site. This is reversed by the intrinsic GTPase activity of Ras proteins which is stimulated by GTPase-activat- ing proteins (GAPs; Fig. 1). In human cells, there are at least nine GEFs and eight GAPs with varied modes of recruitment and regulation [3]; potentially allowing considerable fine control of the location and dura- tion of Ras signalling. Raf and phosphatidylinositol Keywords compartmentalization; GTPase; isoforms; MAP kinase; microdomains; nanoclusters; Raf; Ras; receptor; scaffold Correspondence I. A. Prior, Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown St, Liverpool L69 3BX, UK Fax: +44 151 794 4434 Tel: +44 151 794 5332 E-mail: iprior@liverpool.ac.uk (Received 1 September 2008, revised 16 October 2008, accepted 24 November 2008) doi:10.1111/j.1742-4658.2009.06928.x Differential subcellular compartmentalization of the three main Ras isoforms (H-Ras, N-Ras and K-Ras) is believed to underlie their biological differences. Modulatable interactions between cellular membranes and Ras C-terminal hypervariable region motifs determine differences in trafficking and the relative proportions of each isoform in cell-surface signalling nanoclusters and intracellular endoplasmic reticulum ⁄ Golgi, endosomal and mitochondrial compartments. Ras regulators, effectors and scaffolds are also differentially distributed, potentially enabling preferential coupling to specific signalling pathways in each subcellular location. Here we sum- marize the mechanisms underlying compartment-specific Ras signalling and the outputs generated. Abbreviations ER, endoplasmic reticulum; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factors; HVR, hypervariable region; PtdIns3K, phosphatidylinositol 3-kinase; RTK, receptor tyrosine kinase. FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1817 3-kinase (PtdIns3K) are the archetypal Ras effectors although over 20 others have since been identified, many of which are GEFs and GAPs for other GTPases [4,5]. After 20 years of intensive investigation, most of the potential regulators and effectors of Ras have been identified and characterized; the focus of many laboratories is now switching towards under- standing context-dependent regulation of interactions and outputs within this large signalling web. Ras trafficking and localization H-, K-, and N-Ras share almost complete sequence homology between residues 1–165 encompassing all of the effector and nucleotide-binding motifs (Fig. 1). The isoforms are principally distinguished from each other by the final 23–24 amino acid stretch, the hypervari- able region (HVR), where there is < 15% sequence similarity between Ras proteins. The HVR contains all of the motifs responsible for membrane binding and trafficking of each isoform. After synthesis on cytosolic polysomes, Ras isoforms undergo a series of post- translational modifications to increase their membrane affinity. The cysteine in the C-terminal CAAX motif is farnesylated before the -AAX is proteolytically cleaved and the farnesyl–cysteine is carboxymethylated [5]. The farnesyl group promotes weak interaction with the endoplasmic reticulum (ER) which is stabilized by an adjacent set of motifs that vary among Ras isoforms. This consists of mono- [N-, K(A)-Ras] or di-palmitoy- lation (H-Ras) of cysteines or a hexalysine polybasic sequence [K(B)-Ras]. The second signal motif and farnesylated cysteine shared by all Ras isoforms comprise the targeting domain (Fig. 2); a minimal motif that when fused to GFP displays a superficially equivalent localization as the cognate full length H- and K(B)-Ras proteins [6,7]. Recent data revealed that a third signal motif is neces- sary for the correct localization of mono-palmitoylated N- and K(A)-Ras isoforms. GFP conjugated to the minimal targeting domain of N-Ras is restricted to the Golgi, whereas when the adjacent linker region of the HVR is included the construct localizes to the cell sur- face [7]. When the HVR of the palmitoylated Ras isoforms is compared, there is  70% sequence homol- ogy including a six-residue basic ⁄ hydrophobic patch at the N-terminus of the HVR (Fig. 2). Mutating this sequence increases the amount of endomembranous localization observed, indicating that this motif contributes to cell-surface localization. Similarly, for K(A)-Ras, the basic patch adjacent to the palmitoyl group is sufficient to ensure cell-surface localization [7]. The second signal motif also determines the traffick- ing routes taken by H-, N- and K(B)-Ras to the plasma membrane. K(B)-Ras traffics via a poorly char- acterized Golgi-independent route that in yeast requires class C vps proteins which are normally required to regulate endosome fusion [8–10]. Unlike other small G proteins such as Rabs and Rho proteins, there has been no chaperone such as GDI character- ized for cytosolic Ras trafficking. Although palmitoy- lated Ras isoforms have also been characterized to traffic via Golgi-independent routes in yeast and adipocytes [11,12], in