Ras Superfamily Of GTPases Research Articles

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.

Regulation of Ras Paralog Thermostability by Networks of Buried Ionizable Groups.

Protein folding is governed by a variety of molecular forces including hydrophobic and ionic interactions. Less is known about the molecular determinants of protein stability. Here we used a recently developed computer algorithm (pHinder) to investigate the relationship between buried charge and thermostability. Our analysis revealed that charge networks in the protein core are generally smaller in thermophilic organisms as compared to mesophilic organisms. To experimentally test whether core network size influences protein thermostability, we purified 18 paralogous Ras superfamily GTPases from yeast and determined their melting temperatures (Tm, or temperature at which 50% of the protein is unfolded). This analysis revealed a wide range of Tm values (35-63 °C) that correlated significantly (R = 0.87) with core network size. These results suggest that thermostability depends in part on the arrangement of ionizable side chains within a protein core. An improved capacity to predict protein thermostability may be useful for selecting the best candidates for protein crystallography, the development of protein-based therapeutics, as well as for industrial enzyme applications.

Apoptotic activity of 5-fluorouracil in breast cancer cells transformed by low doses of ionizing α-particle radiation.

Globally, breast cancer in women is the leading cause of cancer death. This fact has generated an interest to obtain insight into breast tumorigenesis and also to develop drugs to control the disease. Ras is a proto-oncogene that is activated as a response to extracellular signals. As a member of the Ras GTPase superfamily, Rho-A is an oncogenic and a critical component of signaling pathways leading to downstream gene regulation. In chemotherapy, apoptosis is the predominant mechanism by which cancer cells die. However, even when the apoptotic machinery remains intact, survival signaling may antagonize the cell death by signals. The aim of this study was to evaluate 5-fluorouracil (5-FU) in cells transformed by low doses of ionizing α-particle radiation, in breast cancer cell lines on these genes, as well as apoptotic activity. We used two cell lines from an in vitro experimental breast cancer model. The MCF-10F and Tumor2 cell lines. MCF-10F was exposed to low doses of high linear energy transfer (LET) α-particles radiation (150 keV/µm). Tumor2, is a malignant and tumorigenic cell line obtained from Alpha5 (60cGy+E/60cGy+E) injected into the nude mice. Results indicated that 5-FU decreased H-ras, Rho-A, p53, Stat1 and increased Bax gene expression in Tumor2 and decreased Rac1, Rho-A, NF-κB and increased Bax and caspase-3 protein expression in Tumor2. 5-FU decreased H-ras, Bcl-xL and NF-κB and increased Bax gene expression. 5-FU decreased Rac1, Rho-A protein expression and increased Bax and caspase-3 protein expression in MDA-MB-231. Flow cytometry indicated 21.5% of cell death in the control MCF-10F and 80% in Tumor2 cell lines. It can be concluded that 5-FU may exert apoptotic activity in breast cancer cells transformed by low doses of ionizing α-particles in vitro regulating genes of Ras family and related to apoptosis such as Bax, Bcl-xL and NF-κB expression.

Zebrafish diras1 Promoted Neurite Outgrowth in Neuro-2a Cells and Maintained Trigeminal Ganglion Neurons In Vivo via Rac1-Dependent Pathway.

The small GTPase Ras superfamily regulates several neuronal functions including neurite outgrowth and neuron proliferation. In this study, zebrafish diras1a and diras1b were identified and were found to be mainly expressed in the central nervous system and dorsal neuron ganglion. Overexpression of green fluorescent protein (GFP)-diras1a or GFP-diras1b triggered neurite outgrowth of Neuro-2a cells. The wild types, but not the C terminus truncated forms, of diras1a and diras1b elevated the protein level of Ras-related C3 botulinum toxin substrate 1 (Rac1) and downregulated Ras homologous member A (RhoA) expression. Glutathione S-transferase (GST) pull-down assay also revealed that diras1a and diras1b enhanced Rac1 activity. Interfering with Rac1, Pak1, or cyclin-dependent kinase 5 (CDK5) activity or with the Arp2/3 inhibitor prevented diras1a and diras1b from mediating the neurite outgrowth effects. In the zebrafish model, knockdown of diras1a and/or diras1b by morpholino antisense oligonucleotides not only reduced axon guidance but also caused the loss of trigeminal ganglion without affecting the precursor markers, such as ngn1 and neuroD. Co-injection with messenger RNA (mRNA) derived from mouse diras1 or constitutively active human Rac1 restored the population of trigeminal ganglion. In conclusion, we provided preliminary evidence that diras1 is involved in neurite outgrowth and maintains the number of trigeminal ganglions through the Rac1-dependent pathway.

