InteractiveFly: GeneBrief

Vav: Biological Overview | References

BIOLOGICAL OVERVIEW

Guided cell migration is a key mechanism for cell positioning in morphogenesis. The current model suggests that the spatially controlled activation of receptor tyrosine kinases (RTKs) by guidance cues limits Rac activity at the leading edge, which is crucial for establishing and maintaining polarized cell protrusions at the front. However, little is known about the mechanisms by which RTKs control the local activation of Rac. Using a multidisciplinary approach, this study identified the GTP exchange factor (GEF) Vav as a key regulator of Rac activity downstream of RTKs in a developmentally regulated cell migration event, that of the Drosophila border cells (BCs). Elimination of the vav gene impairs BC migration. Live imaging analysis reveals that vav is required for the stabilization and maintenance of protrusions at the front of the BC cluster. In addition, activation of the PDGF/VEGF-related receptor (PVR) by its ligand the PDGF/PVF1 factor brings about activation of Vav protein by direct interaction with the intracellular domain of PVR. Finally, FRET analyses demonstrate that Vav is required in BCs for the asymmetric distribution of Rac activity at the front. These results unravel an important role for the Vav proteins as signal transducers that couple signalling downstream of RTKs with local Rac activation during morphogenetic movements (Fernandez-Espartero, 2014).

Directed cell migration plays a crucial role in many normal and pathological processes such as embryo development, immune response, wound healing and tumor metastasis. During development, cells migrate to their final position in response to extracellular stimuli in the microenvironment. To migrate towards or away from a stimulus, individual cells or groups of cells must first achieve direction of migration through the establishment of cell polarity. Guidance cues, such as growth factors, control cell polarization through the regulated recruitment and activation of receptor tyrosine kinases (RTKs) to the leading edge. A key event downstream of RTK signalling in cell migration is the localization of activated Rac at the leading edge. However, little is known about the mechanisms by which external cues regulate Rac activity during cell migration. Rac is activated by GTP exchange factors (GEFs), which facilitate the transition of these GTPases from their inactive (GDP-bound) to their active (GTP-bound) states. Thus, GEFs appear as excellent candidates to regulate the cellular response to extracellular cues during cell migration (Fernandez-Espartero, 2014).

Among the different Rac GEF families characterized so far, the Vav proteins are the only ones known to combine in the same molecule the canonical Dbl (DH) and pleckstrin homology (PH) domains of Rac GEFs and the structural hallmark of tyrosine phosphorylation pathways, the SH2 domain. In addition, Vav activity is regulated by tyrosine phosphorylation in response to stimulation by transmembrane receptors with intrinsic or associated tyrosine kinase activity. These features make Vav proteins ideal candidates to act as signalling transducer molecules coupling growth factor receptors to Rac GTPase activation during cell migration. In fact, a number of cell culture experiments have suggested a role for the Vav proteins in cell migration downstream of growth factor signalling. Thus, the ubiquitously expressed mammalian Vav2 is tyrosine phosphorylated in response to different growth factors, including epidermal (EGF) and platelet-derived (PDGF) growth factors, and its phosphorylation correlates with enhanced Rac activity and migration in some cell types. However, the biological relevance for many of these interactions and the cellular mechanisms by which Vav regulates in vivo cell migration remains to be determined (Fernandez-Espartero, 2014).

The Vav proteins are present in all animal metazoans but not in unicellular organisms. There is a single representative in multicellular invertebrates and urochordata species (such as C. elegans, Drosophila melanogaster and Ciona intestinalis) and usually three representatives in vertebrates. The single Drosophila vav ortholog possesses the same catalytic and regulatory properties as its mammalian counterparts (Couceiro, 2005). In addition, the Drosophila Vav is tyrosine phosphorylated in response to EGF stimulation in S2 cells (Hornstein, 2003; Margolis, 1992). Furthermore, a yeast two hybrid analysis has shown that the SH2-SH3 region of Vav can bind the epidermal growth factor receptor (EGFR) and the intracellular domain of PVR, PVRi, but not a kinase-dead version of PVRi, suggesting that Vav SH2-SH3-HA::PVRi interactions depend on PVR autophosphorylation (Bianco, 2007). Altogether, these results suggest that the role of mammalian Vavs as transducer proteins coupling signalling from growth factors to Rho GTPase activation has been conserved in Drosophila. Thus, this study took advantage of Drosophila to analyse vav contribution to growth factor-induced cell migration in the physiological setting of a multicellular organism (Fernandez-Espartero, 2014).

The migration of the border cells (BCs) in the Drosophila egg chamber represents an excellent model system to study guided cell migration downstream of PVR/EGFR signalling in vivo. Each egg chamber contains one oocyte and 15 nurse cells surrounded by a monolayer of follicle cells (FCs), known as follicular epithelium (FE). The BC cluster is determined at the anterior pole of the FE and it comprises 6-8 outer cells and two anterior polar cells in a central position. BCs delaminate from the anterior FE and migrate posteriorly between the nurse cells until they contact the anterior membrane of the oocyte. BCs use the PVR and the EGFR to read guidance cues, the PDGF-related Pvf1 and the TGFβ-related Gurken, secreted by the oocyte. The Rho GTPase Rac is required for BC migration. The current model proposes that higher levels of Rac activity present in the leading cell determine the direction of migration and that this asymmetric distribution of Rac activity requires guidance receptor input. The unconventional Rac GEF Myoblast city, Mbc, is the only identified downstream signalling effector in this context. However, although genetic analysis have led to propose that the unconventional GEF for Rac, Mbc/DOCK 180, could activate Rac downstream of PVR during BC migration, this has not been formally proven. In addition, Mbc is unlikely to be the only Rac GEF actin downstream of guidance receptors in BCs as the migration phenotype due to complete removal of mbc is not as severe as the loss of both Pvr and Egfr. Thus, other effectors are likely to contribute to the complicated task of guiding BC migration. Many candidate molecules have been tested for their requirement in BC migration, MAPK pathway, PI3K, PLC-gamma, as well as RTK adaptors, such as DOCK, Trio, and Pak, but none of these is individually required (Fernandez-Espartero, 2014).

