Ras signaling from plasma membrane and endomembrane microdomains

Ras proteins are compartmentalized by dynamic interactions with both plasma membrane microdomains and intracellular membranes. The mechanisms underlying Ras compartmentalization involve a series of protein/lipid, lipid/lipid and cytoskeleton interactions, resulting in the generation of discrete microdomains from which Ras operates. Segregation of Ras proteins to these different platforms regulates the formation of Ras signaling complexes and the generation of discrete signal outputs. This temporal and spatial modulation of Ras signal transduction provides a mechanism for the generation of different biological outcomes from different Ras isoforms, as well as flexibility in the signal output from a single activated isoform.     

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.     

Ras signaling on the Golgi

The discovery that Ras proteins are modified by enzymes restricted to the endoplasmic reticulum and Golgi apparatus and that, at steady state, a significant pool of Ras is localized on the Golgi has led to the hypothesis that Ras can become activated on and signal from intracellular membranes. Fluorescent probes capable of showing when and where in living cells Ras becomes activated together with studies of Ras proteins stringently tethered to intracellular membranes have confirmed this hypothesis. Thus, recent studies of Ras have contributed to the rapidly expanding field of compartmentalized signaling.     

Ras pathway signaling on endomembranes

Until recently, the plasma membrane has been considered to be a unique platform from which emanate the signaling events regulating or regulated by Ras and its close relatives. For the past few years, the role of endosomes derived from the plasma membrane as platforms for Ras/mitogen-activated protein kinase signaling has been appreciated. More recently, the cytoplasmic face of the Golgi apparatus and endoplasmic reticulum have been shown to host Ras signaling. The biological implications of compartmentalized signaling are only beginning to emerge.     

Nonconventional trafficking of Ras associated with Ras signal organization

Ras signaling to its downstream effectors appears to include combinations of extracellular-signal-regulated Ras activation at the plasma membrane (PM) and endomembranes, dynamic lateral segregation in the PM, and translocation of Ras from the PM to intracellular compartments. These processes are governed by the C-terminal polybasic farnesyl domain in K-Ras 4B and by the cysteine-palmitoylated C-terminal farnesyl domains in H-Ras and N-Ras. K-Ras 4B has no palmitoylated cysteines. Depalmitoylation/repalmitoylation of H-/N-Ras proteins promotes their cellular redistribution and signaling by mechanisms as yet unknown, possibly involving chaperones. Palmitoylation of H-/N-Ras also promotes their association with 'rasosomes', randomly diffusing nanoparticles that apparently provide a means by which multiple copies of activated Ras and its signal can spread rapidly. Ubiquitination of H-Ras evidently targets it to the endosomes. The polybasic farnesyl domain of K-Ras 4B was shown to act as a target for Ca++/calmodulin, which sequesters the active protein from the PM, thereby facilitating its trafficking to Golgi apparatus and early endosomes. Protein kinase C-dependent phosphorylation of S181 in K-Ras 4B was shown to provide a regulated farnesyl-electrostatic switch on K-Ras 4B, which promotes its translocation to the mitochondria. All these translocation events are characterized by nonconventional trafficking of the farnesyl-modified Ras proteins and seem to govern the selectivity and probably also the robustness of the Ras signal. In this review, we discuss the various modifications and interactions of the farnesylated C-terminus, the trafficking of Ras proteins in the PM and between the PM and the endomembranes, and the relevance of the subcellular localization of Ras for Ras function.     

Interactions of Ras proteins with the plasma membrane and their roles in signaling

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.     

Plasma membrane domain organization regulates EGFR signaling in tumor cells

Macromolecular complexes exhibit reduced diffusion in biological membranes; however, the physiological consequences of this characteristic of plasma membrane domain organization remain elusive. We report that competition between the galectin lattice and oligomerized caveolin-1 microdomains for epidermal growth factor (EGF) receptor (EGFR) recruitment regulates EGFR signaling in tumor cells. In mammary tumor cells deficient for Golgi beta1,6N-acetylglucosaminyltransferase V (Mgat5), a reduction in EGFR binding to the galectin lattice allows an increased association with stable caveolin-1 cell surface microdomains that suppresses EGFR signaling. Depletion of caveolin-1 enhances EGFR diffusion, responsiveness to EGF, and relieves Mgat5 deficiency-imposed restrictions on tumor cell growth. In Mgat5(+/+) tumor cells, EGFR association with the galectin lattice reduces first-order EGFR diffusion rates and promotes receptor interaction with the actin cytoskeleton. Importantly, EGFR association with the lattice opposes sequestration by caveolin-1, overriding its negative regulation of EGFR diffusion and signaling. Therefore, caveolin-1 is a conditional tumor suppressor whose loss is advantageous when beta1,6GlcNAc-branched N-glycans are below a threshold for optimal galectin lattice formation.     

Chaperone-mediated specificity in Ras and Rap signaling

Abstract

Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.     

