Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins

Ras proteins function as critical relay switches that regulate diverse signaling pathways between cell surface receptors and the nucleus. Over the past 2-3 years researchers have identified many components of these pathways that mediate Ras activation and effector function. Among these proteins are several guanine nucleotide exchange factors (GEFs), which are responsible for directly interacting with and activating Ras in response to extracellular stimuli. Analogous GEFs regulate Ras-related proteins that serve other diverse cellular functions. In particular, a growing family of proteins (Dbl homology proteins) has recently been identified, which may function as GEFs for the Rho family of Ras-related proteins. This review summarizes our current knowledge of the structure, biochemistry and biology of Ras and Rho family GEFs. Additionally, we describe mechanisms of GEF activation of Ras in signal transduction and address the potential that deregulated GEFs might contribute to malignant transformation through chronic Ras protein activation.     

Ras proteins control mitochondrial biogenesis and function in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The evolutionarily conserved Ras proteins function as a point of convergence for different signaling pathways in eukaryotes and have been implicated in both aging and cancer development. In Saccharomyces cerevisiae the plasma membrane proteins Ras1 and Ras2 are sensing the nutritional status of the environments, e.g., the abundance and quality of available carbon sources. The cAMP-protein kinase A pathway is the most explored signaling pathway controlled by Ras proteins; it affects a large number of genes, some of which are important to defend the cell against oxidative stress. In addition, recent analysis has shown that the Ras system of yeast is involved in the development of mitochondria and in regulating their activity. As a sensor of environmental status and an effector of mitochondrial activity, Ras serves as a Rosetta stone of cellular energy transduction. This review summarizes the physical and functional involvement of Ras proteins and Ras-dependent signaling pathways in mitochondrial function in S. cerevisiae. Since mitochondria produce harmful reactive oxygen species as an inevitable byproduct and are partly under control of Ras, illuminating these regulatory interactions may improve our understanding of both cancer and aging.     

Targeting protein lipidation in disease

Fatty acids and/or isoprenoids are covalently attached to a variety of disease-related proteins. The distinct chemical properties of each of these hydrophobic moieties allow lipid modification to serve as a mechanism to regulate protein structure, localization and function. This review highlights recent progress in identifying inhibitors of protein lipidation and their effects on human disease. Myristoylation inhibitors have shown promise in blocking the action of human pathogens. Although inhibitors that block prenylation of Ras proteins have not yet been successful for cancer treatment, they may be efficacious in the rare premature aging syndrome progeria. Agents that alter the palmitoylation status of Ras, Wnt and Hh proteins have recently been discovered, and represent the next generation of potential chemotherapeutics.     

A genome-wide Ras-effector interaction network

Here using structural information and protein design tools we have drawn the network of interactions between 20 Ras subfamily proteins with 50 putative Ras binding domains. To validate this network we have cloned six poorly characterized Ras binding domains (RBD) and two Ras proteins (RERG, DiRas1). These, together with previously described RBD domains, Ras and Rap proteins have been analyzed in 70 pull-down experiments. Comparing our interaction network with these and previous pull-down experiments (total of 150 cases) shows a very high accuracy for distinguishing between binders and non-binders ( approximately 0.80). Bioinformatics information was integrated to distinguish those in vitro interactions that are more likely to be relevant in vivo. We proposed several new interactions between Ras family members and effector domains that are of relevance in understanding the physiological role of these proteins. More broadly our results demonstrate that (domain-domain) interaction specificities between members of protein families can be accurately predicted using structural information.     

Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras

Proteins that terminate with a consensus sequence known as CAAX undergo a series of posttranslational modifications that include polyisoprenylation, endoproteolysis, and carboxyl methylation. These modifications render otherwise hydrophilic proteins hydrophobic at their C termini such that they associate with membranes. Whereas prenylation occurs in the cytosol, postprenylation processing is accomplished on the cytoplasmic surface of the endoplasmic reticulum and Golgi apparatus. Among the numerous CAAX proteins encoded in mammalian genomes are many signaling molecules such as monomeric GTPases, including the Ras proteins that play an important role in cancer. In the course of their processing, nascent Ras proteins traffic from their site of synthesis in the cytosol to the endomembrane and then out to the plasma membrane (PM) by at least two pathways. Recently, retrograde pathways have been discovered that deliver mature Ras from the PM back to the Golgi. The Golgi has been identified as a platform upon which Ras can signal. Thus, the subcellular trafficking of Ras proteins has the potential to increase the complexity of Ras signaling by adding a spatial dimension. The complexity of Ras trafficking also affords a wider array of potential targets for the discovery of drugs that might inhibit tumors by interfering with Ras trafficking.     

