The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of RhoGTPases. The structure of the Vav1-DH-PH-CRD/Rac1 complex to 2.6 Å resolution reveals a unique intramolecular network of contacts between the Vav-1 cysteine rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav-1 DH domain for its intimate association with the Switch-II region of Rac1 that is critical for the displacement of the guanine nucleotide. Further, a mutational analysis confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1’s ability to contact a range of RhoGTPases using a novel protein-protein interaction network.
The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.
Vav and Dbl are members of a novel class of oncogene proteins that share significant sequence identity in a approximately 250-amino-acid domain, designated the Dbl homology domain. Although Dbl functions as a guanine nucleotide exchange factor (GEF) and activator of Rho family proteins, recent evidence has demonstrated that Vav functions as a GEF for Ras proteins. Thus, transformation by Vav and Dbl may be a consequence of constitutive activation of Ras and Rho proteins, respectively. To address this possibility, we have compared the transforming activities of Vav and Dbl with that of the Ras GEF, GRF/CDC25. As expected, GRF-transformed cells exhibited the same reduction in actin stress fibers and focal adhesions as Ras-transformed cells. In contrast, Vav- and Dbl-transformed cells showed the same well-developed stress fibers and focal adhesions observed in normal or RhoA(63L)-transformed NIH 3T3 cells. Furthermore, neither Vav- or Dbl-transformed cells exhibited the elevated levels of Ras-GTP (60%) observed with GRF-transformed cells. Finally, GRF, but not Vav or Dbl, induced transcriptional activation from Ras-responsive DNA elements (ets/AP-1, fos promoter, and kappa B). However, like Ras- and GRF-transformed cells, both Vav- and Dbl-transformed cells exhibited constitutively activated mitogen-activated protein kinases (MAPKs) (primarily p42MAPK/ERK2). Since kinase-deficient forms of p42MAPK/ERK2 and p44MAPK/ERK1 inhibited Dbl transformation, MAPK activation may be an important component of its transforming activity. Taken together, our observations indicate that Vav and Dbl transformation is not a consequence of Ras activation and instead may involve the constitutive activation of MAPKs.
Rho GTPases play an important role in cell migration. Here the activity of the Rho GTPase GEF Vav2 is shown to be regulated by a phosphorylation-dependent interaction with PKL (GIT2). PKL is required for Vav2 activation, and, in turn, Vav2 regulates the localization of PKL and β-PIX to focal adhesions and to the leading edge of migrating cells.
The Rho family of GTPases plays an important role in coordinating dynamic changes in the cell migration machinery after integrin engagement with the extracellular matrix. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs). However, the mechanisms by which GEFs and GAPs are spatially and temporally regulated are poorly understood. Here the activity of the proto-oncogene Vav2, a GEF for Rac1, RhoA, and Cdc42, is shown to be regulated by a phosphorylation-dependent interaction with the ArfGAP PKL (GIT2). PKL is required for Vav2 activation downstream of integrin engagement and epidermal growth factor (EGF) stimulation. In turn, Vav2 regulates the subsequent redistribution of PKL and the Rac1 GEF β-PIX to focal adhesions after EGF stimulation, suggesting a feedforward signaling loop that coordinates PKL-dependent Vav2 activation and PKL localization. Of interest, Vav2 is required for the efficient localization of PKL and β-PIX to the leading edge of migrating cells, and knockdown of Vav2 results in a decrease in directional persistence and polarization in migrating cells, suggesting a coordination between PKL/Vav2 signaling and PKL/β-PIX signaling during cell migration.
