RAC/ROP GTPases: 'hubs' for signal integration and diversification in plants

RAC/ROP GTPases are a family of plant-specific signaling molecules solely representing the Ras and Rho family of Ras-related G proteins in plants. RAC/ROPs potentially interact with cell surface-associated signal perception apparatus for a broad range of extracellular stimuli, including hormones, pathogen elicitors and abiotic stress, and mediate diverse cellular pathways in response to these signals. They are also known to interact with multiple effectors, affecting cellular and biochemical systems that regulate actin dynamics, reactive oxygen species production, proteolysis, and gene expression. RAC/ROPs are, thus, ideally suited as integrators for multiple signals and as coordinators of diverse cellular pathways to control growth, differentiation, development and defense responses. Recent findings that suggest how RAC/ROP signaling activity is regulated and how functional specificity can be achieved are discussed here.     

Signaling interplay in Ras superfamily function

Ras proteins function as signaling hubs that are activated by convergent signaling pathways initiated by extracellular stimuli. Activated Ras in turn regulates a diversity of downstream cytoplasmic signaling cascades. Ras proteins are founding members of a large superfamily of small GTPases that have significant sequence and biochemical similarities. Recent observations have established a complex signaling interplay between Ras and other members of the family. A key biochemical mechanism facilitating this crosstalk involves guanine nucleotide exchange factors (GEFs), which serve as regulators and effectors, as well as signaling integrators, of Ras signaling.     

Sensitivity and specificity amplification in signal transduction

Intracellular signal transduction pathways transmit signals from the cell surface to various intracellular destinations, such as cytoskeleton and nucleus through a cascade of protein-protein interactions and activation events, leading to phenotypic changes such as cell proliferation, differentiation, and death. Over the past two decades, numerous signaling proteins and signal transduction pathways have been discovered and characterized. There are two major classes of signaling proteins: phosphoproteins (e.g., mitogen-activated protein kinases) and guanosine triphosphatases (GTPases; e.g., Ras and G proteins). They both function as molecular switches by addition and removal of one or more high-energy phosphate groups. This review discusses developments that seek to quantify the signal transduction processes with kinetic analysis and mathematical modeling of the signaling phosphoproteins and GTPases. These studies have provided insights into the sensitivity and specificity amplification of biological signals in integrated systems.     

Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate

Ras family GTPases (RFGs) regulate signaling pathways that control multiple biological processes. How signaling specificity among the closely related family members is achieved is poorly understood. We have taken a proteomics approach to signaling by RFGs, and we have analyzed interactions of a panel of RFGs with a comprehensive group of known and potential effectors. We have found remarkable differences in the ability of RFGs to regulate the various isoforms of known effector families. We have also identified several proteins as novel effectors of RFGs with differential binding specificities to the various RFGs. We propose that specificity among RFGs is achieved by the differential regulation of combinations of effector families as well as by the selective regulation of different isoforms within an effector family. An understanding of this new level of complexity in the signaling pathways regulated by RFGs is necessary to understand how they carry out their many cellular functions. It will also likely have critical implications in the treatment of human diseases such as cancer.     

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.     

Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains

Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1.     

Ion channel regulation by Ras, Rho, and Rab small GTPases

Regulation of ion channels by heterotrimeric guanosine triphosphatases (GTPases), activated by heptathelical membrane receptors, has been the focus of several recent reviews. In comparison, regulation of ion channels by small monomeric G proteins, activated by cytoplasmic guanine nucleotide exchange factors, has been less well reviewed. Small G proteins, molecular switches that control the activity of cellular and membrane proteins, regulate a wide variety of cell functions. Many upstream regulators and downstream effectors of small G proteins now have been isolated. Their modes of activation and action are understood. Recently, ion channels were recognized as physiologically important effectors of small GTPases. Recent advances in understanding how small G proteins regulate the intracellular trafficking and activity of ion channels are discussed here. We aim to provide critical insight into physiological control of ion channel function and the biological consequences of regulation of these important proteins by small, monomeric G proteins.     

A novel Ras inhibitor,Eri1, engages yeast Ras at the endoplasmic reticulum

Ras oncoproteins are monomeric GTPases that link signals from the cell surface to pathways that regulate cell proliferation and differentiation. Constitutively active mutant forms of Ras are found in ca. 30% of human tumors. Here we report the isolation of a novel gene from Saccharomyces cerevisiae, designated ERI1 (for endoplasmic reticulum-associated Ras inhibitor 1), which behaves genetically as an inhibitor of Ras signaling. ERI1 encodes a 68-amino-acid protein that associates in vivo with GTP-bound Ras in a manner that requires an intact Ras-effector loop, suggesting that Eri1 competes for the same binding site as Ras target proteins. We show that Eri1 localizes primarily to the membrane of the endoplasmic reticulum (ER), where it engages Ras. The recent demonstration that signaling from mammalian Ras is not restricted to the cell surface but can also proceed from the cytoplasmic face of the ER suggests a regulatory function for Eri1 at that membrane.     

