NORMAL GENOMIC

• Bi F, Debreceni B, Zhu K, Salani B, Eva A, Zheng Y
• Autoinhibition mechanism of proto-Dbl.
• Mol Cell Biol. 2001; 21: 1463-74
• Display abstract

• The dbl oncogene encodes a prototype member of the Rho GTPase guanine nucleotide exchange factor (GEF) family. Oncogenic activation of proto-Dbl occurs through truncation of the N-terminal 497 residues. The C-terminal half of proto-Dbl includes residues 498 to 680 and 710 to 815, which fold into the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, respectively, both of which are essential for cell transformation via the Rho GEF activity or cytoskeletal targeting function. Here we have investigated the mechanism of the apparent negative regulation of proto-Dbl imposed by the N-terminal sequences. Deletion of the N-terminal 285 or C-terminal 100 residues of proto-Dbl did not significantly affect either its transforming activity or GEF activity, while removal of the N-terminal 348 amino acids resulted in a significant increase in both transformation and GEF potential. Proto-Dbl displayed a mostly perinuclear distribution pattern, similar to a polypeptide derived from its N-terminal sequences, whereas onco-Dbl colocalized with actin stress fibers, like the PH domain. Coexpression of the N-terminal 482 residues with onco-Dbl resulted in disruption of its cytoskeletal localization and led to inhibition of onco-Dbl transforming activity. The apparent interference with the DH and PH functions by the N-terminal sequences can be rationalized by the observation that the N-terminal 482 residues or a fragment containing residues 286 to 482 binds specifically to the PH domain, limiting the access of Rho GTPases to the catalytic DH domain and masking the intracellular targeting function of the PH domain. Taken together, our findings unveiled an autoinhibitory mode of regulation of proto-Dbl that is mediated by the intramolecular interaction between its N-terminal sequences and PH domain, directly impacting both the GEF function and intracellular distribution.

• Zhang B, Zhang Y, Wang Z, Zheng Y
• The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins.
• J Biol Chem. 2000; 275: 25299-307
• Display abstract

• 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.

• Aghazadeh B, Lowry WE, Huang XY, Rosen MK
• Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation.
• Cell. 2000; 102: 625-33
• Display abstract

• Rho-family GTPases transduce signals from receptors leading to changes in cell shape and motility, mitogenesis, and development. Proteins containing the Dbl homology (DH) domain are responsible for activating Rho GTPases by catalyzing the exchange of GDP for GTP. Receptor-initiated stimulation of Dbl protein Vav exchange activity involves tyrosine phosphorylation. We show through structure determination that the mVav1 DH domain is autoinhibited by an N-terminal extension, which lies in the GTPase interaction site. This extension contains the Tyr174 Src-family kinase recognition site, and phosphorylation or truncation of this peptide results in stimulation of GEF activity. NMR spectroscopy data show that the N-terminal peptide is released from the DH domain and becomes unstructured upon phosphorylation. Thus, tyrosine phosphorylation relieves autoinhibition by exposing the GTPase interaction surface of the DH domain, which is obligatory for Vav activation.

• Solski PA, Abe K, Der CJ
• Analyses of transforming activity of Rho family activators.
• Methods Enzymol. 2000; 325: 425-41

• Rossman KL, Campbell SL
• Bacterial expressed DH and DH/PH domains.
• Methods Enzymol. 2000; 325: 25-38

• Mosteller R, Han J, Das B, Broek D
• Biochemical analysis of regulation of Vav, a guanine-nucleotide exchange factor for Rho family of GTPases.
• Methods Enzymol. 2000; 325: 38-51

• Das B et al.
• Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding.
• J Biol Chem. 2000; 275: 15074-81
• Display abstract