fibroblasts they traffic through the conventional secretory pathway [8,9]. Interestingly, the two palmitoyl groups of H-Ras are not equally necessary for trafficking to the plasma membrane, palmitoylated Cys181 supports cell-surface localization, whereas mono-palmitoylation on Cys184 confines H-Ras to the Golgi [13]. Although based on mutagene- sis studies, these observations are highly relevant to in vivo trafficking because Ras palmitoylation is labile. The measured half life is 10–20% of that of the 21 h half-life of N- and H-Ras, and these cycles of acylation and deacylation are important regulators of global Ras compartmentalization by allowing recycling back to the Golgi complex for re-palmitoylation [14,15]. When de-palmitoylation is inhibited, H-Ras is non-specifi- cally localizes to all endomembranes [16]. Upon reaching the cell surface, each Ras isoform occupies distinct signalling nanoclusters, as discussed in detail below. Activation of upstream receptor tyro- Fig. 1. Ras activation and effectors. Ras is a molecular switch cycling between inactive GDP- and active GTP-bound conformations controlled by GEFs and GAPs. Active Ras stimulates many pathways, notably kinases such as the Raf–MAP kinase cascade and the PtdIns3K–Akt pathway and GEFs for other GTPases. These pathways regulate many cell functions including cell prolifera- tion, differentiation migration and apoptosis. Compartmentalized Ras signalling J. Omerovic and I. A. Prior 1818 FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS sine kinases (RTKs) results in the internalization of up to 60% of the surface receptor population that may be targeted for lysosomal degradation by c-Cbl-dependent ubiquitination [17]. An open question is the extent to which Ras isoforms co-traffic with the activated recep- tor complexes to facilitate signalling from the surface of early ⁄ recycling endosomes. A tiny fraction of H- and N-Ras is ubiquitinated, and ubiquitin–Ras chimeras showed enhanced endosomal localization [18]. However, Ras ubiquitination appears to be uncoupled from receptor activation because it is not dependent on the Ras activation state. Further work is needed to identify the enzymes responsible for ubiquitin turnover on Ras to provide tools for analysing the role of this post-translational modification in Ras biology. In summary, whereas the palmitoylated Ras isoforms traffic from the ER–Golgi to the plasma membrane and endosomes on membranes, the lability of palmitoylation provides a mechanism for cytosolic recycling back to the Golgi complex. The lack of hydrophobic acyl groups on K(B)-Ras facilitates cyto- solic shuttling between the cell surface and intracellular organelles. Although all isoforms have been observed on a variety of organelles, the key difference is the relative proportions of each isoform in each location. Although the cell surface represents the prime location for all isoforms, in many cell types the relative contri- bution to the endomembranous component is typically N ‡ H, K(A) > K(B)-Ras. When combined with observed differences in relative abundance N- and K(B)-Ras are the most abundant isoforms in most cell lines [2], a broad conclusion is that the plasma mem- brane is likely to be more coupled to K-Ras signalling whereas the endomembrane is more coupled to N-Ras signalling pathways. Plasma membrane signalling nanoclusters Although the plasma membrane has traditionally been viewed as a homogeneous organelle, in recent years this model has been updated to incorporate a dense mosaic of signalling domains [19]. These incorporate nanoclusters of proteins and lipids that potentially facilitate signalling by selectively concentrating the components of effector cascades. Several laboratories, using a combination of fractionation, chemical pertur- bation and advanced microscopy techniques, have A B Fig. 2. The Ras HVR and subcellular Ras trafficking. The C-terminal HVR (A) contains membrane-trafficking motifs that regulate the trafficking and nanocluster association of Ras isoforms (B). Membrane interactions can be modulated to allow recycling to the ER ⁄ Golgi or targeting to alternative endo- membranous locations. J. Omerovic and I. A. Prior Compartmentalized Ras signalling FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1819 characterized the association of Ras isoforms with dif- ferent cell-surface nanoclusters that is driven by inter- actions of their HVRs with membrane proteins and lipids [20–22]. These nanoclusters are small (< 15 nm diameter) and short-lived (t 1 ⁄ 2 £ 0.4 s) [23]. K(B)-Ras operates from actin-dependent, choles- terol-independent nanoclusters that are stabilized by galectin-3 and distinct from the H- and N-Ras signal- ling nanoclusters [24,25]. A beneficial feature of the K-Ras polybasic domain is its potential capacity to aggregate the highly charged anionic lipid phosphati- dylinositol-4,5-bisphosphate [26]. Phosphatidylinositol- 4,5-bisphosphate is the substrate for PtdIns3K, a key Ras effector. In