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1.  Evolution and Diversity of the Ras Superfamily of Small GTPases in Prokaryotes 
Genome Biology and Evolution  2014;7(1):57-70.
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.
PMCID: PMC4316618  PMID: 25480683
small GTPase; evolution; GTPase-activating protein; Ras; MglA; signal transduction
2.  Computational Model Explains High Activity and Rapid Cycling of Rho GTPases within Protein Complexes 
PLoS Computational Biology  2006;2(12):e172.
Formation of multiprotein complexes on cellular membranes is critically dependent on the cyclic activation of small GTPases. FRAP-based analyses demonstrate that within protein complexes, some small GTPases cycle nearly three orders of magnitude faster than they would spontaneously cycle in vitro. At the same time, experiments report concomitant excess of the activated, GTP-bound form of GTPases over their inactive form. Intuitively, high activity and rapid turnover are contradictory requirements. How the cells manage to maximize both remains poorly understood. Here, using GTPases of the Rab and Rho families as a prototype, we introduce a computational model of the GTPase cycle. We quantitatively investigate several plausible layouts of the cycling control module that consist of GEFs, GAPs, and GTPase effectors. We explain the existing experimental data and predict how the cycling of GTPases is controlled by the regulatory proteins in vivo. Our model explains distinct and separable roles that the activating GEFs and deactivating GAPs play in the GTPase cycling control. While the activity of GTPase is mainly defined by GEF, the turnover rate is a sole function of GAP. Maximization of the GTPase activity and turnover rate places conflicting requirements on the concentration of GAP. Therefore, to achieve a high activity and turnover rate at once, cells must carefully maintain concentrations of GEFs and GAPs within the optimal range. The values of these optimal concentrations indicate that efficient cycling can be achieved only within dense protein complexes typically assembled on the membrane surfaces. We show that the concentration requirement for GEF can be dramatically reduced by a GEF-activating GTPase effector that can also significantly boost the cycling efficiency. Interestingly, we find that the cycling regimes are only weakly dependent on the concentration of GTPase itself.
A large variety of cellular processes, such as the formation of filopodia or transport vesicles, require that large protein complexes are precisely positioned on intracellular membranes to execute a specific task and then are promptly disassembled to perform their function elsewhere. Small GTPases play a major role in the spatiotemporal control of these complexes. Their function is based on the unique property of cycling between the active GTP-bound state, in which they enable complex formation, and the inactive GDP-bound state, which promotes complex dissolution. Recent experiments based on fluorescence recovery after photobleaching have found that some small GTPases rapidly cycle within protein complexes, causing continuous release and recruitment of the complex components. The seemingly futile cycling is accompanied by a large excess of the active form. This puzzling behavior challenges one's intuition and calls for the application of quantitative methods. Here, Goryachev and Pokhilko use computational modeling to identify regulatory mechanisms that could enable GTPases to cycle with the experimentally observed frequency and efficiency. They show that to achieve high activity and turnover simultaneously, the concentrations of the regulatory molecules that control GTPase cycling should be tightly maintained within the optimal range.
PMCID: PMC1676031  PMID: 17140284
3.  IQGAP1 is a key node within the small GTPase network 
Small GTPases  2013;4(4):199-207.
Coordination of the activity of multiple small GTPases is required for the regulation of many physiological processes, including cell migration. There are now several examples of functional interplay between small GTPase pairs, but the mechanisms that control GTPase activity in time and space are only partially understood. Here, we build on the hypothesis that small GTPases are part of a large, integrated network and propose that key proteins within this network integrate multiple signaling events and coordinate multiple small GTPase activities. Specifically, we identify the scaffolding protein IQGAP1 as a master regulator of multiple small GTPases, including Cdc42, Rac1, Rap1, and RhoA. In addition, we demonstrate that IQGAP1 promotes Arf6 activation downstream of β1 integrin engagement. Furthermore, following literature-curated searches and recent mass spectrometric analysis of IQGAP1-binding partners, we report that IQGAP1 recruits other small GTPases, including RhoC, Rac2, M-Ras, RhoQ, Rab10, and Rab5, small GTPase regulators, including Tiam1, RacGAP1, srGAP2 and HERC1, and small GTPase effectors, including PAK6, N-WASP, several sub-units of the Arp2/3 complex and the formin mDia1. Therefore, we propose that IQGAP1 acts as a small GTPase scaffolding platform within the small GTPase network, and recruits and/or regulates small GTPases, small GTPase regulators and effectors to orchestrate cell behavior. Finally, to identify other putative key regulators of small GTPase crosstalk, we have assembled a small GTPase network using protein-protein interaction databases.
