NORMAL GENOMIC

• Zhang B, Gao Y, Moon SY, Zhang Y, Zheng Y
• Oligomerization of Rac1 gtpase mediated by the carboxyl-terminal polybasic domain.
• J Biol Chem. 2001; 276: 8958-67
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• The Rho family GTPase Rac1 mediates a variety of signal transduction processes leading to activation of NADPH oxidase, actin cytoskeleton reorganization, transcription activation, and stimulation of DNA synthesis. In this study, Rac1 was found to form a reversible monomer and oligomer in both the GDP- and GTP-bound states in vitro and in cells. Mutational analysis and peptide competition experiments showed that the unique C-terminal domain of Rac1 consisting of six consecutive basic residues (amino acids 183-188) is required for the homophilic interaction. Oligomerization of Rac1-GTP led to a self-stimulatory GTPase-activating protein (GAP) activity, resulting in a significantly enhanced intrinsic GTP hydrolysis rate of Rac1-GTP. Deletion or mutation of the polybasic residues drastically decreased its intrinsic GTPase activity and resulted in a loss of the self-stimulatory GAP activity. In the oligomeric state, Rac1 became insensitive to the RhoGAP stimulation, albeit maintaining the responsiveness to the guanine nucleotide exchange factor. The ability of the Rac1 C-terminal mutants to activate the effector p21(cdc42/rac)-activated kinase-1 correlated with their oligomerization states, suggesting that oligomer formation potentiates effector activation. Furthermore, the oligomer-to-monomer transition of Rac1-GDP could be driven effectively by interaction with the Rho guanine nucleotide dissociation inhibitor. Building on previous characterizations of Rac1 interaction with regulatory proteins and effectors, these results suggest that Rac1 may employ yet another means of regulation by cycling between the monomeric and oligomeric states to effectively generate a transient and augmented signal.

• Richnau N, Aspenstrom P
• Rich, a rho gtpase-activating protein domain-containing protein involved in signaling by cdc42 and rac1.
• J Biol Chem. 2001; 276: 35060-70
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• A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting with CIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1.

• Shibata H, Oishi K, Yamagiwa A, Matsumoto M, Mukai H, Ono Y
• PKNbeta interacts with the SH3 Domains of Graf and a Novel Graf Related Protein, Graf 2, Which Are GTPase Activating Proteins for Rho Family.
• J Biochem (Tokyo). 2001; 130: 23-31
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• PKNbeta is a novel isoform of PKNalpha, which is one of the target protein kinases for the small GTPase Rho. By yeast two-hybrid screening of a human embryonic kidney 293 cell cDNA library with the PKNbeta linker region containing proline-rich motifs as a bait, clones encoding Graf (GAP for Rho associated with focal adhesion kinase) and a novel Graf-related protein, termed Graf 2, were isolated. The full length of Graf 2 contains a putative PH domain, a RhoGAP domain, and an SH3 domain as well as Graf. Northern and Western blot analyses demonstrated that Graf 2 is expressed in several tissues, with the highest expression in skeletal muscle. Recombinant Graf 2 exhibited GTPase-activating activity toward the small GTPase RhoA and Cdc42Hs, but not toward Rac1, in vitro. The SH3 domains of Graf and Graf 2 purified from Escherichia coli bound directly to PKNbeta. Graf or Graf 2 was co-immunoprecipitated with PKNbeta in COS-7 cells transiently transfected with Graf or Graf 2 and PKNbeta expression constructs. The catalytically active form of PKNbeta& gr;phosphorylated Graf and Graf 2 in vitro. The interplay of PKNbeta and the GTPase-activating proteins, Graf and Graf 2, may offer a novel mechanism regulating the Rho-mediated signaling.

• Molnar G, Dagher MC, Geiszt M, Settleman J, Ligeti E
• Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins.
• Biochemistry. 2001; 40: 10542-9
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• The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.