Crystal Structure Analysis of Wild Type and Fast Hydrolyzing Mutant of EhRabX3, a Tandem Ras Superfamily GTPase from Entamoeba histolytica

The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8Å resolutions, respectively. Though both G-domains possess “phosphate binding loop containing nucleoside triphosphate hydrolases fold”, only the N-terminal domain binds to guanine nucleotide. The relative orientation of the N-terminal domain and C-terminal domain is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and could allow us to design potential inhibitors for the better treatment of intestinal amebiasis.

Rab from the white shrimp Litopenaeus vannamei: characterization and its regulation upon environmental stress.

With the destruction of the ecological environment, shrimp cultivation in China has been seriously affected by outbreaks of infectious diseases. Rab, which belong to small GTPase Ras superfamily, can regulate multiple steps in eukaryotic vesicle trafficking including vesicle budding, vesicle tethering, and membrane fusion. Knowledge of Rab in shrimp is essential to understanding regulation and detoxification mechanisms of environmental stress. In this study, we analyzed the functions of Rab from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNA of Rab was obtained, which was 751 bp long, with open reading frame encoding 206 amino acids. In this study, for the first time, the gene expression of Rab of L. vannamei was analyzed by quantitative real-time PCR after exposure to five kinds of environmental stresses (bacteria, pH, Cd, salinity and low temperature). The results demonstrate that Rab is sensitive and involved in bacteria, pH, and Cd stress responses and Rab is more sensitive to bacteria than other stresses. Therefore we infer that Rab may have relationship with the anti-stress mechanism induced by environment stress in shrimp and Rab could be used as critical biomarkers for environmental quality assessment.

Rac1: an emerging player in stretch-stimulated glucose transport.

Rac1: an emerging player in stretch-stimulated glucose transport.

A Superfamily Reunion: Conserved Water Analysis of Small GTPases Using the Crystallography Tool DRoP

The Ras superfamily of GTPases is a family of monomeric molecular switches that regulate a variety of cellular processes, such as proliferation, migration, trafficking and vesicle formation. Subfamily classification includes Ras, Rho, Ran, Rab, and Arf/Sar. While the core structure of each small GTPase is conserved, each subfamily interacts with unique effectors that contribute to differing pathway regulation in the cell. The GTP/GDP cycle is a common regulatory feature of signaling output across GTPases. The GTP-bound protein results in an active signaling cascade of interactions in the cell, and the hydrolysis of GTP to GDP turns off the signal. The current research focuses on how water-mediated H-bonding networks differ between the subfamily members with particular attention to their ability to promote intrinsic hydrolysis and communication between distant areas of the GTPases. Structural water molecules contribute to protein-target specificity, enzyme catalysis, and protein dynamics. Detection of Related Solvent Positions (DRoP) is a computational tool that identifies conserved water molecules across multiple Protein Data Bank files. DRoP is employed to study conserved waters in each small GTPase subfamily classification. Several subfamily specific structural water patterns are seen through this analysis, in addition to highly conserved waters belonging to the entire superfamily. The discovery of both unique and common water networks between subfamilies aid in understanding functional differences between small GTPases. This work is funded by the National Science Foundation (NSF MCB-1244203).