Vav proteins are phosphorylation-dependent GDP/GTP exchange factors for Rho/Rac GTPases. Despite intense characterization of mammalian Vav proteins both biochemically and genetically, there is little information regarding the conservation of their biological properties in lower organisms. To approach this issue, we have performed a characterization of the regulatory, catalytic, and functional properties of the single Vav family member of Drosophila melanogaster. These analyses have shown that the intramolecular mechanisms controlling the enzyme activity of mammalian Vav proteins are already present in Drosophila, suggesting that such properties have been set up before the divergence between protostomes and deuterostomes during evolution. We also show that Drosophila and mammalian Vav proteins have similar catalytic specificities. As a consequence, Drosophila Vav can trigger oncogenic transformation, morphological change, and enhanced cell motility in mammalian cells. Gain-of-function studies using transgenic flies support the implication of this protein in cytoskeletal-dependent processes such as embryonic dorsal closure, myoblast fusion, tracheal development, and the migration/guidance of different cell types. These results highlight the important roles of Vav proteins in the signal transduction pathways regulating cytoskeletal dynamics. Moreover, they indicate that the foundations for the regulatory and enzymatic activities of this protein family have been set up very early during evolution.
Vav oncoproteins; Rho/Rac GTPases; GDP/GTP exchange factors; Cell migration; Development; Cytoskeleton; Drosophila
Vav proteins are phosphorylation-dependent Rho/Rac exchange factors that have usually been associated with immune- and cardiovascular-related functions. In this paper, Quevedo et al. demonstrate that Vav3 plays important, although transient, pleiotropic roles during the postnatal development of the cerebellum.
Vav3 is a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases that has been involved in functions related to the hematopoietic system, bone formation, cardiovascular regulation, angiogenesis, and axon guidance. We report here that Vav3 is expressed at high levels in Purkinje and granule cells, suggesting additional roles for this protein in the cerebellum. Consistent with this hypothesis, we demonstrate using Vav3-deficient mice that this protein contributes to Purkinje cell dendritogenesis, the survival of granule cells of the internal granular layer, the timely migration of granule cells of the external granular layer, and to the formation of the cerebellar intercrural fissure. With the exception of the latter defect, the dysfunctions found in Vav3−/− mice only occur at well-defined postnatal developmental stages and disappear, or become ameliorated, in older animals. Vav2-deficient mice do not show any of those defects. Using primary neuronal cultures, we show that Vav3 is important for dendrite branching, but not for primary dendritogenesis, in Purkinje and granule cells. Vav3 function in the cerebellum is functionally relevant, because Vav3−/− mice show marked motor coordination and gaiting deficiencies in the postnatal period. These results indicate that Vav3 function contributes to the timely developmental progression of the cerebellum.
Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor– or epidermal growth factor–dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor–dependent activation of Rac leading to lamellipodia.
Vav; Rac; spreading; integrins; growth factors
The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal–regulated kinase (ERK) and protein kinase D1 (PKD1), and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.
The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay, and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D-NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.
Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unexpectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modulators of Vav2 DH domain function in vivo.
The Vav family is a group of signal transduction molecules that activate Rho/Rac GTPases during cell signaling. Experiments using knockout mice have indicated that the three Vav proteins present in mammals (Vav1, Vav2, and Vav3) are essential for proper signaling responses in hematopoietic cells. However, Vav2 and Vav3 are also highly expressed in nonhematopoietic tissues, suggesting that they may have additional functions outside blood cells. Here, we report that this is the case for Vav2, because the disruption of its locus in mice causes tachycardia, hypertension, and defects in the heart, arterial walls, and kidneys. We also provide physiological and pharmacological evidence demonstrating that the hypertensive condition of Vav2-deficient mice is due to a chronic stimulation of the renin/angiotensin II and sympathetic nervous systems. Together, these results indicate that Vav2 plays crucial roles in the maintenance of cardiovascular homeostasis in mice.
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.
Vav3 is a phosphorylation GDP/GTP exchange factor for Rho/Rac GTPases. Recently, it has been described that Vav3 knockout mice develop hypertension and sympathoexcitation. In this work, we report the neurological cause of this phenotype.
