Nonsteroidal anti-inflammatory drugs alter the spatiotemporal organization of Ras proteins on the plasma membrane

Ras proteins on the inner leaflet of the plasma membrane signal from transient nanoscale proteolipid assemblies called nanoclusters. Interactions between the Ras lipid anchors and plasma membrane phospholipids, cholesterol, and actin cytoskeleton contribute to the formation, stability, and dynamics of Ras nanoclusters. Many small biological molecules are amphiphilic and capable of intercalating into membranes and altering lipid immiscibility. In this study we systematically examined whether amphiphiles such as indomethacin influence Ras protein nanoclustering in intact plasma membrane. We found that indomethacin, a nonsteroidal anti-inflammatory drug, induced profound and complex effects on Ras spatial organization, all likely related to liquid-ordered domain stabilization. Indomethacin enhanced the clustering of H-Ras.GDP and N-Ras.GTP in cholesterol-dependent nanoclusters. Indomethacin also abrogated efficient GTP-dependent lateral segregation of H- and N-Ras between cholesterol-dependent and cholesterol-independent clusters, resulting in mixed heterotypic clusters of Ras proteins that normally are separated spatially. These heterotypic Ras nanoclusters showed impaired Raf recruitment and kinase activation resulting in significantly compromised MAPK signaling. All of the amphiphilic anti-inflammatory agents we tested had similar effects on Ras nanoclustering and signaling. The potency of these effects correlated with the membrane partition coefficients of the individual agents and was independent of COX inhibition. This study shows that biological amphiphiles have wide-ranging effects on plasma membrane heterogeneity and protein nanoclustering, revealing a novel mechanism of drug action that has important consequences for cell signaling.     

Electrostatic interactions positively regulate K-Ras nanocluster formation and function

The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale environments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades.     

Galectin-1 is a novel structural component and a major regulator of h-ras nanoclusters

The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters. Gal-1 is therefore an integral structural component of the H-Ras-signaling nanocluster. Increasing Gal-1 levels increases the stability of H-Ras nanoclusters, leading to enhanced effector recruitment and signal output. Elements in the H-Ras C-terminal hypervariable region and an activated G-domain are required for H-Ras-Gal-1 interaction. Palmitoylation is not required for H-Ras-Gal-1 complex formation, but is required to anchor H-Ras-Gal-1 complexes to the plasma membrane. Our data suggest a mechanism for H-Ras nanoclustering that involves a dual role for Gal-1 as a critical scaffolding protein and a molecular chaperone that contributes to H-Ras trafficking by returning depalmitoylated H-Ras to the Golgi complex for repalmitoylation.     

Aggregation of Lipid-Anchored Full-Length H-Ras in Lipid Bilayers: Simulations with the MARTINI Force Field

Lipid-anchored Ras oncoproteins assemble into transient, nano-sized substructures on the plasma membrane. These substructures, called nanoclusters, were proposed to be crucial for high-fidelity signal transmission in cells. However, the molecular basis of Ras nanoclustering is poorly understood. In this work, we used coarse-grained (CG) molecular dynamics simulations to investigate the molecular mechanism by which full-length H-ras proteins form nanoclusters in a model membrane. We chose two different conformations of H-ras that were proposed to represent the active and inactive state of the protein, and a domain-forming model bilayer made up of di16:0-PC (DPPC), di18:2-PC (DLiPC) and cholesterol. We found that, irrespective of the initial conformation, Ras molecules assembled into a single large aggregate. However, the two binding modes, which are characterized by the different orientation of the G-domain with respect to the membrane, differ in dynamics and organization during and after aggregation. Some of these differences involve regions of Ras that are important for effector/modulator binding, which may partly explain observed differences in the ability of active and inactive H-ras nanoclusters to recruit effectors. The simulations also revealed some limitations in the CG force field to study protein assembly in solution, which we discuss in the context of proposed potential avenues of improvement.     

Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters

Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing.     

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.     

Plasma membrane nanoswitches generate high-fidelity Ras signal transduction

Ras proteins occupy dynamic plasma membrane nanodomains called nanoclusters. The significance of this spatial organization is unknown. Here we show, using in silico and in vivo analyses of mitogen-activated protein (MAP) kinase signalling, that Ras nanoclusters operate as sensitive switches, converting graded ligand inputs into fixed outputs of activated extracellular signal-regulated kinase (ERK). By generating Ras nanoclusters in direct proportion to ligand input, cells build an analogue-digital-analogue circuit relay that transmits a signal across the plasma membrane with high fidelity. Signal transmission is completely dependent on Ras spatial organization and fails if nanoclustering is abrogated. A requirement for high-fidelity signalling may explain the non-random distribution of other plasma membrane signalling complexes.     