• Vav and Sos1 are Dbl family guanine nucleotide exchange factors, which activate Rho family GTPases in response to phosphatidylinositol 3-kinase products. A pleckstrin homology domain adjacent to the catalytic Dbl homology domain via an unknown mechanism mediates the effects of phosphoinositides on guanine nucleotide exchange activity. Here we tested the possibility that phosphatidylinositol 3-kinase substrates and products control an interaction between the pleckstrin homology domain and the Dbl homology domain, thereby explaining the inhibitory effects of phosphatidylinositol 3-kinase substrates and stimulatory effects of the products. Binding studies using isolated fragments of Vav and Sos indicate phosphatidylinositol 3-kinase substrate promotes the binding of the pleckstrin homology domain to the Dbl homology domain and blocks Rac binding to the DH domain, whereas phosphatidylinositol 3-kinase products disrupt the Dbl homology/pleckstrin homology interactions and permit Rac binding. Additionally, Lck phosphorylation of Vav, a known activating event, reduces the affinities between the Vav Dbl homology and pleckstrin homology domains and permits Rac binding. We also show Vav activation in cells, as monitored by phosphorylation of Vav, Vav association with phosphatidylinositol 3,4,5-trisphosphate, and Vav guanine nucleotide exchange activity, is blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest the molecular mechanisms for activation of Vav and Sos1 require disruption of inhibitory intramolecular interactions involving the pleckstrin homology and Dbl homology domains.

• Miki T
• [Malignant transformation and regulation of cell division by a Rho exchange factor ECT2]
• Seikagaku. 2000; 72: 1249-53

• Stam JC, Collard JG
• The DH protein family, exchange factors for Rho-like GTPases.
• Prog Mol Subcell Biol. 1999; 22: 51-83

• Reid T, Bathoorn A, Ahmadian MR, Collard JG
• Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for Cdc42.
• J Biol Chem. 1999; 274: 33587-93
• Display abstract

• Guanine nucleotide exchange factors of the Dbl family regulate the actin cytoskeleton through activation of Rho-like GTPases. At present the Dbl family consists of more than thirty members; many have not been phenotypically or biochemically characterized. Guanine nucleotide exchange factors universally feature a Dbl homology domain followed by a pleckstrin homology domain. Employing data base screening we identified a recently cloned cDNA, KIAA0424, showing substantial sequence homology with Rac activators such as Tiam1, Sos, Vav, and PIX within the catalytic domain. This cDNA appears to be the human homologue of the Ascidian protein Posterior End Mark-2 (PEM-2). We refer to this exchanger as hPEM-2. hPEM-2 encodes a protein of 70 kDa and features an N-terminal src homology 3 domain, followed by tandem Dbl homology and pleckstrin homology domains. The gene is highly expressed in brain and is localized on the human X-chromosome. Employing biochemical activity assays for Rho-like GTPases we found that hPEM-2 specifically activates Cdc42 and not Rac or RhoA. Ectopic expression of hPEM-2 in NIH3T3 fibroblasts revealed a Cdc42 phenotype featuring filopodia formation, followed by cortical actin polymerization and cell rounding. hPEM-2 represents an exchange factor, which may have a role in the regulation of a number of cellular processes through Cdc42.

• Movilla N, Bustelo XR
• Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins.
• Mol Cell Biol. 1999; 19: 7870-85
• Display abstract

• We report here the identification and characterization of a novel Vav family member, Vav-3. Signaling experiments demonstrate that Vav-3 participates in pathways activated by protein tyrosine kinases. Vav-3 promotes the exchange of nucleotides on RhoA, on RhoG and, to a lesser extent, on Rac-1. During this reaction, Vav-3 binds physically to the nucleotide-free states of those GTPases. These functions are stimulated by tyrosine phosphorylation in wild-type Vav-3 and become constitutively activated upon deletion of the entire calponin-homology region. Expression of truncated versions of Vav-3 leads to drastic actin relocalization and to the induction of stress fibers, lamellipodia, and membrane ruffles. Moreover, expression of Vav-3 alters cytokinesis, resulting in the formation of binucleated cells. All of these responses need only the expression of the central region of Vav-3 encompassing the Dbl homology (DH), pleckstrin homology (PH), and zinc finger (ZF) domains but do not require the presence of the C-terminal SH3-SH2-SH3 regions. Studies conducted with Vav-3 proteins containing loss-of-function mutations in the DH, PH, and ZF regions indicate that only the DH and ZF regions are essential for Vav-3 biological activity. Finally, we show that one of the functions of the Vav-3 ZF region is to work coordinately with the catalytic DH region to promote both the binding to GTP-hydrolases and their GDP-GTP nucleotide exchange. These results highlight the role of Vav-3 in signaling and cytoskeletal pathways and identify a novel functional cross-talk between the DH and ZF domains of Vav proteins that is imperative for the binding to, and activation of, Rho GTP-binding proteins.