PMCID: PMC4011815  PMID: 24355937
small GTPase crosstalk; IQGAP1; Ras GTPases; small GTPase network; signal transduction
4.  Bayesian classification of residues associated with protein functional divergence: Arf and Arf-like GTPases 
Biology Direct  2010;5:66.
Certain residues within proteins are highly conserved across very distantly related organisms, yet their (presumably critical) structural or mechanistic roles are completely unknown. To obtain clues regarding such residues within Arf and Arf-like (Arf/Arl) GTPases--which function as on/off switches regulating vesicle trafficking, phospholipid metabolism and cytoskeletal remodeling--I apply a new sampling procedure for comparative sequence analysis, termed multiple category Bayesian Partitioning with Pattern Selection (mcBPPS).
The mcBPPS sampler classified sequences within the entire P-loop GTPase class into multiple categories by identifying those evolutionarily-divergent residues most likely to be responsible for functional specialization. Here I focus on categories of residues that most distinguish various Arf/Arl GTPases from other GTPases. This identified residues whose specific roles have been previously proposed (and in some cases corroborated experimentally and that thus serve as positive controls), as well as several categories of co-conserved residues whose possible roles are first hinted at here. For example, Arf/Arl/Sar GTPases are most distinguished from other GTPases by a conserved aspartate residue within the phosphate binding loop (P-loop) and by co-conserved residues nearby that, together, can form a network of salt-bridge and hydrogen bond interactions centered on the GTPase active site. Residues corresponding to an N-[VI] motif that is conserved within Arf/Arl GTPases may play a role in the interswitch toggle characteristic of the Arf family, whereas other, co-conserved residues may modulate the flexibility of the guanine binding loop. Arl8 GTPases conserve residues that strikingly diverge from those typically found in other Arf/Arl GTPases and that form structural interactions suggestive of a novel interswitch toggle mechanism.
This analysis suggests specific mutagenesis experiments to explore mechanisms underlying GTP hydrolysis, nucleotide exchange and interswitch toggling within Arf/Arl GTPases. More generally, it illustrates how the mcBPPS sampler can complement traditional evolutionary analyses by providing an objective, quantitative and statistically rigorous way to explore protein functional-divergence in molecular detail. Because the sampler classifies the input sequences at the same time, it can be used to generate subgroup profiles, in which functionally-divergent categories of residues are annotated automatically.
This article was reviewed by Frank Eisenhaber, L Aravind and Daniel Gaston (nominated by Eric Bapteste). For the full reviews, go to the Reviewers' comments section.
PMCID: PMC3012027  PMID: 21129209
5.  Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy 
PLoS Biology  2009;7(10):e1000212.
The authors of this research article demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle, and show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein. These movements are of crucial importance for understanding the mechanistic principles of this GAD.
MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlFx structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 Å. With GDP and AlFx, which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlFx requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.
Author Summary
MnmE is an evolutionary conserved G protein that is involved in modification of the wobble U position of certain tRNAs to suppress translational wobbling. Despite high homology between its G domain and the small G protein Ras, MnmE displays entirely different regulatory properties to that of many molecular switch-type G proteins of the Ras superfamily, as its GTPase is activated by nucleotide-dependent homodimerization across the nucleotide-binding site. Here we explore the unusual G domain cycle of the MnmE protein by combining X-ray crystallography with pulse electron paramagnetic resonance (EPR) spectroscopy, which enables distance determinations between spin markers introduced at specific sites within the G domain. We determined the structures of the full-length MnmE dimer in the diphosphate and triphosphate states, which represent distinct steps of the G domain cycle, and demonstrate that the G domain cycle of MnmE comprises large conformational changes and domain movements of up to 18 Å, in which the G domains of the dimeric protein traverse from a GDP-bound open state through an open/closed equilibrium in the triphosphate state to a closed conformation in the transition state, so as to assemble the catalytic machinery.