Spatiotemporal Organization of Ras Signaling: Rasosomes and the Galectin Switch

Summary 1. Ras signaling and oncogenesis depend on the dynamic interplay of Ras with distinctive plasma membrane (PM) microdomains and various intracellular compartments. Such interaction is dictated by individual elements in the carboxy-terminal domain of the Ras proteins, including a farnesyl isoprenoid group, sequences in the hypervariable region (hvr)-linker, and palmitoyl groups in H/N-Ras isoforms.2. The farnesyl group acts as a specific recognition unit that interacts with prenyl-binding pockets in galectin-1 (Gal-1), galectin-3 (Gal-3), and cGMP phosphodiesterase δ. This interaction appears to contribute to the prolongation of Ras signals in the PM, the determination of Ras effector usage, and perhaps also the transport of cytoplasmic Ras. Gal-1 promotes H-Ras signaling to Raf at the expense of phosphoinositide 3-kinase (PI3-K) and Ral guanine nucleotide exchange factor (RalGEF), while galectin-3 promotes K-Ras signaling to both Raf and PI3-K.3. The hvr-linker and the palmitates of H-Ras and N-Ras determine the micro- and macro-localizations of these proteins in the PM and in the Golgi, as well as in ‘rasosomes’, randomly moving nanoparticles that carry palmitoylated Ras proteins and their signal through the cytoplasm.4. The dynamic compartmentalization of Ras proteins contributes to the spatial organization of Ras signaling, promotes redistribution of Ras, and provides an additional level of selectivity to the signal output of this regulatory GTPase.     

Activation of H-ras61L-specific signaling pathways does not require posttranslational processing of H-ras

We have previously demonstrated that H-ras61L retained transforming activity when lacking C-terminal lipid modifications, provided that plasma membrane localization was restored by an N-terminal transmembrane domain. Since several ras-activated pathways contribute to the transformed phenotype, we utilized a novel set of transmembrane domain-anchored H-ras derivatives to examine if lipids are required for activation of any specific signaling pathways. We demonstrate here that H-ras61L-induced activation of the Raf/MEK/MAPK pathway, including recruitment of Raf to the plasma membrane and activation of Raf and MAPK, does not require C-terminal processing of H-ras61L. Biochemical fractionation experiments confirm the localization of TM-ras derivatives to the plasma membrane, as well as the ras-mediated recruitment of c-Raf-1. Changes in the actin cytoskeleton, controlled by H-ras61L-mediated activation of the Rac/ Rho pathway, as well as PI 3-kinase activation, can also occur in the absence of C-terminal lipid modifications. Finally, downstream events, such as the induction of the immediate-early gene c-fos or neurite outgrowth in PC12 cells, are stimulated by the expression of plasma membrane-anchored, nonlipidated H-ras6lL. These results demonstrate that H-ras can be functionally targeted to the plasma membrane using a transmembrane domain sequence and that several signal transduction pathways downstream of H-ras can be activated without the presence of normal lipid modifications.     

Mathematical Modeling of K-Ras Nanocluster Formation on the Plasma Membrane

K-Ras functions as a critical node in the mitogen-activated protein kinase (MAPK) pathway that regulates key cellular functions including proliferation, differentiation, and apoptosis. Following growth factor receptor activation K-Ras.GTP forms nanoclusters on the plasma membrane through interaction with the scaffold protein galectin-3. The generation of nanoclusters is essential for high fidelity signal transduction via the MAPK pathway. To explore the mechanisms underlying K-Ras.GTP nanocluster formation, we developed a mathematical model of K-Ras-galectin-3 interactions. We designed a computational method to calculate protein collision rates based on experimentally determined protein diffusion rates and diffusion mechanisms and used a genetic algorithm to search the values of key model parameters. The optimal estimated model parameters were validated using experimental data. The resulting model accurately replicates critical features of K-Ras nanoclustering, including a fixed ratio of clustered K-Ras.GTP to monomeric K-Ras.GTP that is independent of the concentration of K-Ras.GTP. The model reproduces experimental results showing that the cytosolic level of galectin-3 determines the magnitude of the K-Ras.GTP clustered fraction and illustrates that nanoclustering is regulated by key nonequilibrium processes. Our kinetic model identifies a potential biophysical mechanism for K-Ras nanoclustering and suggests general principles that may be relevant for other plasma-membrane-localized proteins.     