• Rumenapp U, Blomquist A, Schworer G, Schablowski H, Psoma A, Jakobs KH
• Rho-specific binding and guanine nucleotide exchange catalysis by KIAA0380, a dbl family member.
• FEBS Lett. 1999; 459: 313-8
• Display abstract

• Several guanine nucleotide exchange factors (GEFs) for Rho-GTPases have been identified, all of them containing a Dbl homology (DH) and pleckstrin homology (PH) domain, but exhibiting different specificities to the Rho family members, Rho, Rac and Cdc42. We report here that KIAA0380, a protein with a tandem DH/PH domain, an amino-terminal PDZ domain and a regulator of G protein signalling (RGS) homology domain, is a specific GEF for RhoA, but not for Rac1 and Cdc42, as determined by GDP release, guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) binding and protein binding assays. When expressed in J82 cells, DH/PH domain-containing forms of KIAA0380 induced actin stress fibers, whereas expression of the RGS homology domain prevented lysophosphatidic acid (LPA)-induced stress fiber formation.

• Movilla N, Crespo P, Bustelo XR
• Signal transduction elements of TC21, an oncogenic member of the R-Ras subfamily of GTP-binding proteins.
• Oncogene. 1999; 18: 5860-9
• Display abstract

• TC21 is a Ras-like GTPase with high oncogenic potential that is found mutated in some human tumors and overexpressed in breast cancer cell lines. We have conducted cellular and biochemical studies in order to understand the role of this protein in signal transduction and to unveil the signaling elements that participate in the TC21 pathway. Using gene transfer experiments, we demonstrate here that the TC21 oncogene can induce both cellular transformation in mouse fibroblasts and neuronal-like differentiation in rat PC12 cells. Interestingly, the proto-oncogenic version of TC21 shows also a lower, but significant, activity in both biological processes. We also demonstrate that the similarity of the cellular responses induced by TC21 and Ras derive from the utilization of overlapping pathways. Thus, the exchange of guanosine nucleotides in wild type TC21 is catalyzed by Ras exchange factors. Moreover, TC21 binds physically to c-Raf-1 in a GTP-dependent manner. Finally, overexpression of TC21G23V in NIH3T3 cells results in the activation of c-Raf-1 and the MAPK and the JNK branches of serine/threonine cascades. From these results, we conclude that TC21 promotes Ras-like responses in diverse cell types due to the use of overlapping, if not identical, signaling elements of the Ras oncogenic pathway.

• Fischer KD, Tedford K, Penninger JM
• Vav links antigen-receptor signaling to the actin cytoskeleton.
• Semin Immunol. 1998; 10: 317-27
• Display abstract

• The haematopoietic-specific Rho-family guanine-nucleotide exchange factor Vav is a regulator of lymphocyte antigen receptor signaling leading to proliferation of B and T cells, generation of the B1 cell lineage and IL-2 production and maturation in T cells. The specific role it plays in these events, however, has not yet been resolved. Recent findings suggest that Vav is recruited to activated antigen receptors and requires both tyrosine phosphorylation and the presence of activating phospholipids for catalytic activity towards Rho-family GTPases. Studies form vav-deficient mice show that in response to antigen receptor activation, Vav is not essential for activation of JNK kinase pathways, but is required for actin polymerisation and T cell capping. We discuss Vav function in the light of these new findings.

• Cantrell D
• Lymphocyte signalling: a coordinating role for Vav?
• Curr Biol. 1998; 8: 5358-5358
• Display abstract

• Vav1 is a guanine nucleotide exchange factor that selectively activates the Rac1 GTPase and is expressed specifically in haematopoietic cells. Recent work has revealed how Vav1 integrates signals from lymphocyte antigen receptors and costimulatory receptors to control immune function.

