ABSTRACT: The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction.
Project description:A single-molecule fluorescence resonance energy transfer (FRET) method has been developed to observe the activation of the small G protein Ras at the level of individual molecules. KB cells expressing H- or K-Ras fused with YFP (donor) were microinjected with the fluorescent GTP analogue BodipyTR-GTP (acceptor), and the epidermal growth factor-induced binding of BodipyTR-GTP to YFP-(H or K)-Ras was monitored by single-molecule FRET. On activation, Ras diffusion was greatly suppressed/immobilized, suggesting the formation of large, activated Ras-signaling complexes. These complexes may work as platforms for transducing the Ras signal to effector molecules, further suggesting that Ras signal transduction requires more than simple collisions with effector molecules. GAP334-GFP recruited to the membrane was also stationary, suggesting its binding to the signaling complex. The single-molecules FRET method developed here provides a powerful technique to study the signal-transduction mechanisms of various G proteins.
Project description:Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, approximately 30% of the raft protein exists in cholesterol-dependent nanoclusters, with approximately 70% distributed as monomers. The cluster/monomer ratio (number of proteins in clusters/number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster/monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0-7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.
Project description: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.
Project description:The spatial organization of K-Ras proteins into nanoclusters on the plasma membrane is essential for high-fidelity signal transduction. The mechanism underlying K-Ras nanoclustering is unknown. We show here that K-Ras.GTP recruits Galectin-3 (Gal-3) from the cytosol to the plasma membrane where it becomes an integral nanocluster component. Importantly, we show that the cytosolic level of Gal-3 determines the magnitude of K-Ras.GTP nanoclustering and signal output. The beta-sheet layers of the Gal-3 carbohydrate recognition domain contain a hydrophobic pocket that may accommodate the farnesyl group of K-Ras. V125A substitution within this hydrophobic pocket yields a dominant negative Gal-3(V125A) mutant that inhibits K-Ras activity. Gal-3(V125A) interaction with K-Ras.GTP reduces K-Ras.GTP nanocluster formation, which abrogates signal output from the Raf/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) pathway. Gal-3(V125A) negatively regulates cell growth and reduces cellular transformation. Thus, regulation of K-Ras nanocluster formation and signal output by Gal-3 critically depends on the integrity of the Gal-3 hydrophobic pocket. These results show that Gal-3 overexpression in breast cancer cells, which increases K-Ras signal output, represents oncogenic subversion of plasma membrane nanostructure.
Project description:Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output.
Project description:Signaling complexes are localized to distinct plasma-membrane domains which undergo precise spatiotemporal regulation. A crucial link between membrane dynamics and the small GTPase, H-Ras, has been suggested, connecting membrane localization, clustering and scaffolding with its activity and signal transduction. Results of this study suggest a relationship between MPP1 and/or MPP1-dependent plasma-membrane organization and H-Ras activation. Namely, we show here that in HEL cells, MPP1 knock-down lead to the disruption of signaling cascade(s) from the activated insulin receptor. The signal inhibition occurred at the level of H-Ras, as it showed impaired GDP-to-GTP exchange and further interaction with its effector molecule, Raf. Moreover, in these cells H-Ras detergent-resistant membrane localization was not sensitive to insulin treatment which may imply molecular mechanism via which MPP1 affects functions of other proteins which may be connected with functional domain formation. Understanding the link between MPP1 and activation of H-Ras, may provide an important insight into the complexity of Ras related signaling pathways which may become a potential target for associated cancer therapies.
Project description:Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.
Project description:Supramolecular organizing center (SMOC)-mediated signal transduction is an emerging concept in the field of signal transduction that is ushering in a new era. The formation of location-specific, higher-order SMOCs is particularly important for cell death and innate immune signaling processes. Several protein interaction domains, including the death domain (DD) superfamily and the CIDE domain, are representative mediators of SMOC assembly in cell death and innate immune signaling pathways. DD superfamily- and CIDE domain-containing proteins form SMOCs that activate various caspases and provide signaling scaffold platforms. These assemblies can lead to signal transduction and amplification during signaling events. In this review, we summarize recent findings on the molecular basis of DD superfamily- and CIDE domain-mediated SMOC formation.
Project description: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 di160-PC (DPPC), di182-PC (DLiPC), and cholesterol. We found that 4-10 tH molecules assemble into clusters that undergo molecular exchange in the sub-?s to ?s 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.