Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases

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.     

Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation.

Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl-like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl-induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated ("fast-cycling") mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation.     

Leukemia-associated Rho guanine nucleotide exchange factor, a Dbl family protein found mutated in leukemia, causes transformation by activation of RhoA

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) was originally identified as a fusion partner with mixed-lineage leukemia in a patient with acute myeloid leukemia. LARG possesses a tandem Dbl homology and pleckstrin homology domain structure and, consequently, may function as an activator of Rho GTPases. In this study, we demonstrate that LARG is a functional Dbl protein. Expression of LARG in cells caused activation of the serum response factor, a known downstream target of Rho-mediated signaling pathways. Transient overexpression of LARG did not activate the extracellular signal-regulated kinase or c-Jun NH(2)-terminal kinase mitogen-activated protein kinase cascade, suggesting LARG is not an activator of Ras, Rac, or Cdc42. We performed in vitro exchange assays where the isolated Dbl homology (DH) or DH/pleckstrin homology domains of LARG functioned as a strong activator of RhoA, but exhibited no activity toward Rac1 or Cdc42. We found that LARG could complex with RhoA, but not Rac or Cdc42, in vitro, and that expression of LARG caused an increase in the levels of the activated GTP-bound form of RhoA, but not Rac1 or Cdc42, in vivo. Thus, we conclude that LARG is a RhoA-specific guanine nucleotide exchange factor. Finally, like activated RhoA, we determined that LARG cooperated with activated Raf-1 to transform NIH3T3 cells. These data demonstrate that LARG is the first functional Dbl protein mutated in cancer and indicate LARG-mediated activation of RhoA may play a role in the development of human leukemias.     

Differential requirement for Rho family GTPases in an oncogenic insulin-like growth factor-I receptor-induced cell transformation

Insulin-like growth factor I receptor (IGFR) plays an important role in cell growth and transformation. We dissected the downstream signaling pathways of an oncogenic variant of IGFR, Gag-IGFR, called NM1. Loss of function mutants of NM1, Phe-1136 and dS2, that retain kinase activity but are attenuated in their transforming ability were used to identify signaling pathways that are important for transformation of NIH 3T3 cells. MAPK, phospholipase C gamma, and Stat3 were activated to the same extent by NM1 and its two mutants, suggesting that activation of these pathways, individually or in combination, was not sufficient for NM1-induced cell transformation. The mutant dS2 has decreased IRS-1 phosphorylation levels and IRS-1-associated phosphatidylinositol 3'-kinase activity, suggesting that this impairment may be in part responsible for the defectiveness of dS2. We show that Rho family members, RhoA, Rac1, and Cdc42 are activated by NM1, and this activation, particularly RhoA and Cdc42, is attenuated in both mutants of NM1. Dominant negative mutants of Rho, Rac, and Cdc42 inhibited NM1-induced cell transformation, as measured by focus and colony forming ability. Dominant negative Rho most potently inhibited the focus forming activity, whereas Cdc42 was most effective in inhibiting the colony forming ability of NM1-expressing cells. Conversely, constitutively activated (ca) Rho is more effective than ca Rac or ca Cdc42 in rescuing the focus forming ability of the mutants. By contrast, ca Cdc42 is most effective in rescuing the colony forming ability of both mutants.     

Activation of clg, a novel dbl family guanine nucleotide exchange factor gene, by proviral insertion at evi24, a common integration site in B cell and myeloid leukemias

Retroviruses induce leukemia in inbred strains of mice by activating cellular proto-oncogenes and/or inactivating tumor suppressors. The proviral integration sites in these leukemias provide powerful genetic tags for disease gene identification. Here we show that Evi24, a common site of retroviral integration in AKXD B cell and BXH-2 myeloid leukemias, contains a novel Dbl family guanine nucleotide exchange factor gene. We have designated this gene Clg (common-site lymphoma/leukemia guanine nucleotide exchange factor). Proviral integrations on chromosome 7 at Evi24 are located 7.6-10.3 kb upstream of Clg and increased Clg expression 2-5-fold compared with leukemias lacking proviral integrations at Evi24. Clg contains Dbl/pleckstrin homology domains with substantial sequence homology to many Rho family activators, including the transforming Dbl and Dbs/Ost oncogenes. Nucleotide exchange assays indicated that Clg specifically activated nucleotide exchange on Cdc42, but not RhoA or Rac1, in vitro. NIH 3T3 transfection studies showed that overexpression of full-length and carboxyl-terminally truncated forms of Clg morphologically transformed NIH 3T3 cells. This study and studies showing that the human homolog of EVI24 is located in a region of 19q13 frequently amplified in B cell lymphomas and pancreatic and breast cancers implicate Clg and Cdc42 activation in mouse and human cancers.     