PMCID: PMC2749940  PMID: 19806182
6.  Inhibition of Rho and Rac Geranylgeranylation by Atorvastatin Is Critical for Preservation of Endothelial Junction Integrity 
PLoS ONE  2013;8(3):e59233.
Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.
Methods and Results
Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.
In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.
PMCID: PMC3596292  PMID: 23555637
7.  Topological and Functional Properties of the Small GTPases Protein Interaction Network 
PLoS ONE  2012;7(9):e44882.
Small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. A great deal of experimental evidence supports the existence of signaling cascades and feedback loops within and among the small GTPase subfamilies suggesting that these proteins function in a coordinated and cooperative manner. The interplay occurs largely through association with bi-partite regulatory and effector proteins but can also occur through the active form of the small GTPases themselves. In order to understand the connectivity of the small GTPases signaling routes, a systems-level approach that analyzes data describing direct and indirect interactions was used to construct the small GTPases protein interaction network. The data were curated from the Search Tool for the Retrieval of Interacting Genes (STRING) database and include only experimentally validated interactions. The network method enables the conceptualization of the overall structure as well as the underlying organization of the protein-protein interactions. The interaction network described here is comprised of 778 nodes and 1943 edges and has a scale-free topology. Rac1, Cdc42, RhoA, and HRas are identified as the hubs. Ten sub-network motifs are also identified in this study with themes in apoptosis, cell growth/proliferation, vesicle traffic, cell adhesion/junction dynamics, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase response, transcription regulation, receptor-mediated endocytosis, gene silencing, and growth factor signaling. Bottleneck proteins that bridge signaling paths and proteins that overlap in multiple small GTPase networks are described along with the functional annotation of all proteins in the network.
PMCID: PMC3441499  PMID: 23028658
8.  Locking GTPases covalently in their functional states 
Nature Communications  2015;6:7773.
GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase–acryl–nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.
The cellular function of small GTPases is regulated by switching between active (GTP-bound) and inactive (GDP-bound) states. Here the authors develop nucleotide analogues that can be covalently linked to GTPases via a strategically placed cysteine residue to lock the target GTPase in defined activation states.
PMCID: PMC4518245  PMID: 26178622
9.  Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation 
Nucleic Acids Research  2013;41(5):3217-3227.
Ribosome biogenesis GTPase A protein (RbgA) is an essential GTPase required for the biogenesis of the 50S subunit in Bacillus subtilis. Homologs of RbgA are widely distributed in bacteria and eukaryotes and are implicated in ribosome assembly in the mitochondria, chloroplast and cytoplasm. Cells depleted of RbgA accumulate an immature large subunit that is missing key ribosomal proteins. RbgA, unlike many members of the Ras superfamily of GTPases, lacks a defined catalytic residue for carrying out guanosine triphosphate (GTP) hydrolysis. To probe RbgA function in ribosome assembly, we used a combined bioinformatics, genetic and biochemical approach. We identified a RNA-binding domain within the C-terminus of RbgA that is structurally similar to AmiR–NasR Transcription Anti-termination Regulator (ANTAR) domains, which are known to bind structured RNA. Mutation of key residues in the ANTAR domain altered RbgA association with the ribosome. We identified a putative catalytic residue within a highly conserved region of RbgA, His9, which is contained within a similar PGH motif found in elongation factor Tu (EF-Tu) that is required for GTP hydrolysis on interaction with the ribosome. Finally, our results support a model in which the GTPase activity of RbgA directly participates in the maturation of the large subunit rather than solely promoting dissociation of RbgA from the 50S subunit.