• Miki T
• [Oncogenes that activate the Rho family of small GTP-binding proteins]
• Tanpakushitsu Kakusan Koso. 1998; 43: 25-34

• Whitehead IP, Abe K, Gorski JL, Der CJ
• CDC42 and FGD1 cause distinct signaling and transforming activities.
• Mol Cell Biol. 1998; 18: 4689-97
• Display abstract

• Activated forms of different Rho family members (CDC42, Rac1, RhoA, RhoB, and RhoG) have been shown to transform NIH 3T3 cells as well as contribute to Ras transformation. Rho family guanine nucleotide exchange factors (GEFs) (also known as Dbl family proteins) that activate CDC42, Rac1, and RhoA also demonstrate oncogenic potential. The faciogenital dysplasia gene product, FGD1, is a Dbl family member that has recently been shown to function as a CDC42-specific GEF. Mutations within the FGD1 locus cosegregate with faciogenital dysplasia, a multisystemic disorder resulting in extensive growth impairments throughout the skeletal and urogenital systems. Here we demonstrate that FGD1 expression is sufficient to cause tumorigenic transformation of NIH 3T3 fibroblasts. Although both FGD1 and constitutively activated CDC42 cooperated with Raf and showed synergistic focus-forming activity, both quantitative and qualitative differences in their functions were seen. FGD1 and CDC42 also activated common nuclear signaling pathways. However, whereas both showed comparable activation of c-Jun, CDC42 showed stronger activation of serum response factor and FGD1 was consistently a better activator of Elk-1. Although coexpression of FGD1 with specific inhibitors of CDC42 function demonstrated the dependence of FGD1 signaling activity on CDC42 function, FGD1 signaling activities were not always consistent with the direct or exclusive stimulation of CDC42 function. In summary, FGD1 and CDC42 signaling and transformation are distinct, thus suggesting that FGD1 may be mediating some of its biological activities through non-CDC42 targets.

• Kozasa T et al.
• p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13.
• Science. 1998; 280: 2109-11
• Display abstract

• Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.

• Aghazadeh B et al.
• Structure and mutagenesis of the Dbl homology domain.
• Nat Struct Biol. 1998; 5: 1098-107
• Display abstract

• Guanine nucleotide exchange factors in the Dbl family activate Rho GTPases by accelerating dissociation of bound GDP, promoting acquisition of the GTP-bound state. Dbl proteins possess a approximately 200 residue catalytic Dbl-homology (DH) domain, that is arranged in tandem with a C-terminal pleckstrin homology (PH) domain in nearly all cases. Here we report the solution structure of the DH domain of human PAK-interacting exchange protein (betaPIX). The domain is composed of 11 alpha-helices that form a flattened, elongated bundle. The structure explains a large body of mutagenesis data, which, along with sequence comparisons, identify the GTPase interaction site as a surface formed by three conserved helices near the center of one face of the domain. Proximity of the site to the DH C-terminus suggests a means by which PH-ligand interactions may be coupled to DH-GTPase interactions to regulate signaling through the Dbl proteins in vivo.

• Landino LM, Macdonald TL
• Inhibition of the GDP/GTP exchange reaction of ras p21 by aluminum ion.
• J Inorg Biochem. 1997; 66: 99-102
• Display abstract

• In an effort to understand the biochemical mechanisms of aluminum-induced neurotoxicity, we investigated the effects of aluminum ion, Al3+, on the Mg(2+)- and nucleotide-dependent protein, ras p21. Picomolar Al3+ concentrations inhibited the GTPase activity of ras p21 in an Mg(2+)-dependent manner, consistent with an Al3+/Mg2+ competition mechanism. GTPase activity was inhibited by 60% in the presence of 100 microM Mg2+ and 2.9 x 10(-10) M Al3+. Kinetic studies demonstrated that the mode of Al(3+)-induced inhibition of ras p21 GTPase activity changed from competitive to mixed non-competitive as the number of ras p21 turnovers increased. Further dissection of the ras p21 cycle revealed that Mg(2+)-dependent GDP/GTP exchange was the Al(3+)-sensitive step.