Differential regulation of cellular activities by GTPase-activating protein and NF1.

The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies.     

Small GTPases take the stage

Small GTPases are molecular switches that have been adopted to control many eukaryotic cell functions. Starting with the study of the protooncogene Ras in the early 1980s, detailed pathways have been uncovered upstream and downstream of Ras-related GTP binding proteins. Nonetheless, novel members have been discovered at a pace that has outstripped cell biologists, and thus much remains to be established regarding newer family members. Undiscovered functions are still being uncovered for "established" small GTPases such as Ras, Rho, and Ran. The topics covered at this meeting indeed demonstrate that Ras proteins are at the heart of cellular dynamics.     

Mapping Cellular Routes of Ras: A Ubiquitin Trail

The three mammalian Ras isoforms: HRas, NRas and KRas have been widely implicated in the control of cell proliferation, survival, motility and transformation. Although nearly identical with respect to their catalytic and effector-binding properties, HRas, NRas and KRas lead to different biological outcomes in development, cell growth and cancer. This functional distinction is believed to result at least in part from the differential membrane compartmentalization of Ras isoforms. The different distribution of Ras proteins in cellular membranes dictates unique spatio-temporal patterns of activation of effector pathways. This perspective focuses on the factors that control membrane compartmentalization of Ras with an emphasis on a recently discovered novel posttranslational modification of Ras—ubiquitination. The properties of Ras ubiquitination, its contribution to the regulation of Ras intracellular trafficking and finally the influence of Ras ubiquitination on its signaling potential are discussed.     

Many faces of Ras activation

Ras proteins were originally identified as the products of oncogenes capable of inducing cell transformation. Over the last twenty-five years they have been studied in great detail because mutant Ras proteins are associated with many types of human cancer. Wild type Ras proteins play a central role in the regulation of proliferation and differentiation of various cell types. They alternate between an active GTP-bound state and an inactive GDP-bound state. Their activation is catalysed by a specialized group of enzymes known as guanine nucleotide exchange factors (GEFs). To date, four subfamilies of GEF molecules have been identified. Although all of them are able to activate Ras, their structure, tissue expression and regulation are significantly diverse. In this review we will summarize the various mechanisms by which these exchange factors activate Ras.     

Many faces of Ras activation

Ras proteins were originally identified as the products of oncogenes capable of inducing cell transformation. Over the last twenty-five years they have been studied in great detail because mutant Ras proteins are associated with many types of human cancer. Wild type Ras proteins play a central role in the regulation of proliferation and differentiation of various cell types. They alternate between an active GTP-bound state and an inactive GDP-bound state. Their activation is catalysed by a specialized group of enzymes known as guanine nucleotide exchange factors (GEFs). To date, four subfamilies of GEF molecules have been identified. Although all of them are able to activate Ras, their structure, tissue expression and regulation are significantly diverse. In this review we will summarize the various mechanisms by which these exchange factors activate Ras.     

Plasma membrane localization of Ras requires class C Vps proteins and functional mitochondria in Saccharomyces cerevisiae

Ras proteins are synthesized as cytosolic precursors, but then undergo posttranslational lipid addition, membrane association, and subcellular targeting to the plasma membrane. Although the enzymes responsible for farnesyl and palmitoyl lipid addition have been described, the mechanism by which these modifications contribute to the subcellular localization of Ras is not known. Following addition of the farnesyl group, Ras associates with the endoplasmic reticulum (ER), where palmitoylation occurs in Saccharomyces cerevisiae. The subsequent translocation of Ras from the ER to the plasma membrane does not require the classical secretory pathway or a functional Golgi apparatus. Vesicular and nonvesicular transport pathways for Ras proteins have been proposed, but the pathway is not known. Here we describe a genetic screen designed to identify mutants defective in Ras trafficking in S. cerevisiae. The screen implicates, for the first time, the class C VPS complex in Ras trafficking. Vps proteins are best characterized for their role in endosome and vacuole membrane fusion. However, the role of the class C Vps complex in Ras trafficking is distinct from its role in endosome and vacuole vesicle fusion, as a mitochondrial involvement was uncovered. Disruption of class C VPS genes results in mitochondrial defects and an accumulation of Ras proteins on mitochondrial membranes. Ras also fractionates with mitochondria in wild-type cells, where it is detected on the outer mitochondrial membrane by virtue of its sensitivity to protease treatment. These results point to a previously uncharacterized role of mitochondria in the subcellular trafficking of Ras proteins.     