Vav3 is a phosphorylation-dependent activator of Rho/Rac GTPases that has been implicated in hematopoietic, bone, cerebellar, and cardiovascular roles. Consistent with the latter function, Vav3-deficient mice develop hypertension, tachycardia, and renocardiovascular dysfunctions. The cause of those defects remains unknown as yet. Here, we show that Vav3 is expressed in GABAegic neurons of the ventrolateral medulla (VLM), a brainstem area that modulates respiratory rates and, via sympathetic efferents, a large number of physiological circuits controlling blood pressure. On Vav3 loss, GABAergic cells of the caudal VLM cannot innervate properly their postsynaptic targets in the rostral VLM, leading to reduced GABAergic transmission between these two areas. This results in an abnormal regulation of catecholamine blood levels and in improper control of blood pressure and respiration rates to GABAergic signals. By contrast, the reaction of the rostral VLM to excitatory signals is not impaired. Consistent with those observations, we also demonstrate that Vav3 plays important roles in axon branching and growth cone morphology in primary GABAergic cells. Our study discloses an essential and nonredundant role for this Vav family member in axon guidance events in brainstem neurons that control blood pressure and respiratory rates.
We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.
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.
Vav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity.
We found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation.
Our results indicate that increased migration of active Vav1 expressing cells is dependent on Vav1 GEF activity and secretion of an EGF receptor ligand. In addition, activation of ERK downstream of Vav1 is dependent on autocrine EGF receptor stimulation while active Vav1 can stimulate Rac1 and PAK activation independent of ligand binding to the EGF receptor. Thus, stimulation of migration by activated Vav1 involves both EGF receptor-dependent and independent activities induced through the Rho GEF domain of Vav1.
Phagocytosis is the process whereby cells direct the spatially localized, receptor-driven engulfment of particulate materials. It proceeds via remodeling of the actin cytoskeleton and shares many of the core cytoskeletal components involved in adhesion and migration. Small GTPases of the Rho family have been widely implicated in coordinating actin dynamics in response to extracellular signals and during diverse cellular processes, including phagocytosis, yet the mechanisms controlling their recruitment and activation are not known. We show herein that in response to ligation of Fc receptors for IgG (FcγR), the guanine nucleotide exchange factor Vav translocates to nascent phagosomes and catalyzes GTP loading on Rac, but not Cdc42. The Vav-induced Rac activation proceeds independently of Cdc42 function, suggesting distinct roles for each GTPase during engulfment. Moreover, inhibition of Vav exchange activity or of Cdc42 activity does not prevent Rac recruitment to sites of particle attachment. We conclude that Rac is recruited to Fcγ membrane receptors in its inactive, GDP-bound state and that Vav regulates phagocytosis through subsequent catalysis of GDP/GTP exchange on Rac.
Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFκB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways.
The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late “transitional” stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)–induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho–guanine nucleotide exchange factors.
thymocyte; antigen receptor; signal transduction; Ca++; mitogen-activated protein kinase
vav1 has been shown to play a key role in lymphocyte development and activation, but its potential importance in macrophage activation has received little attention. We have previously reported that exposure of macrophages to bacterial lipopolysaccharide (LPS) leads to increased activity of hck and other src-related tyrosine kinases and to the prompt phosphorylation of vav1 on tyrosine. In this study, we tested the role of vav1 in macrophage responses to LPS, focusing on the upregulation of nuclear factor for interleukin-6 expression (NF-IL-6) activity and inducible nitric oxide synthase (iNOS) protein accumulation in RAW-TT10 murine macrophages. We established a series of stable cell lines expressing three mutant forms of vav1 in a tetracycline-regulatable fashion: (i) a form producing a truncated protein, vavC; (ii) a form containing a point mutation in the regulatory tyrosine residue, vavYF174; and (iii) a form with an in-frame deletion of 6 amino acids required for the guanidine nucleotide exchange factor (GEF) activity of vav1 for rac family GTPases, vavGEFmt. Expression of the truncated mutant (but not the other two mutants) has been reported to interfere with T-cell activation. In contrast, we now demonstrate that expression of any of the three mutant forms of vav1 in RAW-TT10 cells consistently inhibited LPS-mediated increases in iNOS protein accumulation and NF-IL-6 activity. These data provide direct evidence for a role for vav1 in LPS-mediated macrophage activation and iNOS production and suggest that vav1 functions in part via activation of NF-IL-6. Furthermore, these findings indicate that the GEF activity of vav1 is required for its ability to mediate macrophage activation by LPS.