The Anti-inflammatory Drug Indomethacin Alters Nanoclustering in Synthetic and Cell Plasma Membranes

The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase seperation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane.     

Ras nanoclusters: molecular structure and assembly

H-, N- and K-ras4B are lipid-anchored, peripheral membrane guanine nucleotide binding proteins. Recent work has shown that Ras proteins are laterally segregated into non-overlapping, dynamic domains of the plasma membrane called nanoclusters. This lateral segregation is important to specify Ras interactions with membrane-associated proteins, effectors and scaffolding proteins and is critical for Ras signal transduction. Here we review biological, in vitro and structural data that provide insight into the molecular basis of how palmitoylated Ras proteins are anchored to the plasma membrane. We explore possible mechanisms for how the interactions of H-ras with a lipid bilayer may drive nanocluster formation.     

GTP-dependent segregation of H-ras from lipid rafts is required for biological activity

Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Here we show that palmitoylation and farnesylation targets H-ras to lipid rafts and caveolae, but that the interaction of H-ras with these membrane subdomains is dynamic. GTP-loading redistributes H-ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-ras. Release of H-ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-ras is located outside rafts irrespective of bound nucleotide. Our studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane.     

Nucleophosmin and Nucleolin Regulate K-Ras Plasma Membrane Interactions and MAPK Signal Transduction

The spatial organization of Ras proteins into nanoclusters on the inner leaflet of the plasma membrane is essential for high fidelity signaling through the MAPK pathway. Here we identify two selective regulators of K-Ras nanoclustering from a proteomic screen for K-Ras interacting proteins. Nucleophosmin (NPM) and nucleolin are predominantly localized to the nucleolus but also have extranuclear functions. We show that a subset of NPM and nucleolin localizes to the inner leaflet of plasma membrane and forms specific complexes with K-Ras but not other Ras isoforms. Active GTP-loaded and inactive GDP-loaded K-Ras both interact with NPM, although NPM-K-Ras binding is increased by growth factor receptor activation. NPM and nucleolin both stabilize K-Ras levels on the plasma membrane, but NPM concurrently increases the clustered fraction of GTP-K-Ras. The increase in nanoclustered GTP-K-Ras in turn enhances signal gain in the MAPK pathway. In summary these results reveal novel extranucleolar functions for NPM and nucleolin as regulators of K-Ras nanocluster formation and activation of the MAPK pathway. The study also identifies a new class of K-Ras nanocluster regulator that operates independently of the structural scaffold galectin-3.Ras proteins are small GTPases that function as molecular switches on the inner leaflet of the plasma membrane, conveying extracellular signals to the cell interior. Ras proteins are critical regulators of signal transduction pathways controlling key cell fates such as cell growth, differentiation, and apoptosis. Deregulation of these pathways results in aberrant cell growth and tumor formation. Mutations that render Ras constitutively active are found in ∼15% of human cancers, making Ras one of the most clinically significant proteins in human carcinogenesis. Oncogenic mutations are most prevalent in the K-Ras gene, accounting for a large proportion of solid tumors including 90% of pancreatic cancer, 50% of colon cancer, and 30% of non-small cell lung cancer (1, 2).The three major Ras isoforms, H-, N-, and K-Ras generate distinct signal outputs in intact cells, signifying specific roles for each isoform. These functional differences stem from significant sequence divergence in the Ras C-terminal 25 amino acids of the hypervariable region (HVR)³ that directs post-translation attachment of different lipid anchors. The minimal membrane anchor of H-Ras comprises two palmitate groups and a farnesyl group, whereas K-Ras is tethered by a farnesyl group and a polybasic domain (3, 4). These minimal anchors, together with flanking protein sequences and the G-domain, interact with lipids and proteins of the plasma membrane, driving the Ras isoforms into spatially and structurally distinct nanodomains on the plasma membrane (5, 6). Ras lateral segregation is further modulated by the activation state of Ras; active GTP-loaded H-Ras is organized in cholesterol-independent nanoclusters, whereas inactive GDP-loaded H-Ras is arrayed in cholesterol-dependent nanoclusters (5, 7–9). Recent work has also shown that GTP-K-Ras clusters into nanodomains that are spatially distinct from GDP-K-Ras, although both types of nanocluster are cholesterol-independent and actin-dependent (7, 9). K-Ras-GTP nanoclustering, however, is regulated by galectin-3, which operates as a nanodomain scaffold (10, 11).Ras-GTP nanoclusters are the sites of Raf/MEK and ERK recruitment to the plasma membrane. Scaffolding all components of the MAPK module within nanoclusters rewires the biochemistry to generate a digital ERKpp output. The operation of Ras-GTP nanoclusters as highly sensitive digital switches is critical to deliver high fidelity signal transmission across the plasma membrane (12–14). A key parameter in epidermal growth factor (EGF) receptor to MAPK signal transmission is the fraction of Ras-GTP that forms nanoclusters; this clustered fraction sets the gain for cellular MAPK signaling (15, 16).NPM (also known as B23) and nucleolin are multifunctional phosphoproteins predominately localized to the nucleolus that play key roles in ribosome biogenesis (17–19). For example, NPM exhibits ribonuclease activity and preferentially cleaves pre-rRNA. NPM and nucleolin also have functions outside of the nucleolus. Both proteins shuttle between the nucleolus and the cytoplasm (20), and this shuttling may allow NPM to operate as molecular chaperone (21). In addition cytosolic NPM is involved in centrosome duplication (22). Like Ras proteins, NPM and nucleolin regulate cell proliferation and transformation and are overexpressed in multiple cancers (23). However, the physiological role of NPM in carcinogenesis remains controversial because it has been described as both an oncogene and a tumor suppressor (23).In this study we identify NPM and nucleolin as proteins that interact specifically with K-Ras but not H-Ras. Furthermore we definitively identify a subset of NPM and nucleolin on the inner leaflet of the plasma membrane where both proteins interact with K-Ras. Importantly, NPM and nucleolin stabilize K-Ras levels on the plasma membrane, leading to an increase in the K-Ras clustered fraction, which amplifies signal output from the MAPK pathway. Combined, our data indicate that NPM and nucleolin play a critical role in signal transduction via the MAPK pathway.     