Photo-Regulation of the Interaction between Ras and Ralgds using GTP Analogues Composed of Photochromic Molecules

Small GTPases of the Ras family have significant roles of signal transduction in eukaryotic cells. The G-proteins transmit the signals from a number of receptors to a variety of effectors. RalGDS is a guanine nucleotide dissociation stimulator for Ral, which is a member of the Ras GTPase superfamily that regulates cellular proliferation, differentiation and transformation by mediating multiple signal transduction pathways. RalGDS can specifically promote the conversion from an inactive GDP-bound state to an active GTP-bound state for Ral. Previously we have demonstrated that the GTPase activity of K-Ras modified with thiol reactive azobenzene derivatives at the functional sites were photo-reversibly controlled upon ultraviolet and visible light irradiation. Aim of this study is to control the interaction of Ras with RalDGS photo-reversibly using photochromic molecules. Recently we found that a fluorescent GTP analogue, 2′(3′)-O-[6-(N-(7- nitrobenz-2- oxa-1,3-diazol-4-yl)amino)hexanoic]-GTP (NBD-GTP) which has a fluorescent probe at 2’ or 3’ position of ribose induced binding of Ras to RalGDS more significantly than regular GTP. NBD-GDP did not induce binding of Ras to RalGDS. The fluorophore NBD might affect as an enhancer of the interaction between Ras and RalGDS. In this study, we synthesized novel photo-responsive GTP analogues that have photochromic molecules at 2’ or 3’ position of ribose, 2′(3′) -O-Azobenzene-EDA-GTP (AB-EDA-GTP), 2′(3′) -O-Spiropyran-EDA-GTP (SP-EDA-GTP). Trans-isomer of AB-EDA-GTP induced more significant binding of Ras to RalGDS than Cis-isomer. On the other hand, SP-EDA-GTP did not show the significant difference between the spiro and merocyanine isomers.

Evolution and diversity of the Ras superfamily of small GTPases in prokaryotes.

The Ras superfamily of small GTPases are single domain nucleotide-dependent molecular switches that act as highly tuned regulators of complex signal transduction pathways. Originally identified in eukaryotes for their roles in fundamental cellular processes including proliferation, motility, polarity, nuclear transport, and vesicle transport, recent studies have revealed that single domain GTPases also control complex functions such as cell polarity, motility, predation, development and antibiotic resistance in bacteria. Here, we used a computational genomics approach to understand the abundance, diversity, and evolution of small GTPases in prokaryotes. We collected 520 small GTPase sequences present in 17% of 1,611 prokaryotic genomes analyzed that cover diverse lineages. We identified two discrete families of small GTPases in prokaryotes that show evidence of three distinct catalytic mechanisms. The MglA family includes MglA homologs, which are typically associated with the MglB GTPase activating protein, whereas members of the Rup (Ras superfamily GTPase of unknown function in prokaryotes) family are not predicted to interact with MglB homologs. System classification and genome context analyses support the involvement of small GTPases in diverse prokaryotic signal transduction pathways including two component systems, laying the foundation for future experimental characterization of these proteins. Phylogenetic analysis of prokaryotic and eukaryotic GTPases supports that the last universal common ancestor contained ancestral MglA and Rup family members. We propose that the MglA family was lost from the ancestral eukaryote and that the Ras superfamily members in extant eukaryotes are the result of vertical and horizontal gene transfer events of ancestral Rup GTPases.

Crystallization and preliminary X-ray analysis of RabX3, a tandem GTPase from Entamoeba histolytica.

Ras superfamily GTPases regulate signalling pathways that control multiple biological processes by modulating the GTP/GDP cycle. Various Rab GTPases, which are the key regulators of vesicular trafficking pathways, play a vital role in the survival and virulence of the enteric parasite Entamoeba histolytica. The Rab GTPases act as binary molecular switches that utilize the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins and thereby regulate a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, nuclear transport and intracellular membrane trafficking in eukaryotes. Entamoeba histolytica RabX3 (EhRabX3) is a unique GTPase in the amoebic genome, the only member in the eukaryotic Ras superfamily that harbours tandem G-domains and shares only 8-16% sequence identity with other GTPases. Recent studies suggested that EhRabX3 binds to a single guanine nucleotide through its N-terminal G-domain (NTD), while the C-terminal G-domain (CTD) plays a potential role in binding of the nucleotide to the NTD. Thus, understanding the intermolecular regulation between the two GTPase domains is expected to reveal valuable information on the overall action of EhRabX3. To provide structural insights into the inclusive action of this unique GTPase, EhRabX3 was crystallized by successive micro-seeding using the vapour-diffusion method. A complete data set was collected to 3.3 Å resolution using a single native EhRabX3 crystal at 100 K on BM14 at the ESRF, Grenoble, France. The crystal belonged to monoclinic space group C2, with unit-cell parameters a=198.6, b=119.3, c=89.2 Å, β=103.1°. Preliminary analysis of the data using the Matthews Probability Calculator suggested the presence of four to six molecules in the asymmetric unit.