• Flatau G, Landraud L, Boquet P, Bruzzone M, Munro P
• Deamidation of RhoA glutamine 63 by the Escherichia coli CNF1 toxin requires a short sequence of the GTPase switch 2 domain.
• Biochem Biophys Res Commun. 2000; 267: 588-92
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• CNF1, a toxin produced by pathogenic Escherichia coli strains, deamidates the RhoA GTP-binding protein glutamine 63 and impairs RhoGAP-mediated GTP hydrolysis resulting in RhoA permanent activation. Using peptides derived from the RhoA sequence, we found that DTAGQEDYDRL (corresponding to RhoA 59-69 residues) was the minimum RhoA-derived peptide which could be deamidated in vitro by the CNF1 catalytic domain (CNF1-Cter). Site-directed mutagenesis outside the RhoA 59-69 sequence had no influence on glutamine 63 deamidation by CNF1-Cter. RhoA proteins with substitutions L57G, D65G, Y66G, or R70G were not affected in their ability to be deamidated by CNF1-Cter, whereas this was abolished by the R68G substitution. Arginine 68 is part of the DYDRL motif that is strictly conserved in Rho, Rac, and Cdc42 but not in other small GTP-binding proteins consistent with the observation that only Rho, Rac, and Cdc42 can be modified by CNF1.

• Zhang H, Seabra MC, Deisenhofer J
• Crystal structure of Rab geranylgeranyltransferase at 2.0 A resolution.
• Structure Fold Des. 2000; 8: 241-51
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• BACKGROUND: Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP). RESULTS: The crystal structure of rat RabGGT at 2.0 A resolution reveals an assembly of four distinct structural modules. The beta subunit forms an alpha-alpha barrel that contains most of the residues in the active site. The alpha subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the alpha subunit binds to the active site in the beta subunit; residue His2alpha directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT. CONCLUSIONS: LRR and Ig domains are often involved in protein-protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the alpha subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102beta and Tyr154beta in FT by Ser48beta and Leu99beta, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes.

• Zhu K, Debreceni B, Li R, Zheng Y
• Identification of Rho GTPase-dependent sites in the Dbl homology domain of oncogenic Dbl that are required for transformation.
• J Biol Chem. 2000; 275: 25993-6001
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• The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the alpha1, alpha6, and alpha9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k(cat) values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.

• Ellis S, Mellor H
• Regulation of endocytic traffic by rho family GTPases.
• Trends Cell Biol. 2000; 10: 85-8
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• Endocytosis is a complicated yet highly efficient process that involves the uptake and processing of cargoes, ranging from small molecules, to activated signalling receptors, to whole microorganisms. Regulation of endocytic pathways is poorly understood. Recent evidence suggests that the Rho GTPase family of signalling proteins is intimately involved in endocytic traffic, providing novel insights into the control mechanisms that govern this process.

• Shiraishi Y et al.
• Cupidin, an isoform of Homer/Vesl, interacts with the actin cytoskeleton and activated rho family small GTPases and is expressed in developing mouse cerebellar granule cells.
• J Neurosci. 1999; 19: 8389-400
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• A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Delta11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin-mGluR1-actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent manner in vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.

• Illenberger D et al.
• Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.
• EMBO J. 1998; 17: 6241-9
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• Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.

• Zhang B, Zheng Y
• Regulation of RhoA GTP hydrolysis by the GTPase-activating proteins p190, p50RhoGAP, Bcr, and 3BP-1.
• Biochemistry. 1998; 37: 5249-57
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• The small GTP-binding protein RhoA becomes inactivated by hydrolyzing bound GTP to GDP through its intrinsic GTPase activity which is further stimulated by a family of Rho GTPase-activating proteins (GAPs). Here we have compared the kinetics of interaction between recombinant RhoA and the RhoGAP domains of p190, p50RhoGAP, Bcr, and 3BP-1. The intrinsic rate of GTP hydrolysis by RhoA is relatively slow when compared to other Rho-family GTPases such as Cdc42 or Rac1 with a rate constant of 0.022 min-1, which can be further stimulated at least 4000-fold by p190 or p50RhoGAP. The RhoGAP domains of Bcr and 3BP-1, which were thought to be inactive toward RhoA, are also found capable of stimulating the GTPase activity of RhoA in a dose-dependent manner. The supreme catalytic activities of p190 and p50RhoGAP toward RhoA reside mostly in their lower Km values (1.79 and 2.83 microM, respectively) which correlate well with their binding affinity for GMP-PNP-bound RhoA (2.18 and 2. 47 microM, respectively), in contrast with Bcr and 3BP-1 which interact with the activated RhoA with much higher Km (89 microM). However, the mechanisms of catalysis by p190 and p50RhoGAP are distinct in at least three aspects: (1) p50RhoGAP displays an effect of product inhibition by binding to the GDP-bound form of RhoA with a Kd of 6 microM in comparison with the Kd for p190 of 33 microM; (2) the Km of p190 increases drastically upon the increase of salt and Mg2+ concentrations, conditions under which only modest changes of Km for p50RhoGAP are observed; and (3) p50RhoGAP remains partially active toward the effector domain mutants of RhoA, Y34K, and T37A, whereas p190 is completely inactive toward Y34K and T37A. These results suggest that there exists a unique mechanism of functional interaction between RhoA and individual RhoGAP which involves distinct structural determinants of the small G-protein to cause the apparent differences in kinetic properties.