• Han J et al.
• Lck regulates Vav activation of members of the Rho family of GTPases.
• Mol Cell Biol. 1997; 17: 1346-53
• Display abstract

• Vav is a member of a family of oncogene proteins that share an approximately 250-amino-acid motif called a Dbl homology domain. Paradoxically, Dbl itself and other proteins containing a Dbl domain catalyze GTP-GDP exchange for Rho family proteins, whereas Vav has been reported to catalyze GTP-GDP exchange for Ras proteins. We present Saccharomyces cerevisiae genetic data, in vitro biochemical data, and animal cell biological data indicating that Vav is a guanine nucleotide exchange factor for Rho-related proteins, but in similar genetic and biochemical experiments we fail to find evidence that Vav is a guanine nucleotide exchange factor for Ras. Further, we present data indicating that the Lck kinase activates the guanine nucleotide exchange factor and transforming activity of Vav.

• Li R, Zheng Y
• Residues of the Rho family GTPases Rho and Cdc42 that specify sensitivity to Dbl-like guanine nucleotide exchange factors.
• J Biol Chem. 1997; 272: 4671-9
• Display abstract

• The Dbl-like guanine nucleotide exchange factor (GEF) Lbc oncoprotein specifically activates the small GTP-binding protein Rho in mammalian fibroblasts to induce transformation and actin stress fiber formation, whereas another Dbl-related molecule, Cdc24, stimulates guanine nucleotide exchange of the Rho family GTPase Cdc42 to elicit effects on both gene induction and actin-based cytoskeleton change in Saccharomyces cerevisiae. To understand the mechanism of these functional interactions, we have taken a biochemical approach to probe the sites on Rho and Cdc42 that are involved in coupling to their respective GEFs, the Lbc and Cdc24 proteins. Point mutations in the switch II region of the small G-proteins, many of which would affect the interaction with GEF in the case of Ras, or a mutation in the switch I region that was identified as a contact site between Rab3A and Rab GEF had little effect on RhoA or Cdc42Hs with regard to the ability to interact with Lbc or Cdc24, suggesting that there exists a unique mechanism of regulation of the Rho family proteins by their GEFs. Analysis of a panel of chimeras made between RhoA and Cdc42Hs, which all maintained the ability to respond to Dbl, their mutual GEF, and to GTPase-activating protein, revealed that at least two distinct sites in each of the GTPases are required for activation by the respective GEFs. Further site-directed mutagenesis studies showed that the conserved residue Tyr32 in the putative effector region of both GTPases (numbered by Cdc42Hs) is critical for binding of the GEFs and that specific recognition for Lbc or Cdc24 is achieved at least in part through residues Lys27 of Rho and Gln116 of Cdc42. Moreover, the loss of GEF responsiveness of a RhoA mutation (D76Q) was found to be caused by the impaired GEF catalysis, not by a change in the GEF binding affinity. Together, these results indicate that multiple sites of the Rho GTPases are involved in the regulation by GEFs, contributing to GEF binding or GEF catalysis, and raise the possibility that activation of each Rho family G-protein by a specific GEF may engage in a distinct mechanism.

• Glaven JA, Whitehead IP, Nomanbhoy T, Kay R, Cerione RA
• Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein.
• J Biol Chem. 1996; 271: 27374-81
• Display abstract

• Lfc and Lsc are two recently identified oncoproteins that contain a Dbl homology domain in tandem with a pleckstrin homology domain and thus share sequence similarity with a number of other growth regulatory proteins including Dbl, Tiam-1, and Lbc. We show here that Lfc and Lsc, like their closest relative Lbc, are highly specific guanine nucleotide exchange factors (GEFs) for Rho, causing a >10-fold stimulation of [3H]GDP dissociation from Rho and a marked stimulation of GDP-[35S]GTPgammas (guanosine 5'-O-(3-thiotriphosphate) exchange. All three proteins (Lbc, Lfc, and Lsc) are able to act catalytically in stimulating the guanine nucleotide exchange activity, such that a single molecule of each of these oncoproteins can activate a number of molecules of Rho. Neither Lfc nor Lsc shows any ability to stimulate GDP dissociation from other related GTP-binding proteins such as Rac, Cdc42, or Ras. Thus Lbc, Lfc, and Lsc appear to represent a subgroup of Dbl-related proteins that function as highly specific GEFs toward Rho and can be distinguished from Dbl, Ost, and Dbs which are less specific and show GEF activity toward both Rho and Cdc42. Consistent with these results, Lbc, Lfc, and Lsc each form tight complexes with the guanine nucleotide-depleted form of Rho and bind weakly to the GDP- and GTPgammaS-bound states. None of these oncoproteins are able to form complexes with Cdc42 or Ras. However, Lfc (but not Lbc nor Lsc) can bind to Rac, and this binding occurs equally well when Rac is nucleotide-depleted or is in the GDP- or GTPgammaS-bound state. These findings raise the possibility that in addition to acting directly as a GEF for Rho, Lfc may play other roles that influence the signaling activities of Rac and/or coordinate the activities of the Rac and Rho proteins.

