Comprehensive Screening for Novel Rab‐Binding Proteins by GST Pull‐Down Assay Using 60 Different Mammalian Rabs‡

The Rab family belongs to the Ras‐like small GTPase superfamily and is implicated in membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs are yet to be identified. In this study, we systematically screened five different cell or tissue lysates for novel Rab effectors by a combination of glutathione S‐transferase (GST) pull‐down assay with 60 different mammalian Rabs and mass spectroscopic analysis. Three of the 21 Rab‐binding proteins we identified, mKIAA1055/TBC1D2B (Rab22‐binding protein), GAPCenA/TBC1D11 (Rab36‐binding protein) and centaurin β2/ACAP2 (Rab35‐binding protein), are GTPase‐activating proteins (GAPs) for Rab or Arf. Although it has recently been proposed that the Rab–GAP (Tre‐2 /Bub2/Cdc16) domain physically interacts with its substrate Rab, these three GAPs interacted with specific Rabs via a domain other than a GAP domain, e.g. centaurin β2 binds GTP‐Rab35 via the ankyrin repeat (ANKR) domain. Although centaurin β2 did not exhibit any Rab35–GAP activity in vitro, the Rab35‐binding ANKR domain of centaurin β2 was found to be required for its plasma membrane localization and regulation of Rab35‐dependent neurite outgrowth of PC12 cells through inactivation of Arf6. These findings suggest a novel mode of interaction between Rab and GAP.     

Optimized small‐molecule pull‐downs define MLBP1 as an acyl‐lipid‐binding protein

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC‐MS to identify candidate binding ligands. We optimized this method using ABA–PYL interactions and show that ABA co‐purifies with wild‐type PYL5 but not a binding site mutant. The K_d of PYL5 for ABA is 1.1 μm , which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37 START domain‐related proteins, which resulted in the identification of ligands that co‐purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co‐purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.     

Structural analysis of protein tyrosine phosphatase 1B reveals potentially druggable allosteric binding sites

Catalytic proteins such as human protein tyrosine phosphatase 1B (PTP1B), with conserved and highly polar active sites, warrant the discovery of druggable nonactive sites, such as allosteric sites, and potentially, therapeutic small molecules that can bind to these sites. Catalyzing the dephosphorylation of numerous substrates, PTP1B is physiologically important in intracellular signal transduction pathways in diverse cell types and tissues. Aberrant PTP1B is associated with obesity, diabetes, cancers, and neurodegenerative disorders. Utilizing clustering methods (based on root mean square deviation, principal component analysis, nonnegative matrix factorization, and independent component analysis), we have examined multiple PTP1B structures. Using the resulting representative structures in different conformational states, we determined consensus clustroids and used them to identify both known and novel binding sites, some of which are potentially allosteric. We report several lead compounds that could potentially bind to the novel PTP1B binding sites and can be further optimized. Considering the possibility for drug repurposing, we discovered homologous binding sites in other proteins, with ligands that could potentially bind to the novel PTP1B binding sites.     

Msb4p, a protein involved in Cdc42p-dependent organization of the actin cytoskeleton, is a Ypt/Rab-specific GAP

Ypt/Rab proteins of the Ras superfamily are regulators of protein transport in exo- and endocytosis. Like Ras and Rho proteins, they have a slow intrinsic GTPase activity that can be accelerated by several orders of magnitude by GTPase-activating proteins (GAP). Here we describe a new member of a family of Ypt/Rab-specific GAPs, Msb4p/Gyp4p, that shares with other Gyp family members significant homology in the catalytic domain, recently identified in Gyp1p and Gyp7p. Purified Msb4p/Gyp4p acts primarily on Sec4p, Ypt6p and Ypt7p and might have a role in polarized secretion.     

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus.

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6-99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.     

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.     

GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila

Typical members of the Ras superfamily of small monomeric GTP-binding proteins function as regulators of diverse processes by cycling between biologically active GTP- and inactive GDP-bound conformations. Proteins that control this cycling include guanine nucleotide exchange factors or GEFs, which activate Ras superfamily members by catalyzing GTP for GDP exchange, and GTPase activating proteins or GAPs, which accelerate the low intrinsic GTP hydrolysis rate of typical Ras superfamily members, thus causing their inactivation. Two among the latter class of proteins have been implicated in common genetic disorders associated with an increased cancer risk, neurofibromatosis-1, and tuberous sclerosis. To facilitate genetic analysis, I surveyed Drosophila and human sequence databases for genes predicting proteins related to GAPs for Ras superfamily members. Remarkably, close to 0.5% of genes in both species (173 human and 64 Drosophila genes) predict proteins related to GAPs for Arf, Rab, Ran, Rap, Ras, Rho, and Sar family GTPases. Information on these genes has been entered into a pair of relational databases, which can be used to identify evolutionary conserved proteins that are likely to serve basic biological functions, and which can be updated when definitive information on the coding potential of both genomes becomes available.     

The small nuclear GTPase Ran: How much does it run?

Ran is one of the most abundant and best conserved of the small GTP binding and hydrolyzing proteins of eukaryotes. It is located predominantly in cell nuclei. Ran is a member of the Ras family of GTPases, which includes the Ras and Ras-like proteins that regulate cell growth and division, the Rho and Rac proteins that regulate cytoskeletal organization and the Rab proteins that regulate vesicular sorting. Ran differs most obviously from other members of the Ras family in both its nuclear localization, and its lack of sites required for post-translational lipid modification. Ran is, however, similar to other Ras family members in requiring a specific guanine nucleotide exchange factor (GEF) and a specific GTPase activating protein (GAP) as stimulators of overall GTPase activity. In this review, the multiple cellular functions of Ran are evaluated with respect to its known biochemistry and molecular interactions.     

植物小G蛋白功能的研究进展

近年来,小G蛋白的调控途径已经成为人们研究细胞信号转导过程的热点问题.小G蛋白家族包括Ras、Rab、Rho、Arf和Ran亚家族,它们起着许多不同的重要细胞生理作用,例如基因表达、细胞骨架重组装、微管的形成以及囊泡和核孔运输机制.这些小G蛋白作为重要的分子开关,具有一个非常保守的功能区域,即I-Ⅳ结构区,它起着关键性作用.从拟南芥(Arabidopsisthaliana)基因组预测分析得出,拟南芥含有93个小G蛋白同源序列,包含Rab、Rho、Arf和Ran亚家族,但没有Ras亚家族.本文主要阐述了迄今在植物中研究小G蛋白各个亚家族功能的最新进展,并对植物、酵母和动物相关的同 源蛋白的生理功能进行比较和推测.     

植物小G 蛋白功能的研究进展

近年来,小G蛋白的调控途径已经成为人们研究细胞信号转导过程的热点问题。小G蛋白家族包括Ras、Rab、Rho、Arf和Ran亚家族,它们起着许多不同的重要细胞生理作用,例如基因表达、细胞骨架重组装、微管的形成以及囊泡和核孔运输机制。这些小G蛋白作为重要的分子开关,具有一个非常保守的功能区域,即I-IV结构区,它起着关键性作用。从拟南芥(Arabidopsis thaliana)基因组预测分析得出,拟南芥含有93个小G蛋白同源序列,包含Rab、Rho、Arf和Ran亚家族,但没有Ras亚家族。本文主要阐述了迄今在植物中研究小G蛋白各个亚家族功能的最新进展,并对植物、酵母和动物相关的同源蛋白的生理功能进行比较和推测。     

Prenylated Rab acceptor protein is a receptor for prenylated small GTPases

Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.     

Conservation and divergence of Grb7 family of Ras-binding domains

Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTR-CNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%–70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.     

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.     

Lineage-specific expansions provide genomic complexity among sea urchin GTPases

In every organism, GTP-binding proteins control many aspects of cell signaling. Here, we examine in silico several GTPase families from the Strongylocentrotus purpuratus genome: the monomeric Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR family, and the "protein biosynthesis" translational GTPases. Identified were 174 GTPases, of which over 90% are expressed in the embryo as shown by tiling array and expressed sequence tag data. Phylogenomic comparisons restricted to Drosophila, Ciona, and humans (protostomes, urochordates, and vertebrates, respectively) revealed both common and unique elements in the expected composition of these families. Galpha and dynamin families contain vertebrate expansions, consistent with whole genome duplications, whereas SRP/SR and translational GTPases are highly conserved. Unexpectedly, Ras superfamily analyses revealed several large (5+) lineage-specific expansions in the sea urchin. For Rho, Rab, Arf, and Ras subfamilies, comparing total human gene numbers to the number of sea urchin genes with vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increase overall numbers such that total sea urchin gene numbers approximate vertebrate gene numbers for each monomeric GTPase family. These findings suggest that lineage-specific expansions may be an important component of genomic evolution in signal transduction.     

