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Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS > NRAS»HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. We suggest that in most cases, being the most abundant Ras isoform correlates with occupying the sweet-spot and that HRAS and NRAS expression is usually insufficient to promote oncogenesis when mutated. However, our results challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage., (© 2023. The Author(s).)

Ras is frequently mutated in cancer, however, there is a lack of consensus in the literature regarding the cancer mutation frequency of Ras, with quoted values varying from 10%-30%. This variability is at least in part due to the selective aggregation of data from different databases and the dominant influence of particular cancer types and particular Ras isoforms within these datasets. To provide a more definitive figure for Ras mutation frequency in cancer, we cross-referenced the data in all major publicly accessible cancer mutation databases to determine reliable mutation frequency values for each Ras isoform in all major cancer types. These percentages were then applied to current U.S. cancer incidence statistics to estimate the number of new patients each year that have Ras-mutant cancers. We find that approximately 19% of patients with cancer harbor Ras mutations, equivalent to approximately 3.4 million new cases per year worldwide. We discuss the Ras isoform and mutation-specific trends evident within the datasets that are relevant to current Ras-targeted therapies., (©2020 American Association for Cancer Research.)

HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies.

RAS proteins are small GTPases that regulate signalling networks that control cellular proliferation and survival. They are frequently mutated in cancer and a commonly occurring group of developmental disorders called RASopathies. We discuss recent findings describing how RAS isoforms and different activating mutations differentially contribute to normal and disease-associated biology and the mechanisms that have been proposed to underpin this., (© 2018 The Author(s).)

Ras proteins are important signalling hubs frequently dysregulated in cancer and in a group of developmental disorders called Rasopathies. Three Ras genes encode four proteins that differentially contribute to these phenotypes. Using quantitative real-time PCR (qRT-PCR) we have measured the gene expression profiles of each of the Ras isoforms in a panel of mouse tissues derived from a full developmental time course spanning embryogenesis through to adulthood. In most tissues and developmental stages we observe a relative contribution of KRas4B > > NRas ≥ KRas4A > HRas to total Ras expression with KRas4B typically representing 60-99% of all Ras transcripts. KRas4A is the most dynamically regulated Ras isoform with significant up-regulation of expression observed pre-term in stomach, intestine, kidney and heart. The expression patterns assist interpretation of the essential role of KRas in development and the preponderance of KRas mutations in cancer.

Background: Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown., Results: Monitoring nucleotide exchange of Ras in permeabilized cells we find, unexpectedly, that the decline of growth factor-induced Ras-GTP levels proceeds in the presence of unabated high nucleotide exchange, pointing to GAP activation as a major mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of high nucleotide exchange. Using pharmacological and genetic approaches we document a raised activity of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin and an involvement of Rsk1 and Rsk2 in the down-regulation of Ras-GTP levels., Conclusions: Our findings show that, in addition to feedback inhibition of Sos, feedback stimulation of the RasGAP neurofibromin enforces termination of the Ras signal in the context of growth-factor signaling. These findings ascribe a precise role to neurofibromin in growth factor-dependent control of Ras activity and illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells play safe to ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange.

Ras proteins are membrane bound signalling hubs that operate from both the cell surface and endomembrane compartments. However, the extent to which intracellular pools of Ras can contribute to cell signalling is debated. To address this, we have performed a global screen of compartmentalised Ras signalling. We find that whilst ER/Golgi- and endosomal-Ras only generate weak outputs, Ras localised to the mitochondria or Golgi significantly and distinctly influence both the abundance and phosphorylation of a wide range of proteins analysed. Our data reveal that ~80% of phosphosites exhibiting large (≥1.5-fold) changes compared to control can be modulated by organellar Ras signalling. The majority of compartmentalised Ras-specific responses are predicted to influence gene expression, RNA splicing and cell proliferation. Our analysis reinforces the concept that compartmentalisation influences Ras signalling and provides detailed insight into the widespread modulation of responses downstream of endomembranous Ras signalling.

Ras proteins operate predominantly from the plasma membrane; however, they have also been localized to most intracellular compartments. Various functions and signaling outputs have been ascribed to endomembranous Ras although systematic comparison and measurement of potential outputs have not yet been carried out. We describe the methodology for isolating and measuring compartment-specific signaling networks using quantitative proteomics. This approach reveals the potential of a subcellular platform for supporting specific outputs and will inform subsequent studies of endogenous isoform-specific Ras signaling., (© 2014 Elsevier Inc. All rights reserved.)

Ras isoforms are membrane bound proteins that differentially localize to the plasma membrane and subcellular compartments within the cell. Whilst the cell surface is the main site for Ras activity the extent to which intracellular pools contribute to Ras function is debated. We have generated Ras chimeras targeting Ras to the ER, Golgi, mitochondria and endosomes to compare the capacity of each of these locations to support activity equivalent to normal Ras function. We find that all locations are capable of regulating the MAP kinase and Akt pathways. Furthermore, whilst endomembranous Ras pools show location-specific competence to support proliferation and transformation, Golgi-Ras is as potent as N-Ras., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras-GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras-GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.

Ras GTPases are important regulators of pathways controlling proliferation, differentiation and transformation. Three ubiquitously expressed almost identical Ras genes are not functionally redundant; this has been attributed to their distinctive trafficking and localization profiles. A palmitoylation cycle controls the correct compartmentalization of H-Ras and N-Ras. We review recent data that reveal how this cycle can be regulated by membrane organization to influence the spatiotemporal signalling of Ras.