• Olson MF, Pasteris NG, Gorski JL, Hall A
• Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases.
• Curr Biol. 1996; 6: 1628-33
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• BACKGROUND: Dbl, a guanine nucleotide exchange factor (GEF) for members of the Rho family of small GTPases, is the prototype of a family of 15 related proteins. The majority of proteins that contain a DH (Dbl homology) domain were isolated as oncogenes in transfection assays, but two members of the DH family, FGD1 (the product of the faciogenital dysplasia or Aarskog-Scott syndrome locus) and Vav, have been shown to be essential for normal embryonic development. Mutations to the FGD1 gene result in a human developmental disorder affecting specific skeletal structures, including elements of the face, cervical vertebrae and distal extremities. Homozygous Vav-/- knockout mice embryos are not viable past the blastocyst stage, indicating an essential role of Vav in embryonic implantation. RESULTS: Here, we show that the microinjection of FGD1 and Vav into Swiss 3T3 fibroblasts induces the polymerization of actin and the assembly of clustered integrin complexes. FGD1 activates Cdc42, whereas Vav activates Rho, Rac and Cdc42. In addition, FGD1 and Vav stimulate the mitogen activated protein kinase cascade that leads to activation of the c-Jun kinase SAPK/JNK1. CONCLUSIONS: We conclude that FGD1 and Vav are regulators of the Rho GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.

• Whitehead IP, Khosravi-Far R, Kirk H, Trigo-Gonzalez G, Der CJ, Kay R
• Expression cloning of lsc, a novel oncogene with structural similarities to the Dbl family of guanine nucleotide exchange factors.
• J Biol Chem. 1996; 271: 18643-50
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• In a screen for genes with oncogenic potential expressed by the murine B6SUtA1 myeloid progenitor cell line, we isolated a 2. 5-kilobase pair cDNA whose expression causes strong morphological transformation and deregulated proliferation of NIH 3T3 cells. The transforming cDNA encodes a truncated protein (designated Lsc) with a region of sequence similarity to the product of the lbc oncogene. This region includes the tandem Dbl homology and pleckstrin homology domains that are hallmarks of the Dbl-like proteins, a family of presumptive or demonstrated guanine nucleotide exchange factors that act on Rho family GTPases. Lsc requires intact Dbl homology and pleckstrin homology domains for its oncogenic activity. The transforming activity of Lsc in NIH 3T3 cells is reduced by cotransfection with p190 (a GTPase activating protein for Rho family GTPases) and the Rho family dominant-negative mutants RhoA(19N), CDC42(17N), and Rac1(17N). These results indicate a role for the Rho family of GTPases in mediating the transforming activity of Lsc and are consistent with the exchange specificities that have been attributed to Dbl family members. The lsc gene is expressed in a variety of tissues and is particularly abundant in hemopoietic tissues (thymus, spleen, and bone marrow). Lsc is a member of a growing family of proteins that may function as activators of Rho family GTPases in a developmental or tissue-specific manner.