Is the model of signal amplification by GPCRs/GEFs activating multiple GTPases relevant to a broad spectrum of heterotrimeric and RAS superfamily GTPases?

Concepts or models of biological processes shape how we think about them, discuss them, and design experiments to test aspects of them. Because of the importance of our models of cell signaling by regulatory GTPases and the desire to extend those models to related signaling modules, I have throughout my career been fascinated by the similarities and differences between the modeling of heterotrimeric G protein and monomeric RAS superfamily GTPases. Recent discussions with colleagues led me to conclude that there is a growing divergence in how researchers model the activation and signaling processes of monomeric and trimeric GTPases and also a surprising lack of consensus within each camp. This series of articles arose in response to these discussions and is intended to spark new ones.

Mechanisms of Nerve Capping Technique in Prevention of Painful Neuroma Formation

Nerve capping techniques have been introduced as a promising treatment modality for the treatment of painful neuroma with varied outcomes; however, its exact mechanism is still unknown. RhoA is one of the members of the RAS superfamily of GTPases that operate as molecular switches and plays an important role in peripheral nerve regeneration. Our aim was to investigate the structural and morphologic mechanisms by which the nerve capping technique prevents the formation of painful neuromas after neuroectomy. We also hoped to provide a theoretical basis for this treatment approach. An aligned nanofiber conduit was used for the capping procedure and the sciatic nerve of Sprague-Dawley rats was selected as the animal model. Behavioral analysis, extent of neuroma formation, histological assessment, expressions of pain markers of substance P and c-fos, molecular biological changes as well as ultrastructural features were investigated and compared with the findings in a no-capping control group. The formation of traumatic neuromas was significantly inhibited in the capping group with relatively “normal” structural and morphological features and no occurrence of autotomy and significantly lower expression of pain markers compared to the no-capping group. The gene expression of RhoA was consistently in a higher level in the capping group within 8 weeks after surgery. This study shows that capping technique will alter the regeneration state of transected nerves and reduce painful neuroma formation, indicating a promising approach for the treatment of painful neuroma. The initiation of the “regenerative brake” induced by structural as well as morphological improvements in the severed nerve is theorized to be most likely a key mechanism for the capping technique in the prevention of painful neuroma formation.

Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation

Ras superfamily GTPase activation and inactivation occur by canonical nucleotide exchange and GTP hydrolysis mechanisms. Despite conservation of active-site residues, the Ras-related Rab GTPase activation pathway differs from Ras and between different Rabs. Analysis of DENND1-Rab35, Rabex-Rab5, TRAPP-Rab1 and DrrA-Rab1 suggests Rabs have the potential for activation by distinct GDP-release pathways. Conserved active-site residues in the Rab switch II region stabilising the nucleotide-free form differentiate these pathways. For DENND1-Rab35 and DrrA-Rab1 the Rab active-site glutamine, often mutated to create constitutively active forms, is involved in GEF mediated GDP-release. By contrast, in Rab5 the switch II aspartate is required for Rabex mediated GDP-release. Furthermore, Rab1 switch II glutamine mutants refractory to activation by DrrA can be activated by TRAPP, showing that a single Rab can be activated by more than one mechanistically distinct GDP-release pathway. These findings highlight plasticity in the activation mechanisms of closely related Rab GTPases. DOI: http://dx.doi.org/10.7554/eLife.01623.001.