addition to 2D lateral diffusion, K-Ras exhibits significant cytosolic exchange due to a mem- brane residency half-life of only a few minutes [27]. This is modulated in vivo by disruption of K(B)-Ras electrostatic interactions with the plasma membrane via protein kinase C-dependent phosphorylation of Ser181 resulting in translocation to mitochondria [28]. The palmitoyl groups of H-Ras specify localization in cholesterol-dependent nanoclusters, however upon activation, H-Ras translocates into new cholesterol- independent nanoclusters [13,22,29]. The activated H-Ras nanoclusters are stabilized by galectin-1 interac- tions with the C-terminal farnesyl group of the HVR [22,30,31]. By contrast, N-Ras moves in the opposite direction when activated [13]; although at present it is unclear whether the H- and N-Ras nanoclusters are actually identical or share a limited number of diag- nostic markers. Recent structural analysis of the interaction of the H-Ras HVR with membranes gave an insight into how Ras activation might translate into differential nano- cluster association. Conformational changes in the N-terminal switch regions of Ras re-orientate the protein’s interaction with the plasma membrane by regulating the membrane interactions of basic residues located in the HVR (GDP-bound Ras) and a4 helix (GTP-bound Ras) [32]. In effect, the changed mem- brane interactions ‘tip over’ the main body of active Ras, potentially facilitating effector and galectin-1 interactions [33]. Although this will promote interac- tions with the active-Ras nanocluster components, the GTP-dependent loss of affinity with the cholesterol- dependent nanoclusters is likely to be due to modulat- ing palmitoyl–membrane interactions [12,33,34]. In addition to characterizing the dynamic associa- tion of Ras with different nanoclusters, the functional consequences of these interactions have been analysed in silico and in vivo. Importantly, if nanocluster forma- tion is inhibited, output is reduced to just 3% of maximal signalling [35]. Nanoclusters are highly sensitive to low signalling inputs, giving them a switch- like behaviour where, apart from a very narrow win- dow, a maximal output is generated over a wide input range [36]. The high abundance but very short life span of Ras signalling domains means that although each nanocluster effectively behaves digitally, the pop- ulation as a whole generates a graded response in which output is proportional to input [35,36]. Given that differential localization of Ras isoforms to distinct signalling nanoclusters is believed to underlie the lack of functional redundancy between Ras isoforms, a key prediction is that effectors will have different affinities for each type of nanocluster. Recent high-resolution microscopy analysis revealed that although both H- and K-Ras can recruit Raf to the plasma membrane, the activated K-Ras nanoclusters retain Raf, whereas activated H-Ras–Raf interactions are transient [24]. These data fit comparative signalling analysis that showed K-Ras to be a proportionally better activator of the Raf-MAPK cascade than H-Ras [37]. In summary, Ras activation-dependent conforma- tional changes in HVR–membrane association result in rapid redistribution to specialized signalling nanoclus- ters stabilized by galectin proteins. The biophysical and biochemical properties of different types of nano- cluster are believed to regulate the variety and time course of effector interactions allowing different outputs. Compartmentalization of accessory proteins and organellar signalling There are few organelles that Ras cannot access; an important question is whether activators, regulators and effectors show similarly compartmentalized distributions. Figure 3 summarizes the locations occu- pied by regulators of Ras signalling. Although all organelles have at least one Ras regulator in residence, the ER ⁄ Golgi and endosomes appear to be hotspots for controlling intracellular Ras signalling. The evidence for this organellar signalling and its func- tional relevance are discussed in later paragraphs, but at this point it is worth highlighting the compartmen- talization of scaffold proteins that are key regulators of Ras–Raf–MAP kinase and Ras–PtdIns3K–Akt signalling. Compartmentalized scaffolds Scaffold proteins contribute to the control of signalling kinetics, cross-talk between pathways and prevention of activation of physiologically irrelevant signals. They Compartmentalized Ras signalling J. Omerovic and I. A. Prior 1820 FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS function by pre-assembling components of signalling cascades in readiness for recruitment to the site of receptor activation or on intracellular organelles. Several Ras pathway scaffolds have been identified, most of which coordinate Raf–MEK–ERK (Raf–MAP kinase) signalling from different locations. These include KSR, AF6 and IQGAP-1 (plasma membrane), b-arrestin (plasma membrane, endosomes, nucleus), p14-MP1 (late