• Bustelo XR
• The VAV family of signal transduction molecules.
• Crit Rev Oncog. 1996; 7: 65-88
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• This review summarizes the current knowledge on the structure, expression, and physiological roles of the Vav family, a novel group of signaling transducers with known representatives in mammalian (Vav and Vav-2) and nematodes (Cel Vav). Vav was the first member of this family identified during the course of gene transfer experiments aimed at characterizing loci involved in human neoplasia. This transforming protein displays a complex array of structural motifs, including calponin-homology, acidic, dbl-homology, pleckstrin-homology, cysteine-rich, SH3, and SH2 domains. After activation of cells with extracellular stimuli, Vav becomes phosphorylated on tyrosine residues and catalyzes the exchange of guanosine nucleotides on the GTP-binding protein Rac-1, thereby allowing the transition of this GTPase from the inactive (GDP-loaded) to the active (GTP-loaded) state. In addition, Vav associates with phosphorylated receptors, protein tyrosine kinases, and intracellular phosphoproteins whose identities are now being determined. Gene targeting experiments indicate that vav gene disruption results in severe signaling defects in lymphoid cells, further reinforcing its role as a key regulator of mitogenic pathways. Vav-2 and C. elegans Vav bear significant structural similarity with Vav, suggesting that they will be important players as well in evolutionarily conserved signal transduction pathways involved in mitogenesis and cellular transformation.

• Boy-Marcotte E, Buu A, Soustelle C, Poullet P, Parmeggiani A, Jacquet M
• The C-terminal part of the CDC25 gene product has Ras-nucleotide exchange activity when present in a chimeric SDC25-CDC25 protein.
• Curr Genet. 1993; 23: 397-401
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• The CDC25 gene from S. cerevisiae encodes an activator of Ras proteins. The C-terminal part of a structurally-related protein encoded by the SDC25 gene is characterised by a Ras-guanine nucleotide exchange activity in vitro whereas the C-terminal part of CDC25 gives no detectable exchange activity. A chimera between the 3' regions of these two genes was constructed by homeologous recombination. This chimeric gene suppresses cdc25 mutations. When expressed in E. coli, the chimeric product is detectable by antibodies directed against the carboxy-terminal CDC25 peptide and has an exchange-factor activity on the Ras2 protein. Therefore, the carboxy-terminal parts of both the CDC25 and the SDC25 gene products are structurally and functionally similar. The CDC25 part of the chimeric protein contains an intrinsic guanine exchange factor which does not require an additional cofactor.

• Heisterkamp N, Kaartinen V, van Soest S, Bokoch GM, Groffen J
• Human ABR encodes a protein with GAPrac activity and homology to the DBL nucleotide exchange factor domain.
• J Biol Chem. 1993; 268: 16903-6
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• We have previously cloned a segment of a gene, ABR, homologous to the BCR gene, which encodes a protein consisting of three distinct functional domains. In the present study, genomic ABR sequences were used to isolate human ABR cDNAs. Surprisingly, the two types of ABR cDNAs identified differed only in their most 5' coding sequences. These are predicted to encode proteins of 93.5 and 92.3 kDa molecular mass. ABR showed a differential expression pattern in various mouse tissues, analogous to that of BCR, and the highest level was found in brain. Similar to BCR, ABR contains a region with homology to DBL, vav, and CDC24, which are likely to or have been shown to encode GTP exchange factors. A domain of ABR with similarity to GAPrho was expressed as a fusion protein in Escherichia coli and was shown to have GAP activity toward rac. Although both ABR and BCR have GAP activity, ABR lacks homology to the serine/threonine kinase domain of BCR. Therefore, ABR is likely to have cellular functions overlapping with but also distinct from those of BCR.

• Hori Y
• Function of the post-translationally modified C-terminal region of rho p21.
• Kobe J Med Sci. 1992; 38: 79-92
• Display abstract

• rhoA p21, a ras p21-like small GTP-binding protein, purified from bovine aortic smooth muscle is similarly modified at its C-terminal region as described for ras p21s. In this study, I examined the role of the post-translational modifications of the C-terminal region of rhoA p21 by comparing bovine rhoA p21 with bacterially-produced rhoA p21 because the bacterial protein was not modified. Bovine rhoA p21 bound to plasma membranes, but bacterial rhoA p21 did not. Both the stimulatory and inhibitory GDP/GTP exchange proteins for bovine rhoA p21 were inactive for bacterial rhoA p21. On the other hand, the GTPase activating protein for bovine rhoA p21 was also active for bacterial rhoA p21. These results indicate that the post-translational modifications of the C-terminal region of bovine rhoA p21, which are absent in bacterial rhoA p21, are essential for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GTPase activating protein.