PMCID: PMC3597669  PMID: 23325847
10.  Computational Analysis of Rho GTPase Cycling 
PLoS Computational Biology  2013;9(1):e1002831.
The Rho family of GTPases control actin organization during diverse cellular responses (migration, cytokinesis and endocytosis). Although the primary members of this family (RhoA, Rac and Cdc42) have different downstream effects on actin remodeling, the basic mechanism involves targeting to the plasma membrane and activation by GTP binding. Our hypothesis is that the details of GTPase cycling between membrane and cytosol are key to the differential upstream regulation of these biochemical switches. Accordingly, we developed a modeling framework to analyze experimental data for these systems. This analysis can reveal details of GDI-mediated cycling and help distinguish between GDI-dependent and -independent mechanisms, including vesicle trafficking and direct association-dissociation of GTPase with membrane molecules. Analysis of experimental data for Rac membrane cycling reveals that the lower apparent affinity of GDI for RacGTP compared to RacGDP can be fully explained by the faster dissociation of the latter from the membrane. Non-dimensional steady-state solutions for membrane fraction of GTPase are presented in multidimensional charts. This methodology is then used to analyze glucose stimulated Rac cycling in pancreatic β-cells. The charts are used to illustrate the effects of GEFs/GAPs and regulated affinities between GTPases and membrane and/or GDI on the amount of membrane bound GTPase. In a similar fashion, the charts can be used as a guide in assessing how targeted modifications may compensate for altered GTPase-GDI balance in disease scenarios.
Author Summary
Among the functions of the small GTPases Rac, RhoA and Cdc42 are the regulation of protein traffic, insulin secretion, cell shape, survival and motility. The last two are important steps for tumor growth and metastasis. The function of these proteins relies on their expression levels, proper membrane localization and activation. In addition, all three proteins compete for the same protein ‘GDI’, which modulates their cycling. These proteins are ubiquitous in mammalian cells, but also studied in simpler in vitro systems and cultured yeast. Here we show, using a series of computational analyses, that for each of these experimental systems the dominant pathway for membrane cycling of GTPases seems to differ. This means that the researcher interested in the physiological function of any of those proteins must make sure that the experimental system is appropriate. We present a methodology to identify the dominant pathways by measuring the apparent membrane dissociation rate of the protein as a function of GDI concentration. We provide charts generated from parametric scans. This analysis is then applied to the Rac-dependent insulin secretion pathway in pancreatic ß-cells, revealing that direct signaling between Rac and the membrane is an essential mechanism that emerges from the data.
PMCID: PMC3542069  PMID: 23326220
11.  A Homogenous Bioluminescent System for Measuring GTPase, GTPase Activating Protein, and Guanine Nucleotide Exchange Factor Activities 
GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators has been challenging due to paucity of convenient assays. In this study, we describe a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Since Mg2+ plays a critical role in influencing the affinity of GTPases with guanosine triphosphate/guanosine diphosphate (GTP/GDP) and the process of nucleotide exchange, manipulating Mg2+ concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP that remains after the GTPase reaction to ATP and detection of the generated ATP using the luciferin/luciferase combination. The GTPase/GAP/GEF-Glo assay system enables monitoring of GTPase, GAP-stimulated GTPase, GAP, and GEF activities. The system can also be used to analyze these proteins when expressed in cells as fusion proteins by performing the assay in a pulldown format. The assays showed minimal false hits upon testing for compound interference using the library of pharmacologically active compounds and its robustness was demonstrated by a high Z′-factor of 0.93 and CV of 2.2%. The assay system has a high dynamic range, formatted in a convenient add–mix–read, and applicable to high-throughput screening.
PMCID: PMC4605356  PMID: 26167953
12.  Positive regulation of Rho GTPase activity by RhoGDIs as a result of their direct interaction with GAPs 
Rho GTPases function as molecular switches in many different signaling pathways and control a wide range of cellular processes. Rho GDP-dissociation inhibitors (RhoGDIs) regulate Rho GTPase signaling and can function as both negative and positive regulators. The role of RhoGDIs as negative regulators of Rho GTPase signaling has been extensively investigated; however, little is known about how RhoGDIs act as positive regulators. Furthermore, it is unclear how this opposing role of GDIs influences the Rho GTPase cycle. We constructed ordinary differential equation models of the Rho GTPase cycle in which RhoGDIs inhibit the regulatory activities of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) by interacting with them directly as well as by sequestering the Rho GTPases. Using this model, we analyzed the role of RhoGDIs in Rho GTPase signaling.
