Systematic functional analysis of the Ras GTPase family unveils a conserved network required for anterograde protein trafficking

Phylogeny is often used to compare entire families of genes/proteins. We previously showed that classification of Caenorhabditis elegans Rho GTPases on the basis of their enzymatic properties was significantly different from sequence alignments. To further develop this concept, we have developed an integrated approach to classify C. elegans small GTPases based on functional data comprising affinity for GTP, sub‐cellular localization, tissue distribution and silencing impact. This analysis led to establish a novel functional classification for small GTPases. To test the relevance of this classification in mammals, we focused our attention on the human orthologs of small GTPases from a specific group comprising arf‐1.2, evl‐20, arl‐1, Y54E10BR.2, unc‐108 and rab‐7. We then tested their involvement in protein secretion and membrane traffic in mammalian systems. Using this approach we identify a novel network containing 18 GTPases, and 23 functionally interacting proteins, conserved between C. elegans and mammals, which is involved in membrane traffic and protein secretion.     

Genome-wide analysis reveals the functional and expressional correlation between RhoGAP and RhoGEF in mouse

Rho GTPases play essential roles in various life activities. Rho GTPase-activating protein (RhoGAP) and Rho guanine nucleotide exchange factor (RhoGEF) are the main regulators of Rho GTPases. RhoGAP, RhoGEF and Rho make up a molecular switch and exert crucial roles in signaling pathways. The genome-wide studies can provide us a comprehensive information of special protein family, but the genome-wide information of RhoGAP and RhoGEF families are still lacking in the mammal lineage. Here, we report the correlations between mouse RhoGAPs and RhoGEFs in gene quantities, evolution, molecular function, and their expression levels in heart embryonic development and cardiovascular medicine treatment at genome-wide scale. Besides, we find that the 3D structures of RhoGAP domains between different species are highly conserved, but that of RhoGEF domains are variable between species. Our present study contributes to a better understanding of the complex regulation mechanisms of RhoGAP and RhoGEF families.     

GEF what? Dock180 and related proteins help Rac to polarize cells in new ways

Rho GTPase activation, which is mediated by guanine nucleotide exchange factors (GEFs), is tightly regulated in time and space. Although Rho GTPases have a significant role in many biological events, they are best known for their ability to restructure the actin cytoskeleton profoundly through the activation of specific downstream effectors. Two distinct families of GEFs for Rho GTPases have been reported so far, based on the features of their catalytic domains: firstly, the classical GEFs, which contain a Dbl homology-pleckstrin homology domain module with GEF activity, and secondly, the Dock180-related GEFs, which contain a Dock homology region-2 domain that catalyzes guanine nucleotide exchange on Rho GTPases. Recent exciting data suggest key roles for the DHR-2 domain-containing GEFs in a wide variety of fundamentally important biological functions, including cell migration, phagocytosis of apoptotic cells, myoblast fusion and neuronal polarization.     

Novel Split-Luciferase-Based Genetically Encoded Biosensors for Noninvasive Visualization of Rho GTPases

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.     

The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences

Many small GTPases in the Ras and Rho families have a C-terminal polybasic region (PBR) comprised of multiple lysines or arginines. The PBR controls diverse functions of these small GTPases, including their ability to associate with membranes, interact with specific proteins, and localize in subcellular compartments. Different signaling pathways mediated by Ras and Rho family members may converge when the small GTPases are directed by their PBRs to shared binding sites in specific proteins or at cell membranes. The PBR promotes the interactions of small GTPases with SmgGDS, which is a nucleocytoplasmic shuttling protein that stimulates guanine nucleotide exchange by small GTPases. The PBR of Rac1 was recently found to have a functional nuclear localization signal (NLS) sequence, which enhances the nuclear accumulation of protein complexes containing SmgGDS and Rac1. Sequence analysis demonstrates that canonical NLS sequences (K-K/R-x-K/R) are present in the PBRs of additional Ras and Rho family members, and are evolutionarily conserved across several phyla. These findings suggest that the PBR regulates the nucleocytoplasmic shuttling of some Ras and Rho family members when they are in protein complexes that are too large to diffuse through nuclear pores. These diverse functions of the PBR indicate its critical role in signaling by Ras and Rho family GTPases.     

Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.     

The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3beta homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.     

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.     

Effect of Mg(2+) on the kinetics of guanine nucleotide binding and hydrolysis by Cdc42

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cell. Mg(2+) ions play key roles in guanine nucleotide binding and in preserving the structural integrity of GTPases. We describe here the kinetics of the interaction of GTP with the Rho family small GTPase Cdc42 in the absence and presence of Mg(2+). In contrast to the cases of Ras and Rab proteins, which require Mg(2+) for the nucleotide binding and intrinsic hydrolysis of GTP, our results show that in the absence of Mg(2+), the binding affinity of GTP to Cdc42 is in the submicromolar concentration, and the Mg(2+) cofactor has only a minor effect on the Cdc42-catalyzed intrinsic hydrolysis rate of GTP. These results suggest that the intrinsic GTPase reaction mechanism of Cdc42 may differ significantly from that of other subfamily members of the Ras superfamily.     