endosomes) and Sef (Golgi) that typi- cally undergo regulated redistribution from the cytosol to these locations [38–43]. A key function of scaffolds is to regulate the kinetics of signalling, an example of this can been seen when the late endosomal MP1 scaffold adaptor protein p14 is knocked-down using RNAi resulting in mislocalization of MP1 to the cytosol [44]. Acute MAP kinase activa- tion that occurs at the plasma membrane was unper- turbed; however, MAP kinase activation following prolonged (10–30 min) epidermal growth factor stimula- tion was significantly reduced. This fits with the model in which prolonged growth factor stimulation results in receptor endocytosis and signalling from endosomes and suggests that activated receptors engage new signal- ling complexes resident within the endocytic system. A final scaffold, Appl1, modulates the Akt signalling pathway and operates from a subpopulation of signal- ling early endosomes [45,46]. Akt has many substrates, however endosomal interaction of Appl1 with Akt speci- fies activation of the GSK3 cell survival pathway in zebrafish [46]. Importantly, when Appl1 was mis-tar- geted to other cellular locations this pathway could not be engaged indicating highly context-dependent signal- ling outputs. Although identified as an effector for the small GTPase Rab5, it will be interesting to see whether endosomal Ras can also activate this signalling complex. Endosomal signalling The initiation points of most signalling cascades are the cell-surface localized RTKs and G-protein-coupled Fig. 3. Compartmentalization of Ras GEFs, GAPs and scaffolds. Ras has been localized to and signals from both cell surface and endomembrane platforms. Ras regulators are also differentially localized, many of the proteins illustrated exhibit regulated recruitment from the cytosol. See text and Omerovic et al. [5] for primary references. J. Omerovic and I. A. Prior Compartmentalized Ras signalling FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS 1821 receptors. A proportion of activated receptors are internalized and sorted through the endosome for recycling back to the surface or delivery to lyso- somes for degradation. Because the tails of early endosomal receptors are still exposed to the cytosol they are also potentially able to initiate signalling cascades if still active. This observation, together with the localization of the Ras GEF, mSos, the activated Raf-MAP kinase cascade and scaffold proteins MP1 and APPL on endosomes, suggests that they are a viable platform for RTK–Ras signalling [47,48]. Several lines of evidence support this idea; first, selective activation of endosomal epidermal growth factor receptor or platelet-derived growth factor receptor stimulated cell proliferation and cell survival, indicating that the cell-signalling machinery on endosomes is functional and capable of generating a biologically relevant output [49,50]. Activation of the full complement of epidermal growth factor receptor effectors requires receptor endocytosis [51]; and inhibition of endocytosis attenuates growth factor stimulated H- and N-Ras but not K-Ras activation of the Raf–MAP kinase pathway [2]. Importantly, whereas acute growth factor stimu- lated MAP kinase activation is primarily believed to emanate from the plasma membrane, endosomes support sustained MAP kinase activation [52,53]. The ability to modulate the kinetics of MAP kinase activation has cell phenotypic consequences; for example, in PC12 cells this results in a switch from proliferation to differentiation [54]. In addition to facilitating endosomal Ras–MAP kinase signalling, the scaffold protein MP-1 also regulates coupling to specific upstream stimuli. This is controlled by the MP-1 interaction partner MORG1 that facilitates lysophosphatidic acid, phorbol ester or serum-depen- dent but not growth factor-dependent MAP kinase activation [55]. The evidence for endosomal Ras pathway signalling conferring signal specificity distinct from that gener- ated from other subcellular signalling platforms is currently limited. However, a clear example is provided by studies of the endosomal Akt scaffold Appl1. During zebrafish development, loss of Appl1 results in apoptosis due to the loss of the Akt–GSK–3b survival signal [46]. In this case, endosomal Akt signalling is an important driver of cell survival in tissues where Appl1 is highly expressed. Presumably the abundance and localization of other co-factors will also be modulated in these cells to ensure that the endosomal microenvi- ronment is favoured for regulating anti-apoptotic signalling. ER ⁄ Golgi signalling Studies of ER ⁄ Golgi signalling provide some of the best evidence for isoform- and compartment-specific Ras signalling having phenotypic consequences. Ras GEFs and GAPs, the Ras effector Rain1 and the scaf- fold Sef have all been localized to the Golgi (Fig. 3) [5,43,56]. Although endogenous Golgi Ras activation has largely proved difficult to visualize [57], an elegant