The model constructed in this study showed that the functions of GEFs and GAPs are integrated into Rho GTPase signaling through the interactions of these regulators with GDIs, and that the negative role of GDIs is to suppress the overall Rho activity by inhibiting GEFs. Furthermore, the positive role of GDIs is to sustain Rho activation by inhibiting GAPs under certain conditions. The interconversion between transient and sustained Rho activation occurs mainly through changes in the affinities of GDIs to GAPs and the concentrations of GAPs.
RhoGDIs positively regulate Rho GTPase signaling primarily by interacting with GAPs and may participate in the switching between transient and sustained signals of the Rho GTPases. These findings enhance our understanding of the physiological roles of RhoGDIs and Rho GTPase signaling.
Electronic supplementary material
The online version of this article (doi:10.1186/s12918-015-0143-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4312443  PMID: 25628036
RhoGDI; Rho GTPases; Ordinary differential equation; GAPs; GEFs
13.  Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases 
Biochimica et biophysica acta  2009;1791(9):850-855.
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.
PMCID: PMC2739575  PMID: 19540930
Phospholipids; Phosphatidic acid; Phospholipase D; Diacylglycerol kinase; Ras; Small GTPases
14.  ROP GTPase-Dependent Actin Microfilaments Promote PIN1 Polarization by Localized Inhibition of Clathrin-Dependent Endocytosis 
PLoS Biology  2012;10(4):e1001299.
A study in leaf epidermal pavement cells reveals that auxin activation of a Rho-like GTPase from plants induces inhibition of endocytosis through the clathrin-mediated pathway by regulating the accumulation of cortical F-actin.
Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis.
Author Summary
Formation of cell polarity is a process of distributing cellular structures or molecules in an asymmetric manner. This process plays an important role in the generation of diverse cell forms and types. In plants, the quintessential hormone auxin is important for diverse physiological functions, including growth and development of cells and organs. To perform these functions, auxin must be transported and localized to specific regions within the plant. This is partially mediated by polar distribution of the PIN-FORMED (PIN) auxin efflux transporters, which transport auxin outside of the cell and allow for the directional short- and long-distance transport of auxin throughout plant tissues and organs. Although auxin itself has been implicated as a signal to regulate PIN polar distribution, how auxin does so remains to be elucidated. We previously showed that auxin promotes the generation of “puzzle-piece” polarity in leaf epidermal pavement cells, which contain interdigitated lobes and indentations, by activating the ROP (Rho-like GTPases from plants) members of the conserved Rho family of small GTPases. Here, we find that auxin-dependent local activation of ROP2 in the lobe region inhibits PIN1 internalization into the endosomal compartments (or endocytosis), leaving higher levels of PIN1 polar distribution in the lobe region. PIN1 internalization is inhibited by altering the actin cytoskeleton through the ROP2 effector protein RIC4, a protein involved in cytoskeletal remodeling. On the basis of our findings, we propose that the Rho GTPase-mediated inhibition of endocytosis of PIN1 provides a self-organizing mechanism for the polar PIN1 distribution. Rho GTPase-based inhibition of endocytosis is also important for the formation of cell polarity in animal cells. Thus, we conclude that Rho GTPase signaling to inhibit endocytosis is a common mechanism for cell polarization in multicellular organisms.
PMCID: PMC3317906  PMID: 22509133
15.  Nf1 RasGAP Inhibition of LIMK2 Mediates a New Cross-Talk between Ras and Rho Pathways 
PLoS ONE  2012;7(10):e47283.
Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways.
Methodology/Principal Findings
In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity: the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition: in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator.
Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases.