• Vincent S, Brouns M, Hart MJ, Settleman J
• Evidence for distinct mechanisms of transition state stabilization of GTPases by fluoride.
• Proc Natl Acad Sci U S A. 1998; 95: 2210-5
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• GTPase-activating proteins (GAPs) function by stabilizing the GTPase transition state. This has been most clearly demonstrated by the formation of a high-affinity complex between various GAPs and GDP-bound GTPases in the presence of aluminum tetrafluoride, which can mimic the gamma-phosphate of GTP. Herein, we report that p190 RhoGAP forms a high-affinity complex with Rho GTPases in the presence of fluoride ions, suggesting that p190 also functions to stabilize the GTPase transition state. However, this Rho-p190 complex does not require aluminum ions or even guanine nucleotide, indicating a distinct role for fluoride that is not consistent with the gamma-phosphate-mimicking hypothesis. These results indicate that it is necessary to reconsider the assumed role of fluoride in stabilizing a variety of other GTPase-GAP interactions where the requirement for aluminum or guanine nucleotide has not yet been addressed.

• Taylor GA et al.
• The inducibly expressed GTPase localizes to the endoplasmic reticulum, independently of GTP binding.
• J Biol Chem. 1997; 272: 10639-45
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• The inducibly expressed GTPase (IGTP) is representative of a newly identified group of interferon gamma-inducible GTPases, whose functions are currently unknown. We have begun to address the cellular function of IGTP by examining its subcellular distribution and its guanine nucleotide binding status. Using immunofluorescence, electron microscopy, and subcellular fractionation, IGTP was localized predominantly to the endoplasmic reticulum of both RAW 264. 7 macrophages and C127 fibroblasts. In the immunostaining experiments, staining of discrete cytoplasmic structures on the periphery of the endoplasmic reticulum was also evident. Using polyethyleneimine-cellulose thin layer chromatography, the guanine nucleotides that complexed to immunoprecipitated IGTP, in both control and interferon gamma-stimulated cells, were 90-95% GTP and 5-10% GDP, suggesting that the protein was in an active state. A mutant IGTP protein was created that had no detectable complexed GTP, and in both subcellular fractionation and IGTP-green fluorescent protein fusion studies, this mutant also localized to the endoplasmic reticulum. These results suggested that the GTP binding status of IGTP is independent of its capacity to localize to the endoplasmic reticulum. Given these results, we propose that IGTP is representative of a new family of endoplasmic reticulum GTPases that may be involved in protein processing or trafficking.

• Bourne HR
• G proteins. The arginine finger strikes again.
• Nature. 1997; 389: 673-4

• Fukui K, Sasaki T, Imazumi K, Matsuura Y, Nakanishi H, Takai Y
• Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily of small G proteins.
• J Biol Chem. 1997; 272: 4655-8
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• The Rab small G protein family, consisting of nearly 30 members, is implicated in intracellular vesicle trafficking. They cycle between the GDP-bound and GTP-bound forms, and the latter is converted to the former by the action of a GTPase activating protein (GAP). No GAP specific for each Rab family member or Rab subfamily has been isolated in mammal. Here we purified a GAP with Rab3A as a substrate from rat brain. The purified protein was specifically active on the Rab3 subfamily members (Rab3A, -B, -C, and -D). Of this subfamily, Rab3A and -C are implicated in Ca2+-dependent exocytosis, particularly in neurotransmitter release. This GAP, named Rab3 GAP, was active on the lipid-modified form, but not on the lipid-unmodified form. Rab3 GAP showed a minimum molecular mass of about 130 kDa on SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a human brain cDNA library, and the isolated cDNA encoded a protein with a Mr of 110,521 and 981 amino acids, which showed no homology to any known protein. The recombinant protein exhibited GAP activity toward the Rab3 subfamily members, and the catalytic domain was located at the C-terminal region. Northern blot analysis indicated that Rab3 GAP was ubiquitously expressed.