八肋游仆虫Rab家族基因克隆和多样性分析

Rab蛋白是在真核细胞内膜泡运输过程中起重要调节作用的一类小分子Ras-like蛋白,为Ras超家族中最大的家族。Rab家族成员在不同的生物中表现出数量的多样性和功能上的分化。为进一步了解Rab蛋白的多样性及其在真核细胞内膜泡运输网络中的功能,本研究利用游仆虫大核染色体特异的端粒结构和基因大小的染色体结构特征,通过简并引物PCR方法从八肋游仆虫(Euplotes octocarinatus)中克隆到9种新的Rab基因,分别为EoRab1A、EoRab2b、EoRab2c、EoRab2d、EoRab6、EoRab7、EoRab2-like、EoRabL2和EoRan(GenBank登陆号为HM371131～HM371139)。序列分析表明,游仆虫中Rab基因家族成员既包括具有维持细胞结构核心功能保守基因,又包括为适应环境而进化出的特殊功能的新基因。     

Exhaustive comparison and classification of ligand‐binding surfaces in proteins

Many proteins function by interacting with other small molecules (ligands). Identification of ligand‐binding sites (LBS) in proteins can therefore help to infer their molecular functions. A comprehensive comparison among local structures of LBSs was previously performed, in order to understand their relationships and to classify their structural motifs. However, similar exhaustive comparison among local surfaces of LBSs (patches) has never been performed, due to computational complexity. To enhance our understanding of LBSs, it is worth performing such comparisons among patches and classifying them based on similarities of their surface configurations and electrostatic potentials. In this study, we first developed a rapid method to compare two patches. We then clustered patches corresponding to the same PDB chemical component identifier for a ligand, and selected a representative patch from each cluster. We subsequently exhaustively as compared the representative patches and clustered them using similarity score, PatSim. Finally, the resultant PatSim scores were compared with similarities of atomic structures of the LBSs and those of the ligand‐binding protein sequences and functions. Consequently, we classified the patches into ～2000 well‐characterized clusters. We found that about 63% of these clusters are used in identical protein folds, although about 25% of the clusters are conserved in distantly related proteins and even in proteins with cross‐fold similarity. Furthermore, we showed that patches with higher PatSim score have potential to be involved in similar biological processes.     

Merging chemical and biological space: Structural mapping of enzyme binding pocket space

Structure‐based drug design tries to mutually map pharmacological space populated by putative target proteins onto chemical space comprising possible small molecule drug candidates. Both spaces are connected where proteins and ligands recognize each other: in the binding pockets. Therefore, it is highly relevant to study the properties of the space composed by all possible binding cavities. In the present contribution, a global mapping of protein cavity space is presented by extracting consensus cavities from individual members of protein families and clustering them in terms of their shape and exposed physicochemical properties. Discovered similarities indicate common binding epitopes in binding pockets independent of any possibly given similarity in sequence and fold space. Unexpected links between remote targets indicate possible cross‐reactivity of ligands and suggest putative side effects. The global clustering of cavity space is compared to a similar clustering of sequence and fold space and compared to chemical ligand space spanned by the chemical properties of small molecules found in binding pockets of crystalline complexes. The overall similarity architecture of sequence, fold, and cavity space differs significantly. Similarities in cavity space can be mapped best to similarities in ligand binding space indicating possible cross‐reactivities. Most cross‐reactivities affect co‐factor and other endogenous ligand binding sites. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

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

Identification and Characterization of RBEL1 Subfamily of GTPases in the Ras Superfamily Involved in Cell Growth Regulation

Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236–302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.The Ras superfamily is known to comprise five structurally distinct subfamilies of small GTPases, including Ras, Rho, Rab, Sar1/Arf, and Ran, and each subfamily of these GTPases possess distinct functions in the regulation of a variety of cellular processes such as cell proliferation, cell differentiation, cytoskeletal organization, protein transport, and trafficking (1–4). The Ras subfamily of GTPases (N-, H-, and K-Ras) function predominantly in relaying signals from receptors at the plasma membrane and modulating cell signaling pathways that regulate cell proliferation, differentiation, and survival (5). Ran GTPase, on other hand, is a key regulator of nucleocytoplasmic transport that regulates protein transport across the nuclear pore complex (6, 7). The Rab subfamily is the largest subfamily among the Ras superfamily and contains more than 60 members. The key functions of the Rab GTPases are to regulate protein exocytic and endocytic pathways and modulate intracellular protein transport/trafficking (8–13).In general, the Ras superfamily GTPases cycle between an active GTP-bound state and an inactive GDP-bound state. There are five N-terminal motifs involved in the binding and hydrolysis of GTP that are highly conserved among all GTPases: G1 (GXXXXGK(S/T)), G2 (T), G3 (DXXG), G4 ((N/T)(K/Q)XD), and G5 (EXSAX). Each sequence has particular functions involved in binding nucleotides (GTP or GDP) and facilitating hydrolysis (4, 14, 15). In general, the intrinsic GTPase activity (converting GTP to GDP) and exchange of GDP for GTP are slow processes for these GTPases and thus require regulatory proteins such as GTPase-activating proteins and GDP/GTP exchange factors to facilitate these processes (16–18).For the last two decades, the Ras superfamily has been a major focus in the cancer field as many of the members are either mutated or dysregulated in cancer. The founding members of the Ras superfamily, H-Ras and K-Ras, were first identified as viral oncogenes (1, 4). Later studies demonstrated that mutations of the Ras proteins (H-, N-, and K-Ras) occur frequently in human cancers, and the mutations identified are mostly clustered within the GTP-binding domains of the proteins thus locking Ras proteins in a GTP-bound configuration. GTP-bound Ras is constitutively active; it constantly activates its effector proteins to transduce cell proliferative signals (1, 4). Unlike Ras subfamily genes, mutations occurring in Rab and Rab-like genes are less common, yet alterations in gene expression of a number of Rab genes have been reported in multiple human malignancies. For example, Rab25 overexpression has been linked to prostate cancer progression (19). Rab2 overexpression has been found in lung adenomas and adenocarcinomas (20). In addition, alterations in Rab gene expression have also been linked to cancer drug resistance. For instance, resistance to the anticancer drug doxorubicin in MCF-7 cells has been linked with reduced expression of Rab6C, and introduction of exogenous Rab6C restores drug sensitivity (21).We have recently reported the identification two novel Ras superfamily GTPases, RBEL1A and RBEL1B (22). RBEL1A and RBEL1B are two splice variants of the RBEL1 gene and are highly homologous to the Rab and Ran GTPases within their N-terminal GTP-binding domains (22). Our studies show that both RBEL1A and -B predominantly bind to GTP. A single point mutation (T57N) in the GTP-binding domain of RBEL1A and -B abolishes their ability to bind to both GTP and GDP. Both RBEL1A and RBEL1B localize in the nucleus as well as in the cytosol. Whereas RBEL1A is predominantly cytosolic, RBEL1B is primarily nuclear. Interestingly, our studies also suggested that nucleotide (GTP or GDP)-binding could be important for the nuclear distribution of RBEL1B, because the nucleotide binding-deficient mutant form (T57N) of RBEL1B did not reside in the nucleus but rather became largely cytosolic (22).In our continuous efforts to fully elucidate the function of RBEL1, we have identified two additional splice variants that we have named RBEL1C and RBEL1D. Here we report further characterization of all four RBEL1 splice variants in terms of their GTPase activities, subcellular localizations, regulations, and potential functions. Our results indicate that RBEL1 GTPases, although sharing some common features with other Ras superfamily members, also harbor unique characteristics that are significantly different from other Ras superfamily GTPases. Based on our findings, we suggest that RBEL1 proteins appear to form a novel subfamily of GTPases within the Ras superfamily.