Signaling from Ras to Rac and beyond: not just a matter of GEFs

Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase-->activator--> GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.     

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.     

COMBINE analysis of the specificity of binding of Ras proteins to their effectors

The small guanosine triphosphate (GTP)-binding proteins of the Ras family are involved in many cellular pathways leading to cell growth, differentiation, and apoptosis. Understanding the interaction of Ras with other proteins is of importance not only for studying signalling mechanisms but also, because of their medical relevance as targets, for anticancer therapy. To study their selectivity and specificity, which are essential to their signal transfer function, we performed COMparative BINding Energy (COMBINE) analysis for 122 different wild-type and mutant complexes between the Ras proteins, Ras and Rap, and their effectors, Raf and RalGDS. The COMBINE models highlighted the amino acid residues responsible for subtle differences in binding of the same effector to the two different Ras proteins, as well as more significant differences in the binding of the two different effectors (RalGDS and Raf) to Ras. The study revealed that E37, D38, and D57 in Ras are nonspecific hot spots at its effector interface, important for stabilization of both the RalGDS-Ras and Raf-Ras complexes. The electrostatic interaction between a GTP analogue and the effector, either Raf or RalGDS, also stabilizes these complexes. The Raf-Ras complexes are specifically stabilized by S39, Y40, and D54, and RalGDS-Ras complexes by E31 and D33. Binding of a small molecule in the vicinity of one of these groups of amino acid residues could increase discrimination between the Raf-Ras and RalGDS-Ras complexes. Despite the different size of the RalGDS-Ras and Raf-Ras complexes, we succeeded in building COMBINE models for one type of complex that were also predictive for the other type of protein complex. Further, using system-specific models trained with only five complexes selected according to the results of principal component analysis, we were able to predict binding affinities for the other mutants of the particular Ras-effector complex. As the COMBINE analysis method is able to explicitly reveal the amino acid residues that have most influence on binding affinity, it is a valuable aid for protein design.     

Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth

Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only approximately 20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.     

Pathogens reWritE Rho's rules

Many bacterial pathogens use a specialized "type III" secretion system to deliver virulence effector proteins into host mammalian cells. In this issue of Cell, Alto et al. (2006) describe a new family of effectors that share a WxxxE sequence motif. These effectors directly stimulate host signaling pathways by mimicking activated Ras-like cellular GTPases.     

Phosphorylated guanine nucleotide exchange factor C3G,induced by pervanadate and Src family kinases localizes to the Golgi and subcortical actin cytoskeleton

Background  

The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.     

Biochemical characterization of the Ras-related GTPases Rit and Rin.

We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k(off) values for GTP than GDP in the presence of 10 mM Mg(2+). These GTP dissociation rates are 5- to 10-fold faster than most Ras-like GTPases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins suggest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.     

Signal transduction gRABs attention

Rab proteins are small GTPases involved in the regulation of vesicular membrane traffic. Research done in the past years has demonstrated that some of these proteins are under the control of signal transduction pathways. Still, several recent papers point out to a new unexpected role for this family of Ras-related proteins, as potential regulators of intracellular signaling pathways. In particular, several evidence indicate that members of the Rab family of small GTPases, through their effectors, are key molecules participating to the regulation of numerous signal transduction pathways profoundly influencing cell proliferation, cell nutrition, innate immune response, fragmentation of compartments during mitosis and apoptosis. Even more surprisingly, direct involvement of Rab proteins in signaling to the nucleus has been demonstrated. This review will focus on aspects of Rab proteins function connected to signal transduction and, in particular, connections between membrane traffic and other cell pathways will be examined.     

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.     

PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.     

RalGDS family members couple Ras to Ral signalling and that's not all

Ras proteins function as molecular switches that are activated in response to signalling pathways initiated by various extracellular stimuli and subsequently bind to numerous effector proteins leading to the activation of several signalling cascades within the cell. Ras and Ras-related proteins belong to a large superfamily of small GTPases characterized by significant sequence and function similarities. Several evidence indicate the existence of complex signalling networks that link Ras with its relatives in the family. A key role in this cross-talk is played by guanine nucleotide exchange factors (GEFs) that serve both as regulators and as effectors of Ras family proteins. The members of the RalGDS family, RalGDS, RGL, RGL2/Rlf and RGL3, can interact with activated Ras through their Ras Binding Domain (RBD), but may function as effectors for other Ras family members. They possess a REM-CDC25 homology region like RasGEFs, but specifically activate only RalA and RalB and not Ras or other Ras-related small GTPases. In this review we provide an update on this recently discovered family of GEFs, highlighting their crucial role in coupling activated Ras to activation of Ral, thus regulating several fundamental cell processes, and also discussing some evidence supporting Ras-independent additional functions of RalGDS proteins.