• Threadgill R, Bobb K, Ghosh A
• Regulation of dendritic growth and remodeling by Rho, Rac, and Cdc42.
• Neuron. 1997; 19: 625-34
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• The acquisition of cell type-specific morphologies is a central feature of neuronal differentiation and has important consequences for nervous system function. To begin to identify the underlying molecular mechanisms, we have explored the role of Rho-related GTPases in the dendritic development of cortical neurons. Expression of dominant negative mutants of Rac or Cdc42, the Rho-inhibitory molecule C3 transferase, or the GTPase-activating protein RhoGAP p190 causes a marked reduction in the number of primary dendrites in nonpyramidal (multipolar) neurons and in the number of basal dendrites in neurons with pyramidal morphologies. Conversely, the expression of constitutively active mutants of Rho, Rac, or Cdc42 leads to an increase in the number of primary and basal dendrites. In cortical cultures, as in vivo, dendritic remodeling leads to an apparent transformation from pyramidal to nonpyramidal morphologies over time. Strikingly, this shift in favor of nonpyramidal morphologies is also inhibited by the expression of dominant negative mutants of Cdc42 and Rac and by RhoGAP p190. These observations indicate that Rho, Rac, and Cdc42 play a central role in dendritic development and suggest that differential activation of Rho-related GTPases may contribute to the generation of morphological diversity in the developing cortex.

• Schmidt G, Sehr P, Wilm M, Selzer J, Mann M, Aktories K
• Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1.
• Nature. 1997; 387: 725-9
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• The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases, which act as molecular switches in diverse signal-transduction processes. Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation or glucosylation. Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necrotizing factors 1 and 2 (CNF1 and 2). These toxins induce actin assembly and multinucleation in culture cells. Here we show that treatment of RhoA with CNF1 inhibits the intrinsic GTPase activity of RhoA and completely blocks GTPase activity stimulated by the Rho-GTPase-activating protein (rhoGAP). Analysis by mass spectrometry and amino-acid sequencing of proteolytic peptides derived from CNF1-treated RhoA indicate that CNF1 induces deamidation of a glutamine residue at position 63 (Gln 63) to give constitutively active Rho protein.

• Backlund PS Jr
• Post-translational processing of RhoA. Carboxyl methylation of the carboxyl-terminal prenylcysteine increases the half-life of Rhoa.
• J Biol Chem. 1997; 272: 33175-80
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• RhoA and related GTP-binding proteins are modified post-translationally at their carboxyl terminus to form a prenylcysteine methyl ester. The synthesis and post-translational modification of RhoA and Cdc42 were examined in the RAW264 macrophage cell line, and the effect of carboxyl methylation on protein turnover was determined. Cells were labeled with [35S]cysteine, and RhoA or Cdc42 was immunoprecipitated with specific antibodies. Both RhoA and Cdc42 were methylated rapidly in control cells, with little accumulation of unmethylated protein. Carboxyl methylation of RhoA was inhibited by incubation of cells with a carbocyclic adenosine analog, 3-deazaaristeromycin, resulting in the accumulation of unmethylated RhoA. Under these conditions, Cdc42 methylation was inhibited only partially. When methylation was inhibited, the RhoA half-life decreased from 31 to 12 h, and the Cdc42 half-life decreased from 15 to 11 h. The increased degradation of unmethylated RhoA demonstrates a novel function for carboxyl-terminal prenylcysteine carboxyl methylation in protecting RhoA and related proteins from degradation.