Mechanisms of signal transduction by Ras.

The role of Ras in the transduction of signals that control cell growth is undisputed. However, the identity of the Ras signalling pathway remains unknown. Evidence is mounting that Ras can receive signals from different cell surface receptors most likely via a common intermediate, GAP. A new insight into the possible function of Ras is provided by the recent findings that certain ligands can induce the coordinated redistribution of Ras and cell surface receptors. The next challenge is to identify the specific targets for the action of Ras.     

Electron microscopic imaging of Ras signaling domains

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.     

Prospective of Ras signaling in stem cells

The Ras signaling pathway plays a predominant role during development and controls diverse biological process in all eukaryotic cells. It is a member of the large family of GTPases proteins that binds and hydrolyzes GTP. Ras is a lipid-anchored protein on the intracellular membrane compartments, and cycles between inactive GDP-bound and the signaling competent GTP-bound conformation. Studies have demonstrated Ras to be a central regulator in signal transduction pathways responding to diverse extracellular and intracellular stimuli. Much progress has been made towards delineating specific genes involved in the process of pluripotency and differentiation of stem cells. Here, we discuss recent aspects of Ras signaling pathways in mediating stem cell properties.     

Mechanisms of Ras Protein Targeting in Mammalian Cells

Syncollin is a 16-kDa protein that is associated with the luminal surface of the zymogen granule membrane in the pancreatic acinar cell. Detergent-solubilized, purified syncollin migrates on sucrose density gradients as a large (120-kDa) protein, suggesting that it exists naturally as a homo-oligomer. In this study, we investigated the structure of the syncollin oligomer. Chemical cross-linking of syncollin produced a ladder of bands, the sizes of which are consistent with discrete species from monomers up to hexamers. Electron microscopy of negatively stained syncollin revealed doughnut-shaped structures of outer diameter 10 nm and inner diameter 3 nm. Atomic force microscopy (AFM) of syncollin on mica supports at pH 7.6 showed particles of molecular volume 155 nm(3). Smaller particles were observed either at alkaline pH (11.0), or in the presence of a reducing agent (dithiothreitol), conditions that cause dissociation of the oligomer. AFM imaging of syncollin attached to supported lipid bilayers again revealed doughnut-shaped structures (outer diameter 31 nm, inner diameter 6 nm) protruding 1 nm from the bilayer. Finally, addition of syncollin to liposomes rendered them permeable to the water-soluble fluorescent probe 5(6)-carboxyfluorescein. These results are discussed in relation to the possible physiological role of syncollin.     

hSef co-localizes and interacts with Ras in the inhibition of Ras/MAPK signaling pathway

To study the mechanism of the inhibitory effects of Sef (similar expression to fgf genes) on Ras/mitogen-activated protein kinase (MAPK) signaling pathway, we observed cellular localization of this protein. Immunofluorescent staining results show that Sef locates in the vesicles of the cytoplasm without bFGF treatment but co-localizes with Ras on the plasma membrane (PM) in response to bFGF stimulation. The coimmunoprecipitation assay demonstrates that Sef interacts with Ras or RasG12V, respectively. We observed that Sef inhibited FGF induced, but not RasG12V mediated, signal transduction. We propose that Sef interacted with Ras in the inhibition of Ras/MAPK signaling pathway.     

Choose your own path: specificity in Ras GTPase signaling

The Ras superfamily of small G proteins contributes importantly to numerous cellular and physiological processes (M. F. Olsen and R. Marais, Semin. Immunol., 2000, 12, 63). This family comprises a large class of proteins (more than 150) which all share a common enzymatic function: hydrolysis of the gamma-phosphate of guanosine triphosphate (GTP) to create the products guanosine diphosphate (GDP) and inorganic phosphate (Y. Takai, T. Sasaki and T. Matozaki, Physiol. Rev., 2001, 81, 153). For this reason Ras family proteins, which include the Ras, Rho, Arf/Sara, Ran and Rab subfamilies, are classified as GTPases (G. W. Reuther and C. J. Der, Curr. Opin. Cell Biol., 2000, 12, 157). Guanine nucleotide coupling is a key regulator of enzymatic function; thus, Ras family GTPases participate in signal transduction. Ras signaling depends on binding to effectors. Many of the known effectors can bind to multiple Ras isotypes, often leading to common cellular outcomes, but each Ras isotype also engages specific effector pathways to mediate unique functions. Further, each Ras isotype can propagate multiple signaling pathways, indicating the presence of cellular determinants which allow for promiscuity in Ras-effector interactions while also maintaining specificity. Small distinctions in sequence, structure, and/or cellular regulation contribute to these differences in Ras-effector binding and subsequent cellular effects. A major focus of investigation in the Ras signaling field is identifying the determinants of these individualized functions. This review will attempt to summarize the current state of understanding of this question (with a particular focus on the Ras subfamily) and the approaches being taken to address it, and will discuss prospective areas for future investigation.