PMCID: PMC3474823  PMID: 23082153
16.  Signaling Through Rho GTPase Pathway as Viable Drug Target 
Current medicinal chemistry  2009;16(11):1355-1365.
Signaling through the Rho family of small GTPases has been increasingly investigated for their involvement in a wide variety of diseases such as cardiovascular, pulmonary, and neurological disorders as well as cancer. Rho GTPases are a subfamily of the Ras superfamily proteins which play essential roles in a number of biological processes, especially in the regulation of cell shape change, cytokinesis, cell adhesion, and cell migration. Many of these processes demonstrate a common theme: the rapid and dynamic reorganization of actin cytoskeleton of which Rho signaling has now emerged as a major switch control. The involvement of dynamic changes of Rho GTPases in disease states underscores the need to produce effective inhibitors for their therapeutic applications. Fasudil and Y-27632, with many newer additions, are two classes of widely used chemical compounds that inhibit Rho kinase (ROCK), an important downstream effector of RhoA subfamily GTPases. These inhibitors have been successful in many preclinical studies, indicating the potential benefit of clinical Rho pathway inhibition. On the other hand, except for Rac1 inhibitor NSC23766, there are few effective inhibitors directly targeting Rho GTPases, likely due to the lack of optimal structural information on individual Rho-RhoGEF, Rho-RhoGAP, or Rho-RhoGDI interaction to achieve specificity. Recently, LM11A-31 and other derivatives of peptide mimetic ligands for p75 neutrotrophoin receptor (p75NTR) show promising effects upstream of Rho GTPase signaling in neuronal regeneration. CCG-1423, a chemical compound showing profiles of inhibiting downstream of RhoA, is a further attempt for the development of novel pharmacological tools to disrupt Rho signaling pathway in cancer. Because of a rapidly growing number of studies deciphering the role of the Rho proteins in many diseases, specific and potent pharmaceutical modulators of various steps of Rho GTPase signaling pathway are critically needed to target for therapeutic intervention in cardiovascular disease, neurological disorders, and cancer progression.
PMCID: PMC3829470  PMID: 19355891
Rho GTPase; ROCK; Y-27632; fasudil; LM11A-31; NSC23766; CCG-1423
17.  Hematopoietic-specific Rho GTPases Rac2 and RhoH and human blood disorders 
Experimental cell research  2013;319(15):2375-2383.
The small guanosine triphosphotases (GTPases) Rho proteins are members of the Ras-like superfamily. Similar to Ras, most Rho GTPases cycle between active GTP-bound, and inactive GDP-bound conformations and act as molecular switches that control multiple cellular functions. While most Rho GTPases are expressed widely, the expression of Rac2 and RhoH are restricted to hematopoietic cells. RhoH is an atypical GTPase that lacks GTPase activity and remains in the active conformation. The generation of mouse knock-out lines has led to new understanding of the functions of both of these proteins in blood cells. The phenotype of these mice also led to the identification of mutations in human RAC2 and RHOH genes and the role of these proteins in immunodeficiency diseases. This review outlines the basic biology of Rho GTPases, focusing on Rac and RhoH and summarizes human diseases associated with mutations of these genes.
PMCID: PMC3997055  PMID: 23850828
Rho GTPases; Rac; RhoH; cytoskeleton; hematopoiesis
18.  Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner 
Molecular and Cellular Biology  1998;18(12):7444-7454.
The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.
PMCID: PMC109325  PMID: 9819430
19.  Novel Split-Luciferase-Based Genetically Encoded Biosensors for Noninvasive Visualization of Rho GTPases 
PLoS ONE  2013;8(4):e62230.