Rho GTPases and their regulators in neuronal functions and development

Neurons are specialized cell types which send out processes in order to communicate with other cells, which can be immediate neighbors or whose cell bodies are far distant. Neuronal morphology as in all cells is determined in large part through the regulation of the cytoskeleton. One of the key regulators of the actin cytoskeleton is the Rho family of GTPases. The Rho GTPases function as molecular switches to turn on or off downstream biochemical pathways depending on the stimuli. Their activities and their regulation are controlled by many other proteins such as the guanine nucleotide exchange factors and the GTPase-activating proteins. The activities of some of the Rho family members are reported to be antagonistic to one another. In general, Rac and Cdc42 promote neurite outgrowth while RhoA stimulates retraction. The balance of these opposing activities of the different Rho GTPases is crucial for the morphology and function of the neurons.     

Cooperative activation of PI3K by Ras and Rho family small GTPases

Phosphoinositide 3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of cell polarization, motility, and chemotaxis. They influence each other's activities by direct and indirect feedback processes that are only partially understood. Here, we show that 21 small GTPase homologs activate PI3K. Using a microscopy-based binding assay, we show that K-Ras, H-Ras, and five homologous Ras family small GTPases function upstream of PI3K by directly binding the PI3K catalytic subunit, p110. In contrast, several Rho family small GTPases activated PI3K by an indirect cooperative positive feedback that required a combination of Rac, CDC42, and RhoG small GTPase activities. Thus, a distributed network of Ras and Rho family small GTPases induces and reinforces PI3K activity, explaining past challenges to elucidate the specific relevance of different small GTPases in regulating PI3K and controlling cell polarization and chemotaxis.     

RHO GTPase Signaling for Axon Extension: Is Prenylation Important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.     

Protein–protein‐interactions in a multiplexed,miniaturized format a functional analysis of Rho GTPase activation and inhibition

A miniaturized, bead‐based protein–protein‐interaction assay was developed to study the interaction of Rho GTPases with regulatory proteins. The setup, which uses only minute amounts of sample, was used to analyze small molecules that inhibit the interaction between Rho GTPases and RhoGDIα. Prenylcysteine analogues and the replacement of GDP by non‐hydrolysable GTP analogues prevented the formation of Rho GTPase‐RhoGDIα complexes in a concentration‐dependent manner.     

Rho GAPs and GEFs

Within blood vessels, endothelial cell–cell and cell–matrix adhesions are crucial to preserve barrier function, and these adhesions are tightly controlled during vascular development, angiogenesis, and transendothelial migration of inflammatory cells. Endothelial cellular signaling that occurs via the family of Rho GTPases coordinates these cell adhesion structures through cytoskeletal remodelling. In turn, Rho GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). To understand how endothelial cells initiate changes in the activity of Rho GTPases, and thereby regulate cell adhesion, we will discuss the role of Rho GAPs and GEFs in vascular biology. Many potentially important Rho regulators have not been studied in detail in endothelial cells. We therefore will first overview which GAPs and GEFs are highly expressed in endothelium, based on comparative gene expression analysis of human endothelial cells compared with other tissue cell types. Subsequently, we discuss the relevance of Rho GAPs and GEFs for endothelial cell adhesion in vascular homeostasis and disease.     

Mutations in FGD4 encoding the Rho GDP/GTP exchange factor FRABIN cause autosomal recessive Charcot-Marie-Tooth type 4H

Charcot-Marie-Tooth (CMT) disorders are a clinically and genetically heterogeneous group of hereditary motor and sensory neuropathies characterized by muscle weakness and wasting, foot and hand deformities, and electrophysiological changes. The CMT4H subtype is an autosomal recessive demyelinating form of CMT that was recently mapped to a 15.8-Mb region at chromosome 12p11.21-q13.11, in two consanguineous families of Mediterranean origin, by homozygosity mapping. We report here the identification of mutations in FGD4, encoding FGD4 or FRABIN (FGD1-related F-actin binding protein), in both families. FRABIN is a GDP/GTP nucleotide exchange factor (GEF), specific to Cdc42, a member of the Rho family of small guanosine triphosphate (GTP)-binding proteins (Rho GTPases). Rho GTPases play a key role in regulating signal-transduction pathways in eukaryotes. In particular, they have a pivotal role in mediating actin cytoskeleton changes during cell migration, morphogenesis, polarization, and division. Consistent with these reported functions, expression of truncated FRABIN mutants in rat primary motoneurons and rat Schwann cells induced significantly fewer microspikes than expression of wild-type FRABIN. To our knowledge, this is the first report of mutations in a Rho GEF protein being involved in CMT.     

Structural basis for the selective activation of Rho GTPases by Dbl exchange factors

Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo.     

Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs,GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.     

IgA1 protease from Neisseria gonorrhoeae inhibits TNFalpha-mediated apoptosis of human monocytic cells

The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity. In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF. We validated the YEA by activating GTPases by previously reported GEFs. We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-actin stress fiber assembly in fibroblasts, characteristic of RhoA activation.     

Analysis of RhoA and Rho GEF activity in whole cells and the cell nucleus

We have recently shown that a fraction of the total cellular pool of the small GTPase RhoA resides in the nucleus, and that the nuclear guanine nucleotide exchange factor (GEF) Net1 has a role in the regulation of its activity. In this protocol, we describe a method to measure both the activities of the nuclear pools of RhoA and Rho GEFs. This process required the development of a nuclear isolation protocol that is both fast and virtually free of cytosolic and membrane contaminants, as well as a redesign of existing RhoA and Rho GEF activity assays so that they work in nuclear samples. This protocol can be also used for other Rho GTPases and Rho GEFs, which have also been found in the nucleus. Completion of the procedure, including nuclear isolation and RhoA or Rho GEF activity assay, takes 1 h 40 min. We also include details of how to perform a basic assay of whole-cell extracts.