Requirement for Akt-mediated survival in cell transformation by the dbl oncogene

The dbl oncogene product is the founding member of a large family of oncogenic proteins that function by activating the small GTP-binding proteins Cdc42, Rac and Rho. Through its substrate GTPases, Dbl transduces proliferative signals from cell-surface receptors to diverse cellular effectors and signaling pathways. The mechanisms by which these multiple signals are integrated, as well as their relative contribution to Dbl-induced cell transformation, are presently poorly understood. We investigated the role of the survival regulators PI3-kinase and Akt in Dbl-induced cell transformation. We found that Dbl induced the phosphorylation of Akt on threonine 308, through the GTPases Rac and Cdc42 and in a PI3-kinase dependent manner. Pharmacological or biochemical interference with this pathway lead to a marked, dose-dependent inhibition of the focus formation activity exhibited by Dbl-expressing cells. Dbl expression stimulated the phosphorylation of the anti-apoptotic Akt substrate Bad, and caused a marked decrease in basal levels of apoptosis. Finally, we found that activated Cdc42 existed in cells in complex with phosphoionositide-dependent kinase-1 (PDK1), the downstream mediator of PI3-kinase action. The data indicate that Dbl signaling stimulate the formation of a novel survival complex, through which anti-apoptotic signals are generated and propagated.     

Vav transformation requires activation of multiple GTPases and regulation of gene expression

Although Vav can act as a guanine nucleotide exchange factor for RhoA, Rac1, and Cdc42, its transforming activity has been ascribed primarily to its ability to activate Rac1. However, because activated Vav, but not Rac-specific guanine nucleotide exchange factors, exhibits very potent focus-forming transforming activity when assayed in NIH 3T3 cells, Vav transforming activity must also involve activation of Rac-independent pathways. In this study, we determined the involvement of other Rho family proteins and their signaling pathways in Vav transformation. We found that RhoA, Rac1, and Cdc42 functions are all required for Vav transforming activity. Furthermore, we determined that Vav activation of nuclear factor-kappaB and the Jun NH2-terminal kinase mitogen-activated protein kinase (MAPK) is necessary for full transformation by Vav, whereas p38 MAPK does not seem to play an important role. We also determined that Vav is a weak activator of Elk-1 via a Ras- and MAPK/extracellular signal-regulated kinase kinase-dependent pathway, and this activity was essential for Vav transformation. Thus, we conclude that full Vav transforming activation is mediated by the activation of multiple small GTPases and their subsequent activation of signaling pathways that regulate changes in gene expression. Because Vav is activated by the epidermal growth factor receptor and other tyrosine kinases involved in cancer development, defining the role of aberrant Vav signaling may identify activities of receptor tyrosine kinases important for human oncogenesis.     

Activation of Dbl restores migration in polyamine-depleted intestinal epithelial cells via Rho-GTPases

Integrin binding to the extracellular matrix (ECM) activated Rho GTPases, Src, and focal adhesion kinase in intestinal epithelial cells (IEC)-6. Polyamine depletion inhibited activities of Rac1, RhoA, and Cdc42 and thereby migration. However, constitutively active (CA) Rac1 expression abolished the inhibitory effect of polyamine depletion, indicating that polyamines are involved in a process upstream of Rac1. In the present study, we examined the role of polyamines in the regulation of the guanine nucleotide exchange factor, diffuse B-cell lymphoma (Dbl), for Rho GTPases. Polyamine depletion decreased the level as well as the activation of Dbl protein. Dbl knockdown by siRNA altered cytoskeletal structure and decreased Rac1 activity and migration. Cells expressing CA-Dbl increased migration, Rac1 activity, and proliferation. CA-Dbl restored migration in polyamine-depleted cells by activating RhoA, Rac1, and Cdc42. CA-Dbl caused extensive reorganization of the F-actin cortex into stress fibers. Inhibition of Rac1 by NSC23766 significantly decreased migration of vector-transfected cells and CA-Dbl-transfected cells. However, the inhibition of migration was significantly higher in the vector-transfected cells compared with that seen in the CA-Dbl-transfected cells. Dbl localized in the perinuclear region in polyamine-depleted cells, whereas it localized with the stress fibers in control cells. CA-Dbl localized with stress fibers in both the control and polyamine-depleted cells. These results suggest that polyamines regulate the activation of Dbl, a membrane-proximal process upstream of Rac1.     

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

The ERM proteins (ezrin, radixin, and moesin) are a group of band 4.1-related proteins that are proposed to function as membrane/cytoskeletal linkers. Previous biochemical studies have implicated RhoA in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. We show that lysophosphatidic acid stimulation of serum-starved NIH3T3 cells resulted in relocalization of radixin into apical membrane/actin protrusions, which was blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, was sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment led to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin revealed that RhoA activity is necessary and sufficient for their phosphorylation. These findings reveal a novel function of RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins.     

Src kinase regulates the activation of a novel FGD-1-related Cdc42 guanine nucleotide exchange factor in the signaling pathway from the endothelin A receptor to JNK

Small GTPases act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state. Upon stimulation with extracellular signals, guanine-nucleotide exchange factors (GEFs) stimulate the exchange of GDP to GTP to shift toward the active forms of small GTPases, recognizing the downstream targets. Here we show that KIAA0793, containing substantial sequence homology with the catalytic Dbl homology domain of the faciogenital dysplasia gene product (FGD1), is a specific GEF for Cdc42. We, therefore, tentatively named it FRG (FGD1-related Cdc42-GEF). Src kinase directly phosphorylates and activates FRG, as Vav family GEFs. Additionally, FRG is involved in the signaling pathway from the endothelin A receptor to c-Jun N-terminal kinase, resulting in the inhibition of cell motility. These results suggest that FRG is a member of Cdc42-GEF and plays an important role in the signaling pathway downstream of G protein-coupled receptors.     

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.