• Ludbrook SB, Eccleston JF, Strom M
• Cloning and characterization of a rhoGAP homolog from Dictyostelium discoideum.
• J Biol Chem. 1997; 272: 15682-6
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• Small GTPases interact with a variety of proteins that affect nucleotide binding and cleavage. GTPase activating proteins (GAPs) are one class of these proteins that act by accelerating the intrinsic GTPase rate resulting in the formation of the biologically inactive GDP-bound form of the GTPase. For the Rho subfamily of GTPases, there is a growing number of proteins with rhoGAP activity that are identifiable by a homologous region of about 150 amino acids. We have exploited this homology using the polymerase chain reaction to clone the first rhoGAP homolog, called DdRacGAP, from the slime mold Dictyostelium discoideum. The GAP domain of DdRacGAP (amino acids 1-212), when expressed and purified from Escherichia coli, is active on both Dictyostelium and human Rho family GTPases but not human Ras. The full-length protein is 1356 amino acids in length and has several interesting homologies in addition to the GAP domain, including an SH3 domain, a dbl homology domain, and a pleckstrin homology domain.

• Olenik C, Barth H, Just I, Aktories K, Meyer DK
• Gene expression of the small GTP-binding proteins RhoA, RhoB, Rac1, and Cdc42 in adult rat brain.
• Brain Res Mol Brain Res. 1997; 52: 263-9
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• GTPases of the Rho subfamily, i.e. Rho, Rac and Cdc42, are molecular switches in various signaling pathways. Best characterized are their functions in the regulation of the actin cytoskeleton. In neuronal cell lines they are involved in the mechanisms leading to synapse formation and plasticity. It is still unknown whether they have respective functions in the mammalian CNS. In this case, they should be present in the adult brain, especially in areas known for their synaptic remodeling. We have studied the expression of the Rho GTPases in adult rat brain with in situ hybridization and Western blot analysis. High amounts of RhoA, RhoB, Rac1 and Cdc42 mRNAs were detected in neurons of the hippocampus, i.e. in pyramidal cells of the CA1-CA4 regions as well as in granule cells of the dentate gyrus and in hilar cells. Also in cerebellum, Purkinje and granular cells expressed the four mRNAs. Strong gene expression was also found in brainstem, thalamus and neocortex. Using Western blot analysis, RhoA and Cdc42 proteins were detected in hippocampus, cerebellum, thalamus and neocortex. It is concluded that GTPases of the Rho family play a role in the regulation of cellular functions in the adult brain.

• Danley DE, Chuang TH, Bokoch GM
• Defective Rho GTPase regulation by IL-1 beta-converting enzyme-mediated cleavage of D4 GDP dissociation inhibitor.
• J Immunol. 1996; 157: 500-3
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• GTPases of the Rho family regulate many aspects of inflammatory cell activity, including motility, formation of toxic oxygen metabolites, and generation of proinflammatory cytokines. Defective regulation of such signaling pathways leads to a variety of acute and chronic inflammatory disorders, although the mechanisms by which this occurs have not been well defined. We describe in this work specific proteolytic cleavage of D4 GDI, a critical regulator of Rho GTPase activity in inflammatory leukocytes, by IL-1 beta-converting enzyme (ICE). Cleavage of D4 GDI by ICE occurs at Asp55, leading to the formation of the truncated D4 that is unable to effectively bind and regulate GTPases of the Rho family. Our data suggest that activation of ICE protease(s) at inflammatory sites leads to defective Rho GTPase regulation. Release of these critical regulatory proteins may contribute substantially to the inflammatory response at these sites, exacerbating and perpetuating the resulting tissue damage.

• Nur-e-Kamal MS
• Overexpression and functional analysis of a mitogen-inducible nuclear GTPase activating protein, Spa-1.
• Int J Biochem Cell Biol. 1996; 28: 1241-7
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• The two Ras-related GTPases called Rap1 and Rsr1, which share 50% sequence identity with Ras GTPases are known to be activated by two distinct mammalian GAPs, i.e. cytosolic GAP3c of 55 kDa and membrane-bound GAP3m of 85 kDa. Recently we have cloned a gene encoding a 68 kDa (p68) protein product, which is associated with chromosomes during interphase. The N-terminal 190 amino acids share 43% sequence identity with the second half of the GTPase activating domain (residues 210-397) of GAP3m. The N-terminal fragment of 209 amino acids of Spa-1 (called Span-N) was overproduced in E. coli as a glutathione S-transferase (GST) fusion protein and affinity purified. Rap1 and Rsr1 GTPase stimulatory activity of Spa-1 was tested and compared with GAP3m. Spa-1 preferentially stimulates Rsr1 GTPase rather than Rap1 GTPase, while GAP3m has a preference for Rap1 GTPase. This suggests that although Spa-1 and GAP3m stimulate GTPase of Rap1 family members, they differ in affinity for them. By mutational analysis it was also found that amino acid residues 10-183 are enough for Rap GAP activity of Spa-1.