Previous pharmacologic and genetic studies have demonstrated a critical role for the low molecular weight GTP-binding protein RhoA in the regulation of cell-mediated killing by cytotoxic lymphocytes. However, a specific Rho family guanine nucleotide exchange factor (GEF) that activates this critical regulator of cellular cytotoxicity has not been identified. In this study, we provide evidence that the Rho family GEF, Vav-2, is present in cytotoxic lymphocytes, and becomes tyrosine phosphorylated after the cross-linking of activating receptors on cytotoxic lymphocytes and during the generation of cell-mediated killing. In addition, we show that overexpression of Vav-2 in cytotoxic lymphocytes enhances cellular cytotoxicity, and this enhancement requires a functional Dbl homology and Src homology 2 domain. Interestingly, the pleckstrin homology domain of Vav-2 was found to be required for enhancement of killing through some, but not all activating receptors on cytotoxic lymphocytes. Lastly, although Vav and Vav-2 share significant structural homology, only Vav is able to enhance nuclear factor of activated T cells–activator protein 1–mediated gene transcription downstream of the T cell receptor. These data demonstrate that Vav-2, a Rho family GEF, differs from Vav in the control of certain lymphocyte functions and participates in the control of cell-mediated killing by cytotoxic lymphocytes.
natural killer cell; cytotoxic T cell; Vav-2; RhoA; signal transduction
Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5′ regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.
We have investigated the role of Vav2, a reported Rac1/Cdc42 GEF, on the development of Xenopus spinal neurons in vitro and in vivo. Both gain and loss of Vav2 function inhibited the rate neurite extension on laminin (LN), while only GFP-Vav2 over-expression enhanced process formation and branching. Vav2 over-expression protected neurons from RhoA-mediated growth cone collapse, similar to constitutively active Rac1, suggesting Vav2 activates Rac1 in spinal neurons. Enhanced branching on LN required both Vav2 GEF activity and N-terminal tyrosine residues, but protection from RhoA-mediated collapse only required GEF activity. Interestingly, wild-type spinal neurons exhibited increased branching on the cell adhesion molecule L1, which required Vav2 GEF function, but not N-terminal tyrosine residues. Finally, we find that Vav2 differentially affects Rohon-Beard peripheral and central process extension, but promotes neurite branching of commissural interneurons near the ventral midline. Together, we suggest that balanced Vav2 activity is necessary for optimal neurite outgrowth and promotes branching by targeting GEF activity to branch points.
axon pathfinding; Xenopus; GEF; Rac1; L1; commissural interneuron; growth cone
Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.
Vav1 is expressed exclusively in hematopoietic cells and is required for T cell development and activation. Vav1-deficient mice show thymic hypocellularity due to a partial block during thymocyte development at the DN3 stage and between the double positive (DP) and single positive (SP) transition. Vav1 has been shown to play a significant role in several non-hematopoietic tumors but its role in leukemogenesis is unknown. To address this question, we investigated the role of Vav1 in retrovirus-induced T cell leukemogenesis. Infection of Vav1-deficient mice with the Moloney strain of murine leukemia virus (M-MuLV) significantly affected tumor phenotype without modulating tumor incidence or latency. M-MuLV-infected Vav1-deficient mice showed reduced splenomegaly, higher hematocrit levels and hypertrophic thymi. Notably, Vav1-deficient mice with M-MuLV leukemias presented with markedly lower TCRβ/CD3 levels, indicating that transformation occurred at an earlier stage of T cell development than in WT mice. Thus, impaired T cell development modulates the outcome of retrovirus-induced T cell leukemias, demonstrating a link between T cell development and T cell leukemogenesis.
leukemia; murine leukemia virus; retrovirus; T cell; Vav