Exchange factors of the RasGRP family mediate Ras activation in the Golgi

H-Ras and N-Ras become activated both at the plasma membrane and in endomembrane structures such as the Golgi apparatus. This compartmentalized activation is relevant from a signaling standpoint, because effector molecules can become activated differently depending on the region of the cell where Ras proteins are activated. An unsolved question in this new regulatory mechanism is the understanding of how Ras proteins become activated in endomembranes. To approach this problem, we have studied the subcellular distribution and activities of a number of Ras guanosine nucleotide exchange factors. Our results indicate that Ras activation at the plasma membrane and endoplasmic reticulum is an unspecific process that can be achieved by most Ras activators. In contrast, GTP loading of Ras at the Golgi is only induced by members of the Ras guanosine nucleotide releasing protein family. In agreement with these observations, Ras guanosine nucleotide releasing proteins are the only Ras activators showing localization in the Golgi. These results indicate that the compartmentalized activation of effector pathways by Ras proteins depends not only on the specific localization of the GTPases but also in the availability of GDP/GTP exchange factors capable of activating Ras proteins in specific subcellular compartments.     

Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions. Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation. To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made. The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase. The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown. This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.     

Ras-mediated cell proliferation and cell death: some clues from the interleukin-2 receptor system

Oncoproteins of the Ras family have been extensively studied because of their implication in human cancer. Their roles have been primarily assigned to the commandment of cell proliferation and suppression of apoptosis, which has also been demonstrated by the involvement of Ras activation in the signal transduction pathways triggered by most cytokine receptors. Nevertheless, the functions of Ras proteins have been extended in the last years by the findings showing that they can also act as promoters or enhancers of apoptosis in various systems and conditions. These considerations have raised the issue as to how the signals delivered by Ras are regulated and translated in terms of cellular responses, suggesting that signal complementation may direct the final fate of cells. As an example, the interleukin-2 receptor system may represent a useful model in which the meaning of Ras signals may be evaluated in terms of interactions with other simultaneous signalling events, since knowledge of the biochemical events triggered by the interaction of interleukin-2 with its cell surface receptor in lymphocytes has allowed the proposal of a complete signalling model arranged in three independent channels, one of which is mediated by Ras.This work was supported by grants from CICYT and Pharmacia-Upjohn.     

PIRLs: a novel class of plant intracellular leucine-rich repeat proteins

Leucine-rich repeat (LRR) proteins feature tandem leucine-rich motifs that form a protein-protein interaction domain. Plants contain diverse classes of LRR proteins, many of which take part in signal transduction. We have identified a novel family of nine Arabidopsis LRR proteins that, based on predicted intracellular location and LRR motif consensus sequence, are related to Ras-binding LRR proteins found in signaling complexes in animals and yeast. This new class has been named plant intracellular Ras group-related LRR proteins (PIRLs). We have characterized PIRL cDNAs, rigorously defined gene and protein annotations, investigated gene family evolution and surveyed mRNA expression. While LRR regions suggested a relationship to Ras group LRR proteins, outside of their LRR domains PIRLs differed from Ras group proteins, exhibiting N- and C-terminal regions containing low complexity stretches and clusters of charged amino acids. PIRL genes grouped into three subfamilies based on sequence relationships and gene structures. Related gene pairs and dispersed chromosomal locations suggested family expansion by ancestral genomic or segmental duplications. Expression surveys revealed that all PIRL mRNAs are actively transcribed, with three expressed differentially in leaves, roots or flowers. These results define PIRLs as a distinct, plant-specific class of intracellular LRR proteins that probably mediate protein interactions, possibly in the context of signal transduction. T-DNA knock-out mutants have been isolated as a starting point for systematic functional analysis of this intriguing family.     

Membrane binding of lipidated Ras peptides and proteins — The structural point of view

Biological membranes are interesting interfaces, at which important biological processes occur. In addition to integral membrane proteins, a number of proteins bind to the membrane surface and associate with it. Posttranslational lipid modification is one important mechanism, by which soluble molecules develop a propensity towards the membrane and reversibly bind to it. Membrane binding by insertion of hydrophobic lipid moieties is relevant for up to 10% of all cellular proteins. A particular interesting lipid-modified protein is the small GTPase Ras, which plays a key role in cellular signal transduction. Until recently, the structural basis for membrane binding of Ras was not well-defined. However, with the advent of new synthesis techniques and the advancement of several biophysical methods, a number of structural and dynamical features about membrane binding of Ras proteins have been revealed. This review will summarize the chemical biology of Ras and discuss in more detail the biophysical and structural features of the membrane bound C-terminus of the protein.