• Vojtek AB, Cooper JA
• Rho family members: activators of MAP kinase cascades.
• Cell. 1995; 82: 527-9

• Burbelo PD et al.
• p190-B, a new member of the Rho GAP family, and Rho are induced to cluster after integrin cross-linking.
• J Biol Chem. 1995; 270: 30919-26
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• p120GAP forms distinct complexes with two phosphoproteins, p62 and p190. Here we have cloned a cDNA encoding a protein with 51% amino acid identity to p190 (hereafter designated p190-A) and have designated it p190-B. The N-terminal portion of p190-B contained several motifs characteristic of a GTPase domain, while its C terminus contained a Rho GAP domain. A recombinant Rho GAP domain polypeptide showed GAP activity for RhoA, Rac1, and G25K/CDC42Hs. Immunoprecipitation and immunofluorescence studies demonstrated that p190-B protein was expressed in a variety of cells and was localized diffusely in the cytoplasm and in fibrillar patterns that co-localized with the alpha 5 beta 1 integrin receptor for fibronectin. Adhesion of fibronectin-coated latex beads to cells resulted in recruitment of significant amounts of p190-B and Rho to the plasma membrane beneath the site of bead binding. In contrast, beads coated with polylysine or concanavalin A were unable to recruit p190-B or Rho. Additionally, anti-beta 1 or anti-alpha 5 integrin antibody-coated beads were also able to recruit large amounts of p190-B and Rho. These results identify a novel second member of the p190 family and establish the existence of a novel transmembrane link between integrins and a new protein p190-B and Rho.

• Diekmann D, Hall A
• In vitro binding assay for interactions of Rho and Rac with GTPase-activating proteins and effectors.
• Methods Enzymol. 1995; 256: 207-15

• Klebe C, Bischoff FR, Ponstingl H, Wittinghofer A
• Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.
• Biochemistry. 1995; 34: 639-47
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• The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein). The intrinsic GTPase of Ran is one-tenth of the rate of p21ras and is even lower in the Ran(Q69L) mutant. Under saturating conditions the rate constant for the RanGAP1 stimulated GTPase reaction is 2.1 s-1 at 25 degrees C, which is a 10(5)-fold stimulation, whereas RanGAP1 has no effect on Ran(Q69L). The intrinsic guanine nucleotide dissociation rates of Ran are also very low and are likewise increased 10(5)-fold by the exchange factor RCC1. Methods to describe the reaction kinetically are presented. The Ran(T24N) mutant, which is analogous to the S17N mutant of p21ras, has decreased relative affinities for both GDP/GTP and favors GDP binding. However, it was found to interact almost normally with RCC1. The combination of these properties leads to stabilization of the Ran(T24N)-RCC1 complex and may result in vivo in depletion of RCC1 available for stimulating guanine nucleotide exchange.

• Scherle P, Behrens T, Staudt LM
• Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes.
• Proc Natl Acad Sci U S A. 1993; 90: 7568-72
• Display abstract

• The Ras-related small GTP-binding proteins are involved in diverse cellular events, including cell signaling, proliferation, cytoskeletal organization, and secretion. The interconversion of the active, GTP-bound form of the protein to the inactive, GDP-bound form is influenced by two types of regulatory proteins, those that alter the intrinsic GTPase activity of the GTP-binding protein and those that affect the rate of GDP/GTP exchange. By utilizing a subtractive hybridization approach, we have isolated a human gene encoding Ly-GDI, a protein that has striking homology to the product of a previously cloned gene, Rho-GDI, which inhibits GDP/GTP exchange on the Rho family of GTPases. In contrast to Rho-GDI, which is ubiquitously expressed, Ly-GDI is expressed only in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. Stimulation of T lymphocytes with phorbol ester leads to phosphorylation of Ly-GDI, suggesting an involvement of Ly-GDI in lymphocyte activation pathways. Cell type-specific regulators of the Ras-like GTP-binding proteins may provide one mechanism by which different cell types respond uniquely to signals transduced through the same cell surface receptor or may provide a way by which the GTP-binding proteins can be uniquely engaged by tissue-restricted receptors.