bystander fluorescence technique revealed that delayed and sustained endogenous Golgi Ras activation could be stimulated by growth factors [58]. Ras activation on the Golgi is mediated by calcium ⁄ diacylglycerol (Ca 2+ ⁄ DAG)-dependent stimulation of RasGRP1 [59], and on the ER by lysophosphatidic acid-dependent stimulation of RasGRF1 and Ras- GRF2 [60]. Although most studies have relied on overexpression of Ras or Ras modulators to study compartmentalized Ras activation and signalling, recent work by Philips and colleagues revealed the different wiring involved in activating Ras on the cell surface and Golgi in T cells [61]. T-cell receptor activation stimulates Golgi-Ras, however, co-stimula- tion with the integrin LFA-1 also activated Ras on the plasma membrane by raising the local concentration of DAG and phosphatidic acid to also activate cell surface localized RasGRP1 [61]. Therefore, whereas Ras and the GEF RasGRP1 are found in two compartments, their organelle-specific activation can be precisely regulated by different extracellular ligands inducing localized concentrations of second messengers. The difficulty in observing Golgi Ras activation compared with plasma membrane Ras activation could lead to the conclusion that it represents a minor inconsequential component of global Ras signalling. However, the cell is unlikely to waste resources on redundant signalling pathways. Moreover, strong evidence for physiologically relevant Golgi Ras signal- ling recently emerged in studies of thymocyte selection which revealed that the endogenous Golgi Ras pool is necessary for positive thymocyte selection whereas the plasma membrane specifies negative selection [62]. Finally, the Golgi also interestingly provides a site for negative regulation of Ras signalling and stimula- tion of cell proliferation and differentiation via the action of RKTG. This is a seven transmembrane protein that sequesters cytosolic Raf to the Golgi, competitively inhibiting interaction with activated Ras and the MAP kinase cascade [63]. On a more general point, it is also tempting to speculate that negative regulation of Ras pathways via phosphatases may also be highly compartmentalized. Compartmentalized Ras signalling J. Omerovic and I. A. Prior 1822 FEBS Journal 276 (2009) 1817–1825 ª 2009 The Authors Journal compilation ª 2009 FEBS Other compartmentalized Ras signalling Although the ER ⁄ Golgi and endosomes have been strongly identified as sites of Ras signalling, both the mitochondria and nucleus have also been shown to contain specific Ras isoforms. An H-Ras splice variant lacking the C-terminal HVR, p19 ras localizes to the nucleus and cytosol where it regulates the activity of the tumour suppressor p73 [64]. Mitochondrial Ras signalling by N-Ras and K(B)-Ras has also been characterized. Phosphorylation of Ser181 within the K(B)-Ras HVR destabilizes the electrostatic interac- tions of the polybasic domain with the plasma membrane and promotes redistribution to mitochon- dria where it induces Bcl-X L -dependent apoptosis [28]. N-Ras and K-Ras also play a role in maintaining normal mitochondrial morphology and function [65]. Conclusion Compartmentalized Ras signalling enables a spectrum of outputs to be tuned from a minimal core-signalling machinery by modulating access to a variety of activat- ing and effector proteins. These are evolutionarily conserved mechanisms because in yeast one Ras protein controls two outputs related to mating or morphology via signalling from the plasma membrane or endomembrane respectively [66]. In mammalian systems we are still at an early stage in the identifica- tion and validation of phenotypes regulated by local- ized Ras signalling. In future the major challenge remains to understand what output each compartment is capable of controlling and how this is achieved. A key problem lies in the tools available to investigate this that typically utilize overexpression and therefore potential distortion of information flow through signalling pathways. Also, differences in compartmen- talized signalling are likely to mostly involve the same pathways stimulated to different extents in different locations; i.e. only by looking across a wide range of potential pathways are we likely to understand what is really happening. Novel large-scale quantitative screen- ing approaches and new tools that locally stimulate or inhibit the endogenous Ras pathway represent ideals that many groups are working towards combining to try to understand this important phenomenon. Acknowledgements IAP is a Royal Society University Research Fellow; work in our laboratory is funded by Cancer Research UK, the Wellcome Trust and the North West Cancer Research Fund. References 1 Karnoub AE & Weinberg RA (2008) Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 9, 517–531. 2 Omerovic J, Hammond DE, Clague MJ & Prior IA (2008) Ras isoform abundance and signalling in human cancer cell lines. 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