Insights into the GTP/GDP Cycle of RabX3, a Novel GTPase from Entamoeba histolytica with Tandem G-Domains

Members of the small GTPase Ras superfamily regulate a host of systems through their ability to catalyze the GTP/GDP cycle. All family members reported thus far possess a single GTPase domain with a P-loop containing a nucleoside triphosphate hydrolase fold. Here for the first time we report a novel member from Entamoeba histolytica, EhRabX3, which harbors two GTPase domains in tandem and exhibits unique biochemical properties. A combination of biochemical and microcalorimetric studies revealed that EhRabX3 binds to a single guanine nucleotide through its N-terminal domain. Unlike most of the members of the Ras superfamily, the dissociation of the nucleotide from EhRabX3 is independent of Mg(2+), perhaps indicating a novel mechanism of nucleotide exchange by this protein. We found that EhRabX3 is extremely sluggish in hydrolyzing GTP, and that could be attributed to its atypical nucleotide binding pocket. It harbors substitutions at two positions that confer oncogenicity to Ras because of impaired GTP hydrolysis. Engineering these residues into the conserved counterparts enhanced their GTPase activity by at least 20-fold. In contrast to most of the members of the Ras superfamily, EhRabX3 lacks the prenylation motif. Using indirect immunofluorescence and biochemical fractionation, we demonstrated that the protein is distributed all over the cytosol in amoebic trophozoites. Collectively, this unique ancient GTPase exhibits a striking evolutionary divergence from the other members of the superfamily.

Down-Regulation of C9orf86 in Human Breast Cancer Cells Inhibits Cell Proliferation, Invasion and Tumor Growth and Correlates with Survival of Breast Cancer Patients

C9orf86 which is a novel subfamily within the Ras superfamily of GTPases, is overexpressed in the majority of primary breast tumors. Few functional studies have focused on the C9orf86 protein; therefore, in this study, we explored the role of C9orf86 in breast carcinogenesis. In our study, we found that silencing of C9orf86 by siRNA in MCF-7 and SK-BR-3 cells resulted in suppressed cell proliferation as well as in vitro cell invasion capabilities. Moreover, knockdown of C9orf86 inhibited tumor growth in nude mice. Cell cycle and apoptotic assays showed that the anti-proliferative effect of C9orf86-siRNA was mediated by arresting cells in the G1 phase and promoting apoptosis. In addition, we found that patients with high levels of C9orf86 expression showed a significant trend towards worse survival compared to patients with low C9orf86 expression (P = 0.002). These results provide evidence that C9orf86 represents a novel and clinically useful biomarker for BC patients and plays an important role during the progression of BC.

Imprinted Chromatin around DIRAS3 Regulates Alternative Splicing of GNG12-AS1, a Long Noncoding RNA

Imprinted gene clusters are regulated by long noncoding RNAs (lncRNAs), CCCTC binding factor (CTCF)-mediated boundaries, and DNA methylation. DIRAS3 (also known as ARH1 or NOEY1) is an imprinted gene encoding a protein belonging to the RAS superfamily of GTPases and is located within an intron of a lncRNA called GNG12-AS1. In this study, we investigated whether GNG12-AS1 is imprinted and coregulated with DIRAS3. We report that GNG12-AS1 is coexpressed with DIRAS3 in several tissues and coordinately downregulated with DIRAS3 in breast cancers. GNG12-AS1 has several splice variants, all of which initiate from a single transcription start site. In placenta tissue and normal cell lines, GNG12-AS1 is biallelically expressed but some isoforms are allele-specifically spliced. Cohesin plays a role in allele-specific splicing of GNG12-AS1. In breast cancer cell lines with loss of DIRAS3 imprinting, DIRAS3 and GNG12-AS1 are silenced in cis and the remaining GNG12-AS1 transcripts are predominantly monoallelic. The GNG12-AS1 locus, which includes DIRAS3, provides an example of imprinted cotranscriptional splicing and a potential model system for studying the long-range effects of CTCF-cohesin binding on splicing and transcriptional interference.

Abstract 2900: Phylogenetic classification, structural evolution, and functional divergence of the Ras superfamily of GTPases as tracked by the Conserved Domain Database (CDD)..