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway

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.     

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics.     

Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase

Ras proteins activate diverse effector molecules. Depending on the cellular context, Ras activation may have different biological consequences: induction of cell proliferation, senescence, survival, or death. Augmentation and selective activation of particular effector molecules may underlie various Ras actions. In fact, Ras effector-loop mutants interacting with distinctive effectors provide evidence for such selectivity. Interactions of active Ras with escort proteins, such as galectin-1, could also direct Ras selectivity. Here we show that in comparison with Ras transfectants, H-Ras/galectin-1 or K-Ras4B/galectin-1 co-transfectants exhibit enhanced and prolonged epidermal growth factor (EGF)-stimulated increases in Ras-GTP, Raf-1 activity, and active extracellular signal-regulated kinase. Galectin-1 antisense RNA inhibited these EGF responses. Conversely, Ras and galectin-1 co-transfection inhibited the EGF-stimulated increase in phosphoinositide 3-kinase (PI3K) activity. Galectin-1 transfection also inhibited Ras(G12V)-induced PI3K but not Raf-1 activity. Galectin-1 co-immunoprecipitated with Ras(G12V) or with Ras(G12V/T35S) that activate Raf-1 but not with Ras(G12V/Y40C) that activates PI3K. Thus, galectin-1 binds active Ras and diverts its signal to Raf-1 at the expense of PI3K. This demonstrates a novel mechanism controlling the duration and selectivity of the Ras signal. Ras gains selectivity when it is associated with galectin-1, mimicking the selectivity of Ras(T35S), which activates Raf-1 but not PI3K.     

Effect of H-ras proteins on the activity of polyphosphoinositide phospholipase C in HL60 membranes

The present study was undertaken to investigate whether purified ras proteins can affect the activity of polyphosphoinositide specific phospholipase C in a cell-free membrane system. For this purpose we used homogenous preparations of the proto-oncogenic (H-ras(gly 12)) and the oncogenic (H-ras(val 12)) forms of the human H-ras proteins and membranes prepared from the human leukemic HL60 cells. We demonstrate that both the proto-oncogenic and the oncogenic form of H-ras proteins stimulate phospholipase C activity only when coupled to non-hydrolysable analogues of GTP.     

N-Ras Forms Dimers at POPC Membranes

Ras is a central regulator of cellular signaling pathways. It is mutated in 20–30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between β2 and β3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants.     

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.