• Markby DW, Onrust R, Bourne HR
• Separate GTP binding and GTPase activating domains of a G alpha subunit.
• Science. 1993; 262: 1895-901
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• Most members of the guanosine triphosphatase (GTPase) superfamily hydrolyze guanosine triphosphate (GTP) quite slowly unless stimulated by a GTPase activating protein or GAP. The alpha subunits (G alpha) of the heterotrimeric G proteins hydrolyze GTP much more rapidly and contain an approximately 120-residue insert not found in other GTPases. Interactions between a G alpha insert domain and a G alpha GTP-binding core domain, both expressed as recombinant proteins, show that the insert acts biochemically as a GAP. The results suggest a general mechanism for GAP-dependent hydrolysis of GTP by other GTPases.

• Ridley AJ et al.
• rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo.
• EMBO J. 1993; 12: 5151-60
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• rho family GTPases link extracellular signals to changes in the organization of cytoskeletal actin. Serum stimulation of quiescent Swiss 3T3 fibroblasts leads to rho-dependent actin stress fibre formation and focal adhesions, whilst several growth factors initiate signalling pathways leading to rac-dependent actin polymerization at the plasma membrane, and membrane ruffling. The product of the breakpoint cluster region gene bcr, rho GTPase accelerating protein (rhoGAP) and rasGAP-associated p190 share structurally related rho GAP domains, and possess GAP activity for rho family members in vitro. We have directly compared the activities of the isolated GAP domains of these three proteins in regulating different rho family GTPases, both by in vitro assays and by microinjection, to address their possible physiologic functions. We show that bcr accelerates the GTPase activity of rac, but not rho in vitro, and inhibits rac-mediated membrane ruffling, but not rho-mediated stress fibre formation, after microinjection into Swiss 3T3 fibroblasts. In vitro, rhoGAP has a striking preference for G25K as a substrate, whilst p190GAP has marked preferential activity for rho. Furthermore, p190 preferentially inhibits rho-mediated stress fibre formation in vivo. Our data suggest that p190, rhoGAP and bcr play distinct roles in signalling pathways mediated through different rho family GTPases.

• McCormick F
• GTP-binding proteins as oncogenes in human tumors.
• Environ Health Perspect. 1991; 93: 17-8

• Diekmann D et al.
• Bcr encodes a GTPase-activating protein for p21rac.
• Nature. 1991; 351: 400-2
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• More than thirty small guanine nucleotide-binding proteins related to the ras-encoded oncoprotein, termed Ras or p21ras, are known. They regulate many fundamental processes in all eukaryotic cells, such as growth, vesicle traffic and cytoskeletal organization. GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis of Ras-related proteins, leading to down-regulation of the active GTP-bound form. For p21ras, two GAP proteins are known, rasGAP and the neurofibromatosis (NF1) gene product. There is evidence that rasGAP may also be a target protein for regulation by Ras and be involved in downstream signalling. We have purified a GAP protein for p21rho, which is involved in the regulation of the actin cytoskeleton. Partial sequencing of rhoGAP reveals significant homology with the product of the bcr (breakpoint cluster region) gene, the translocation breakpoint in Philadelphia chromosome-positive chronic myeloid leukaemias. We show here that the carboxy-terminal domains of the bcr-encoded protein (Bcr) and of a Bcr-related protein, n-chimaerin, are both GAP proteins for the Ras-related GTP-binding protein, p21rac. This result suggest that Bcr could be a target for regulation by Rac and has important new implications for the role of bcr translocations in leukaemia.

• Schweighoffer F, Rey I, Barlat I, Soubigou P, Mayaux JF, Tocque B
• Rap gene products mobilize a different metabolic pathway than P21 ras proteins.
• Adv Second Messenger Phosphoprotein Res. 1990; 24: 329-34

• Tsai MH, Hall A, Stacey DW
• Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins.
• Mol Cell Biol. 1989; 9: 5260-4
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• Certain lipids were found to inhibit the interaction between rho and R-ras proteins and their respective GTPase-activating proteins (GAP). Inhibitory lipids were similar for each protein but differed significantly from those previously found to inhibit the interaction between ras protein and GAP activity. These data raise the possibility that ras and related proteins are controlled biologically by interactions between lipids and GAP molecules.