Abstract The Conserved Domain Database (CDD, http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) is part of NCBI's Entrez query and retrieval system and is a collection of annotated multiple sequence alignment (MSA) models representing ancient conserved protein domains, basic units of protein function and protein evolution.These MSA models are available as position specific score matrices (PSSMs) for fast identification of conserved domains in protein sequences via Reverse Position Specific -BLAST (RPS-BLAST), a variation of the commonly used PSI-BLAST. The domain models can be viewed and examined interactively using NCBI's CDTree/Cn3D software applications. Conserved functional annotations such as key catalytic and ligand-binding sites are displayed on protein structures sharing significant homology to the query, thereby inferring the underlying mechanism(s) of the protein domain family. Here, we present the current, functionally annotated, evolutionary classification of the Ras GTPase superfamily, including the circularly permuted Ras-like domain, and its downstream effectors such as RasGAP and RasGEF. Using CDTree/Cn3D tools, subfamilies in diverse organisms were inferred from the MSAs, phylogenetic trees, and domain architectures of the Ras superfamily. Furthermore, several subfamilies comprising of only uncharacterized protein sequences have their functions annotated by inference from the molecular and cellular functions of known related proteins having 3D structure, and based on the available literature. We demonstrate the utility of CDTree/Cn3D in studying molecular evolution of protein domain families, and in accelerating the pace of biological discovery by enabling researchers to obtain valuable clues regarding as-yet-unidentified molecular properties and mechanisms. CDTree/Cn3D software is freely available for download via the CDD website. Citation Format: James S. Song, Narmada Thanki, Myra K. Derbyshire, Marc Gwadz, Noreen R. Gonzales, Aron Marchler-Bauer, Stephen H. Bryant. Phylogenetic classification, structural evolution, and functional divergence of the Ras superfamily of GTPases as tracked by the Conserved Domain Database (CDD). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2900. doi:10.1158/1538-7445.AM2013-2900

Roles of Rho GTPases in Intracellular Transport and Cellular Transformation

Rho family GTPases belong to the Ras GTPase superfamily and transduce intracellular signals known to regulate a variety of cellular processes, including cell polarity, morphogenesis, migration, apoptosis, vesicle trafficking, viral transport and cellular transformation. The three best-characterized Rho family members are Cdc42, RhoA and Rac1. Cdc42 regulates endocytosis, the transport between the endoplasmic reticulum and Golgi apparatus, post-Golgi transport and exocytosis. Cdc42 influences trafficking through interaction with Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, leading to changes in actin dynamics. Rac1 mediates endocytic and exocytic vesicle trafficking by interaction with its effectors, PI3kinase, synaptojanin 2, IQGAP1 and phospholipase D1. RhoA participates in the regulation of endocytosis through controlling its downstream target, Rho kinase. Interestingly, these GTPases play important roles at different stages of viral protein and genome transport in infected host cells. Importantly, dysregulation of Cdc42, Rac1 and RhoA leads to numerous disorders, including malignant transformation. In some cases, hyperactivation of Rho GTPases is required for cellular transformation. In this article, we review a number of findings related to Rho GTPase function in intracellular transport and cellular transformation.

Membrane-Dependent Modulation of the mTOR Activator Rheb: NMR Observations of a GTPase Tethered to a Lipid-Bilayer Nanodisc

Like most Ras superfamily proteins, the GTPase domain of Ras homologue enriched in brain (Rheb) is tethered to cellular membranes through a prenylated cysteine in a flexible C-terminal region; however, little is known about how Rheb or other GTPases interact with the membrane or how this environment may affect their GTPase functions. We used NMR methods to characterize Rheb tethered to nanodiscs, monodisperse protein-encapsulated lipid bilayers with a diameter of 10 nm. Membrane conjugation markedly reduced the rate of intrinsic nucleotide exchange, while GTP hydrolysis was unchanged. NMR measurements revealed that the GTPase domain interacts transiently with the surface of the bilayer in two distinct preferred orientations, which are determined by the bound nucleotide. We propose models of membrane-dependent signal regulation by Rheb that shed light on previously unexplained in vivo properties of this GTPase. The study presented provides a general approach for direct experimental investigation of membrane-dependent properties of other Ras-superfamily GTPases.