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.     

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.     

The Efficacy of Raf Kinase Recruitment to the GTPase H-ras Depends on H-ras Membrane Conformer-specific Nanoclustering

Solution structures and biochemical data have provided a wealth of mechanistic insight into Ras GTPases. However, information on how much the membrane organization of these lipid-modified proteins impacts on their signaling is still scarce. Ras proteins are organized into membrane nanoclusters, which are necessary for Ras-MAPK signaling. Using quantitative conventional and super-resolution fluorescence methods, as well as mathematical modeling, we investigated nanoclustering of H-ras helix α4 and hypervariable region mutants that have different bona fide conformations on the membrane. By following the emergence of conformer-specific nanoclusters in the plasma membrane of mammalian cells, we found that conformers impart distinct nanoclustering responses depending on the cytoplasmic levels of the nanocluster scaffold galectin-1. Computational modeling revealed that complexes containing H-ras conformers and galectin-1 affect both the number and lifetime of nanoclusters and thus determine the specific Raf effector recruitment. Our results show that mutations in Ras can affect its nanoclustering response and thus allosterically effector recruitment and downstream signaling. We postulate that cancer- and developmental disease-linked mutations that are associated with the Ras membrane conformation may exhibit so far unrecognized Ras nanoclustering and therefore signaling alterations.     

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.     

Binding of Gbetagamma subunits to cRaf1 downregulates G-protein-coupled receptor signalling.

Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP-Galpha and Gbetagamma subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP-Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the beta subunit of heterotrimeric G proteins in vitro, and with the betagamma subunit complex in vivo [6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gbeta1gamma2, and that this association in mammalian cells is significantly enhanced when active p21(Ras) is present or when cRaf1 is otherwise targeted to the membrane. Association with Gbeta1gamma2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gbetagamma-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cbeta (PLCbeta) by sequestration of Gbetagamma subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gbetagamma, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21(Ras).     

Activation of the MAPK module from different spatial locations generates distinct system outputs

The Ras/Raf/MEK/ERK (MAPK) pathway directs multiple cell fate decisions within a single cell. How different system outputs are generated is unknown. Here we explore whether activating the MAPK module from different membrane environments can rewire system output. We identify two classes of nanoscale environment within the plasma membrane. The first, which corresponds to nanoclusters occupied by GTP-loaded H-, N- or K-Ras, supports Raf activation and amplifies low Raf kinase input to generate a digital ERKpp output. The second class, which corresponds to nanoclusters occupied by GDP-loaded Ras, cannot activate Raf and therefore does not activate the MAPK module, illustrating how lateral segregation on plasma membrane influences signal output. The MAPK module is activated at the Golgi, but in striking contrast to the plasma membrane, ERKpp output is analog. Different modes of Raf activation precisely correlate with these different ERKpp system outputs. Intriguingly, the Golgi contains two distinct membrane environments that generate ERKpp, but only one is competent to drive PC12 cell differentiation. The MAPK module is not activated from the ER. Taken together these data clearly demonstrate that the different nanoscale environments available to Ras generate distinct circuit configurations for the MAPK module, bestowing cells with a simple mechanism to generate multiple system outputs from a single cascade.     

Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.     

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.     

Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration

Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MEK-ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and beta-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.     

Organization of the integrin LFA-1 in nanoclusters regulates its activity

The beta2-integrin LFA-1 facilitates extravasation of monocytes (MOs) into the underlying tissues, where MOs can differentiate into dendritic cells (DCs). Although DCs express LFA-1, unlike MOs, they cannot bind to ICAM-1. We hypothesized that an altered integrin organization on the DC plasma membrane might cause this effect and investigated the relationship between membrane organization and function of LFA-1 on MOs and DCs. High-resolution mapping of LFA-1 surface distribution revealed that on MOs LFA-1 function is associated with a distribution in well-defined nanoclusters (100-150-nm diameter). Interestingly, a fraction of these nanoclusters contains primed LFA-1 molecules expressing the specific activation-dependent L16-epitope. Live imaging of MO-T-cell conjugates showed that only these primed nanoclusters are dynamically recruited to the cellular interface forming micrometer-sized assemblies engaged in ligand binding and linked to talin. We conclude that besides affinity regulation, LFA-1 function is controlled by at least three different avidity patterns: random distributed inactive molecules, well-defined ligand-independent proactive nanoclusters, and ligand-triggered micrometer-sized macroclusters.     

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.     

Binding of Gβγ subunits to cRaf1 downregulates G-protein-coupled receptor signalling

Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP–Gα and Gβγ subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP–Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the β subunit of heterotrimeric G proteins in vitro, and with the βγ subunit complex in vivo[6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gβ1γ2, and that this association in mammalian cells is significantly enhanced when active p21^Ras is present or when cRaf1 is otherwise targeted to the membrane. Association with Gβ1γ2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gβγ-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cβ (PLCβ) by sequestration of Gβγ subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gβγ, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21^Ras.     

Ras signalling on the endoplasmic reticulum and the Golgi

Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.     

Oligomerization and nanocluster organization render specificity

Nanoclusters are anchored to membranes, either within them or in the cytoplasm latched onto the cytoskeleton, whose reorganization can regulate their activity. Nanoclusters have been viewed in terms of cooperativity and activation; here we perceive nanocluster organization from a conformational standpoint. This leads us to suggest that while single molecules encode activity, nanoclusters induce specificity, and that this is their main evolutionary aim. Distinct, isoform‐specific nanocluster organization can drive the preferred effector (and ligand) interactions and thereby designate signalling pathways. The absence of detailed structural information across the nanocluster, due to size and dynamics, hinders an in‐depth grasp of its mechanistic features; however, available data already capture some of the principles and their functional ‘raison d'être’. Collectively, clustering lends stability and reduces the likelihood of proteolytic cleavage; it also increases the effective local concentration and enables efficient cooperative activation. However, clustering does not determine the ability of the single molecule to function. Drugs targeting nanoclusters can attenuate activity by hampering cooperativity; however, this may not perturb activation and signalling, which originate from the molecules themselves, and as such, are likely to endure. What then is the major role of nanoclustering? Assuming that single molecules evolved first, with a subsequent increase in cellular complexity and emergence of highly similar isoform variants, evolution faced the threat of signalling promiscuity. We reason that this potential risk was thwarted by oligomerization and clustering; clustering confers higher specificity, and a concomitant extra layer of cellular control. In our Ras example, signalling will be more accurate as a dimer than as a monomer, where its isomer specificity could be compromised.     

Ras proteins: different signals from different locations

Ras signalling has classically been thought to occur exclusively at the inner surface of a relatively uniform plasma membrane. Recent studies have shown that Ras proteins interact dynamically with specific microdomains of the plasma membrane as well as with other internal cell membranes. These different membrane microenvironments modulate Ras signal output and highlight the complex interplay between Ras location and function.