The hypothesis that lipid rafts exist in plasma membranes and have crucial biological functions remains controversial. The lateral heterogeneity of proteins in the plasma membrane is undisputed, but ...the contribution of cholesterol-dependent lipid assemblies to this complex, non-random organization promotes vigorous debate. In the light of recent studies with model membranes, computational modelling and innovative cell biology, I propose an updated model of lipid rafts that readily accommodates diverse views on plasma-membrane micro-organization.
Ras proteins assemble into transient nanoclusters on the plasma membrane. Nanoclusters are the sites of Ras effector recruitment and activation and are therefore essential for signal transmission. ...The dynamics of nanocluster formation and disassembly result in interesting emergent properties including high-fidelity signal transmission. More recently the lipid structure of Ras nanoclusters has been reported and shown to contribute to isoform-specific Ras signaling. In addition specific lipids play critical roles in mediating the formation, stability and dynamics of Ras nanoclusters. In consequence the spatiotemporal organization of these lipids has emerged as important and novel regulators of Ras function. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
•Ras proteins assemble into transient nanoclusters on the plasma membrane.•Nanoclusters are the sites of Ras effector recruitment and activation.•Ras nanoclusters have distinct lipid composition.•Lipids mediate spatial cross talk and lateral segregation between Ras nanoclusters.•Ras nanoclustering and MAPK signaling are highly transient.
K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15–20% ...of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1–3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy.
K-Ras is targeted to the plasma membrane by a C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain. We used quantitative spatial imaging and atomistic ...molecular dynamics simulations to examine molecular details of K-Ras plasma membrane binding. We found that the K-Ras anchor binds selected plasma membrane anionic lipids with defined head groups and lipid side chains. The precise amino acid sequence and prenyl group define a combinatorial code for lipid binding that extends beyond simple electrostatics; within this code lysine and arginine residues are non-equivalent and prenyl chain length modifies nascent polybasic domain lipid preferences. The code is realized by distinct dynamic tertiary structures of the anchor on the plasma membrane that govern amino acid side-chain-lipid interactions. An important consequence of this specificity is the ability of such anchors when aggregated to sort subsets of phospholipids into nanoclusters with defined lipid compositions that determine K-Ras signaling output.
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•The K-Ras anchor encodes previously unsuspected anionic lipid binding specificity•Lipid selectivity is realized by defined conformational structures of the anchor•Subtle changes to anchor sequence or prenylation profoundly alter lipid specificity•Lipid sorting capacity of the anchor is a key determinant of K-Ras signal output
Selective interactions with lipids shape Ras signaling output through formation of nanoclusters.
The cell plasma membrane (PM) is a highly dynamic and heterogeneous lipid environment, driven by complex hydrophobic and electrostatic interactions among the hundreds of types of lipid species. ...Although the biophysical processes governing lipid lateral segregation in the cell PM have been established in vitro, biological implications of lipid heterogeneity are poorly understood. Of particular interest is how membrane proteins potentially utilize transient spatial clustering of PM lipids to regulate function. This review focuses on a lipid‐anchored small GTPase K‐Ras as an example to explore how its C‐terminal membrane‐anchoring domain, consisting of a contiguous hexa‐lysine polybasic domain and an adjacent farnesyl anchor, possesses a complex coding mechanism for highly selective lipid sorting on the PM. How this lipid specificity modulates K‐Ras signal transmission will also be discussed.
Plasma membrane (PM) is a highly heterogeneous environment and contains various nano‐domains with distinct local lipid composition. Lipid‐anchored small GTPases Ras proteins use their C‐terminal membrane anchoring features for specific lipid sorting. In particular, K‐Ras has a C‐terminal hexa‐lysine polybasic domain and an adjacent farnesyl anchor, which together allow the capability to selectively sort acidic phosphatidylserine lipids in the inner leaflet of the PM. This summarizes the latest findings in K‐Ras‐selective lipid sorting and the potential biological implications.
Mutations of rat sarcoma virus (RAS) oncogenes (HRAS, KRAS and NRAS) can contribute to the development of cancers and genetic disorders (RASopathies). The spatiotemporal organization of RAS is an ...important property that warrants further investigation. In order to function, wild‐type or oncogenic mutants of RAS must be localized to the inner leaflet of the plasma membrane (PM), which is driven by interactions between their C‐terminal membrane‐anchoring domains and PM lipids. The isoform‐specific RAS–lipid interactions promote the formation of nanoclusters on the PM. As main sites for effector recruitment, these nanoclusters are biologically important. Since the spatial distribution of lipids is sensitive to changing environments, such as mechanical and electrical perturbations, RAS nanoclusters act as transducers to convert external stimuli to intracellular mitogenic signalling. As such, effective inhibition of RAS oncogenesis requires consideration of the complex interplay between RAS nanoclusters and various cell surface and extracellular stimuli. In this review, we discuss in detail how, by sorting specific lipids in the PM, RAS nanoclusters act as transducers to convert external stimuli into intracellular signalling.
Rat sarcoma virus proteins are highly mutated in human cancer. The main signalling platforms of RAS, called nanoclusters, are located on the inner leaflet of the plasma membrane and comprise distinct lipid and protein contents. Here, we summarize the latest findings on how the distinct lipid contents of RAS nanoclusters actively participate in the transient regulations of RAS function and pathology.
Signalling ballet in space and time Kholodenko, Boris N; Hancock, John F; Kolch, Walter
Nature reviews. Molecular cell biology,
201006, 2010-Jun, 2010-6-00, 20100601, Letnik:
11, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Although we have amassed extensive catalogues of signalling network components, our understanding of the spatiotemporal control of emergent network structures has lagged behind. Dynamic behaviour is ...starting to be explored throughout the genome, but analysis of spatial behaviours is still confined to individual proteins. The challenge is to reveal how cells integrate temporal and spatial information to determine specific biological functions. Key findings are the discovery of molecular signalling machines such as Ras nanoclusters, spatial activity gradients and flexible network circuitries that involve transcriptional feedback. They reveal design principles of spatiotemporal organization that are crucial for network function and cell fate decisions.
RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the ...effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.
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.
Recent experiments have shown that membrane-bound Ras proteins form transient, nanoscale signaling platforms that play a crucial role in high-fidelity signal transmission. However, a detailed ...characterization of these dynamic proteolipid substructures by high-resolution experimental techniques remains elusive. Here we use extensive semiatomic simulations to reveal the molecular basis for the formation and domain-specific distribution of Ras nanoclusters. As model systems, we chose the triply lipidated membrane targeting motif of H-ras (tH) and a large bilayer made up of di16:0-PC (DPPC), di18:2-PC (DLiPC), and cholesterol. We found that 4–10 tH molecules assemble into clusters that undergo molecular exchange in the sub-μs to μs time scale, depending on the simulation temperature and hence the stability of lipid domains. Driven by the opposite preference of tH palmitoyls and farnesyl for ordered and disordered membrane domains, clustered tH molecules segregate to the boundary of lipid domains. Additionally, a systematic analysis of depalmitoylated and defarnesylated tH variants allowed us to decipher the role of individual lipid modifications in domain-specific nanocluster localization and thereby explain why homologous Ras isoforms form nonoverlapping nanoclusters. Moreover, the localization of tH nanoclusters at domain boundaries resulted in a significantly lower line tension and increased membrane curvature. Taken together, these results provide a unique mechanistic insight into how protein assembly promoted by lipid-modification modulates bilayer shape to generate functional signaling platforms.
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