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes., (Copyright © 2011. Published by Elsevier Ltd.)

The trafficking, membrane localization, and lipid raft association of Ras proteins, which are crucial oncogenic mediators, dictate their isoform-specific biological responses. Accordingly, their spatiotemporal dynamics are tightly regulated. While extensively studied for H- and K-Ras, such information on N-Ras, an etiological oncogenic factor, is limited. Here, we report a novel mechanism regulating the activation-dependent spatiotemporal organization of N-Ras, its modulation by biologically relevant stimuli, and isoform-specific effects on signaling. We combined patching/immobilization of another membrane protein with fluorescence recovery after photobleaching (patch-FRAP) and FRAP beam size analysis to investigate N-Ras membrane interactions. Clustering of raft-associated proteins, either glycosylphosphatidylinositol-anchored influenza virus hemagglutinin (HA-GPI) or fibronectin receptors, selectively enhanced the plasma membrane-cytoplasm exchange of N-Ras-GTP (preferentially associated with raft domains) in a cholesterol-dependent manner. Electron microscopy (EM) analysis showed N-Ras-GTP localization in cholesterol-sensitive clusters, from which it preferentially detached upon HA-GPI cross-linking. HA-GPI clustering enhanced the Golgi compartment (GC) accumulation and signaling of epidermal growth factor (EGF)-stimulated N-Ras-GTP. Notably, the cross-linking-mediated enhancement of N-Ras-GTP exchange and GC accumulation depended strictly on depalmitoylation. We propose that the N-Ras activation pattern (e.g., by EGF) is altered by raft protein clustering, which enhances N-Ras-GTP raft localization and depalmitoylation, entailing its exchange and GC accumulation following repalmitoylation. This mechanism demonstrates a functional signaling role for the activation-dependent differential association of Ras isoforms with raft nanodomains.

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 summarize the mechanisms underlying compartment-specific Ras signalling and the outputs generated.

Ras proteins have become paradigms for isoform- and compartment-specific signaling. Recent work has shown that Ras isoforms are differentially distributed within cell surface signaling nanoclusters and on endomembranous compartments. The critical feature regulating Ras protein localization and isoform-specific functions is the C-terminal hypervariable region (HVR). In this review we discuss the differential post-translational modifications and reversible targeting functions of Ras isoform HVR motifs. We describe how compartmentalized Ras signaling has specific functional consequences and how cell surface signaling nanoclusters generate precise signaling outputs.

The ubiquitously expressed major Ras isoforms: H-, K- and N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N- and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically K> or =N>>H. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.

RAS isoforms have been proposed to exhibit differing biological outputs due to differences in their relative occupancy of cellular organelles and signalling microdomains. The membrane binding and targeting motifs of RAS are encoded by the C-terminal hypervariable region (HVR), and the precise localisation depends upon interactions between the HVR and the host membrane. Classic studies revealed that all RAS proteins rely on farnesylation and either palmitoylation or a polybasic stretch for stable binding to membranes. We now show that, for N-RAS and Ki-RAS4A, mono-palmitoylation and farnesylation are not sufficient for specifying stable cell-surface localisation. A third motif that is present within the linker domain of all palmitoylated RAS HVRs is necessary for stabilising localisation to the plasma membrane. This motif comprises acidic residues that stabilise palmitoylation and basic amino acids that are likely to interact electrostatically with acidic phospholipids enriched at the cell surface. Importantly, altered localisation is achieved without changes in palmitoylation status. Our data provide a mechanism for distinct HVR membrane interactions controlling subcellular distribution. In the context of the full-length RAS proteins, this is likely to be of crucial importance for controlling signalling output and engagement with different pools of effectors.

Ras GTPases mediate a wide variety of cellular processes by converting a multitude of extracellular stimuli into specific biological responses including proliferation, differentiation and survival. In mammalian cells, three ras genes encode four Ras isoforms (H-Ras, K-Ras4A, K-Ras4B and N-Ras) that are highly homologous but functionally distinct. Differences between the isoforms, including their post-translational modifications and intracellular sorting, mean that Ras has emerged as an important model system of compartmentalised signalling and membrane biology. Ras isoforms in different subcellular locations are proposed to recruit distinct upstream and downstream accessory proteins and activate multiple signalling pathways. Here, we summarise data relating to isoform-specific signalling, its role in disease and the mechanisms promoting compartmentalised signalling. Further understanding of this field will reveal the role of Ras signalling in development, cellular homeostasis and cancer and may suggest new therapeutic approaches.

Ras isoform-specific signaling from the plasma membrane appears to be regulated by interactions with distinct functional microdomains. We have developed protocols allowing the generation of 2-D spatial maps describing cell surface microdomain distributions. The combined electron microscopic (EM)-statistics approach provides nanometer scale resolution allowing both inner and outer leaflet domains to be visualized and cross-correlated with each other or with a protein of interest. In particular, the technique has allowed the interaction of Ras isoforms with signaling microdomains and proteins regulating these compartments to be screened. By allowing detailed monitoring of cell surface organization and compartmentalization, the approach has widespread potential for studies of plasma membrane-dependent cell biology, including regulated signaling and membrane trafficking.

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype.

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effectors and exchange factors.

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.