Rho family GTPases are critical regulators of many important cellular processes and the dysregulation of their activities is implicated in a variety of human diseases including oncogenesis and propagation of malignancy. The traditional methods, such as “pull-down” or two-hybrid procedures, are poorly suited to dynamically evaluate the activity of Rho GTPases, especially in living mammalian cells. To provide a novel alternative approach to analyzing Rho GTPase-associated signaling pathways in vivo, we developed a series of bioluminescent biosensors based on the genetically engineered firefly luciferase. These split-luciferase-based biosensors enable non-invasive visualization and quantification of the activity of Rho GTPases in living subjects. The strategy is to reasonably split the gene of firefly luciferase protein into two inactive fragments and then respectively fuse the two fragments to Rho GTPase and the GTPase-binding domain (GBD) of the specific effector. Upon Rho GTPase interacting with the binding domain in a GTP-dependent manner, these two luciferase fragments are brought into close proximity, leading to luciferase reconstitution and photon production in the presence of the substrate. Using these bimolecular luminescence complementation (BiLC) biosensors, we successfully visualized and quantified the activities of the three best characterized Rho GTPases by measuring the luminescence in living cells. We also experimentally investigated the sensitivity of these Rho GTPase biosensors to upstream regulatory proteins and extracellular ligands without lysing cells and doing labor-intensive works. By virtue of the unique functional characteristics of bioluminescence imaging, the BiLC-based biosensors provide an enormous potential for in vivo imaging of Rho GTPase signaling pathways and high-throughput screening of therapeutic drugs targeted to Rho GTPases and (or) upstream molecules in the near future.
PMCID: PMC3627919  PMID: 23614039
20.  Regulating Rac in the Nervous System: Molecular Function and Disease Implication of Rac GEFs and GAPs 
BioMed Research International  2015;2015:632450.
Rho family GTPases, including RhoA, Rac1, and Cdc42 as the most studied members, are master regulators of actin cytoskeletal organization. Rho GTPases control various aspects of the nervous system and are associated with a number of neuropsychiatric and neurodegenerative diseases. The activity of Rho GTPases is controlled by two families of regulators, guanine nucleotide exchange factors (GEFs) as the activators and GTPase-activating proteins (GAPs) as the inhibitors. Through coordinated regulation by GEFs and GAPs, Rho GTPases act as converging signaling molecules that convey different upstream signals in the nervous system. So far, more than 70 members of either GEFs or GAPs of Rho GTPases have been identified in mammals, but only a small subset of them have well-known functions. Thus, characterization of important GEFs and GAPs in the nervous system is crucial for the understanding of spatiotemporal dynamics of Rho GTPase activity in different neuronal functions. In this review, we summarize the current understanding of GEFs and GAPs for Rac1, with emphasis on the molecular function and disease implication of these regulators in the nervous system.
PMCID: PMC4388020  PMID: 25879033
21.  Activation of PAK by a bacterial type III effector EspG reveals alternative mechanisms of GTPase pathway regulation 
Small GTPases  2011;2(4):217-221.
Small Rho GTPases regulate a diverse range of cellular behavior within a cell. Their ability to function as molecular switches in response to a bound nucleotide state allows them to regulate multiple dynamic processes, including cytoskeleton organization and cellular adhesion. Because the activation of downstream Rho GTPase signaling pathways relies on conserved structural features of target effector proteins (i.e., CRIB domain), these pathways are particularly vulnerable to microbial pathogenic attack. Here, we discuss new findings for how the bacterial virulence factor EspG from EHEC O157:H7 exploits a CRIB-independent activation mechanism of the Rho GTPase effector PAK. We also compare this mechanism to that of EHEC EspFU, a bacterial virulence factor that directly activates N-WASP. While both virulence factors break the inhibitory interaction between the autoinhibitory and activity-bearing domains of PAK or WASP, the underlying mechanics are very distinct from endogenous Cdc42/Rac GTPase regulation. The ability of bacterial proteins to identify novel regulatory principles of host signaling enzymes highlights the multi-level nature of protein activation, and makes them effective tools to study mammalian Rho GTPase signaling pathways.
PMCID: PMC3225911  PMID: 22145094
EPEC; EHEC; EspG; EspFU; PAK; type III effector; WASP; N-WASP; toxin
22.  Developmental expression of three small GTPases in the mouse eye 
Molecular Vision  2007;13:1144-1153.
The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
PMCID: PMC2779149  PMID: 17653061
23.  GAPs 
Cellular Logistics  2011;1(2):49-51.
Whether your passion is to understand and reverse disease processes or “simply” a better understanding of how cells work, anyone wishing to understand cell regulation today must have a detailed and accurate understanding of regulatory GTPase mechanisms and their application to specific pathways. This is becoming increasingly difficult as the details of signaling by members of different families of GTPases and their regulators expand. But this is all the more reason to continually ask, which aspects of GTPase signaling are distinct to a GTPase or its subfamily and which are conserved throughout the superfamily? We each have slightly different views of the key aspects of GTPase signaling that are derived from the main GTPases studied in our own labs; e.g., translocation onto a membrane is an essential and integral aspect of Arf activation but not of other GTPases. However, one aspect of GTPase signaling that I had come to believe to be widespread and of general importance is not universally accepted. In fact, through my conversations at the recent FASEB summer research conference on “Arf Family GTPases” and reading of the literature in a graduate tutorial class, I realized that it is not known or accepted by the majority of researchers. The question is the role of GTPase activating proteins (GAPs) in signaling. Are they “pure” terminators of signaling or do they serve effector functions?
PMCID: PMC3116585  PMID: 21686252
Arf; GAP; Arf GAP; RGS; ArfGAP1
24.  A RAC/CDC-42–Independent GIT/PIX/PAK Signaling Pathway Mediates Cell Migration in C. elegans 
PLoS Genetics  2008;4(11):e1000269.
P21 activated kinase (PAK), PAK interacting exchange factor (PIX), and G protein coupled receptor kinase interactor (GIT) compose a highly conserved signaling module controlling cell migrations, immune system signaling, and the formation of the mammalian nervous system. Traditionally, this signaling module is thought to facilitate the function of RAC and CDC-42 GTPases by allowing for the recruitment of a GTPase effector (PAK), a GTPase activator (PIX), and a scaffolding protein (GIT) as a regulated signaling unit to specific subcellular locations. Instead, we report here that this signaling module functions independently of RAC/CDC-42 GTPases in vivo to control the cell shape and migration of the distal tip cells (DTCs) during morphogenesis of the Caenorhabditis elegans gonad. In addition, this RAC/CDC-42–independent PAK pathway functions in parallel to a classical GTPase/PAK pathway to control the guidance aspect of DTC migration. Among the C. elegans PAKs, only PAK-1 functions in the GIT/PIX/PAK pathway independently of RAC/CDC42 GTPases, while both PAK-1 and MAX-2 are redundantly utilized in the GTPase/PAK pathway. Both RAC/CDC42–dependent and –independent PAK pathways function with the integrin receptors, suggesting that signaling through integrins can control the morphology, movement, and guidance of DTC through discrete pathways. Collectively, our results define a new signaling capacity for the GIT/PIX/PAK module that is likely to be conserved in vertebrates and demonstrate that PAK family members, which are redundantly utilized as GTPase effectors, can act non-redundantly in pathways independent of these GTPases.
Author Summary
Cell migration is essential for the development and maintenance of metazoan tissue. A migrating cell must navigate through complex environments and properly interpret the signals present in its path. This cellular movement is accomplished through transduction of the signals into directed reorganization of the cellular structure. Among the most important molecules that orchestrate signals from the exterior of the cells into cellular movement are the small GTPases, which function in intracellular signal transduction cascades. We have studied the interactions between GTPases, their effectors, and the environmental signals during cellular migrations in C. elegans. We have found that while some GTPases do control the guidance of these migrating cells, a certain highly conserved complex of proteins thought to be involved in mediating GTPase signaling during cellular migrations in fact functions independently of these GTPases to specifically control the structure and movement of the migrating cells. These results have revealed an unexpected role of a well-known and highly conserved signaling complex, which is particularly important since members of this complex are associated with human mental retardation. Our results may imply that the disease phenotype is likely more complex than previously thought and may in fact occur from disruption of this novel pathway.
PMCID: PMC2581894  PMID: 19023419
25.  New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution 
In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes.
X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido)-triphosphate), a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom.
For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.
PMCID: PMC2987813  PMID: 20973973

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