Vav proteins were initially involved in lymphocyte ontology (Bustelo, 2000; Turner, 2002). Only recently, cell culture experiments have implicated these proteins in cell migration events downstream of guidance factors. Interestingly, Vav proteins can either promote or inhibit cell migration. In macrophages, Vav is required for macrophage colony-stimulating factor-induced chemotaxis (Vedham, 2005). In human peripheral blood lymphocytes, Vav is involved in the migratory response to the chemokine stromal cell-derived factor-1 (Vicente-Manzanares, 2005). Conversely, in Schwann cells, Vav2 is required to inhibit cell migration downstream of the brain-derived neurotrophic factor and ephrinA5 (Afshari, 2010; Yamauchi, 2004). In spite of the knowledge gained from cell culture experiments, the biological relevance for many of the above interactions has remained elusive. In recent years, Vav proteins have started to emerge as critical Rho GEFs acting downstream of RTKs in diverse biological processes (Cowan, 2005; Hunter, 2006). Analysis of Vav2^-/- Vav3^-/- mice revealed retinogeniculate axonal projection defects (Cowan, 2005) and impaired ephrin-A1-induced migration during angiogenesis (Hunter, 2006), suggesting a role for Vav in axonal targeting and angiogenesis downstream of Eph receptors in vivo. This study has shown that Vav can act downstream of growth factors receptors to promote BC migration in the developing Drosophila ovary, supporting a role of this family of GEFs in transducing signals from RTKs to regulate cell migration during development (Fernandez-Espartero, 2014).

Analysis of the cellular mechanisms by which Vav regulates cell migration in vertebrates is hampered by the inaccessibility of the cells and the difficulty of visualizing them in their natural environment within the embryo. Thus, it is not yet clear how Vav proteins regulate cell migration downstream of RTKs during development. In this study, by analysing cell movement in their physiological environment, it has been possible to show that Vav is required to control the length, stabilization and life of front cellular protrusions. In addition, disruption of Vav function in vivo was found to result in a decrease in Rac activity at the leading edge. Defective signalling downstream of EGFR/PVR results in defects in the dynamics of cellular protrusion and Rac activation, which are very similar to those observed in vav^-/- BCs (Poukkula, 2011; Wang, 2010). In addition, this study found that ectopic activation of Vav in BCs, as it is the case for PVR/EGFR and Rac, causes non-polarized massive F-actin accumulation. Thus, it is suggested that one of the roles of Vav in directed cell migration downstream of EGF/PVF signals is to remodel the actin cytoskeleton via Rac activation, hence promoting the formation and stabilization of cellular protrusions in the direction of migration. Studies in cultured neurons, have shown that the main role for mouse Vav2 during axonal repulsion is to mediate a Rac-dependent endocytosis of ephrin-Eph (Cowan, 2005). Although endocytosis has been normally shown to be involved in attenuation of RTKs signalling, in BCs it has been proposed to ensure RTKs recycling to regions of higher signalling, thus promoting directed BC movement (Jekely, 2005). This is based on the fact that elimination in BCs of the ubiquitin ligase Cbl, which has been shown to regulate RTK endocytosis, leads to delocalized RTK signal and migration defects. In this context, another possible role for Vav downstream of EGFR/PVR could be to mediate RTK endocytosis, as it is the case during axonal repulsion. Further analysis will be needed to fully explore the molecular and cellular mechanisms by which Vav proteins regulate cell migration in vivo in other developmental contexts (Fernandez-Espartero, 2014).

BC migration is a complex event and activation of EGF/PDGF receptors will most likely engage different GEFs to affect the distinct cytoskeletal changes necessary to accomplish it. In fact, the migration phenotype of BCs mutant for vav is less severe than that of BCs double mutant for both EGFR and PVR. In addition, although reducing Vav function decreases the asymmetry in Rac activity between front and back present in wild-type clusters, it does not eliminate it, as it happens when the function of both guidance receptors is compromised. All these results suggest that there are other GEFs besides Vav that could act downstream of EGFR and PVR to activate Rac. Previous analysis have implicated the Rac exchange factor Mbc/DOCK180 and its cofactor ELMO on BC migration. In this context, Vav and the Mbc/ELMO complex could act synergistically as GEFs to mediate Rac activation to a precise level and/or to a precise location. This awaits the validation of the Mbc/ELMO complex as a GEF for Rac in BCs. In the future, it will be important to determine how the different GEFs contribute to Rac activation, which specific downstream effectors of Rac they activate, and ultimately what cellular aspects of the migration process they control (Fernandez-Espartero, 2014).

In summary, this work demonstrates that Vav functions downstream of RTKs to control directed cell migration during development. Furthermore, this study has unravelled the cellular and molecular mechanism by which Vav regulates cell migration in the developing Drosophila egg chamber: binding of PDGF/EGF to their receptors would induce Vav activation through tyrosine phosphorylation and its association with the activated receptors. This would lead to an increase in Rac activity at the leading edge of migrating cells, which promotes the stabilization and growth of the cellular front extensions, thus controlling directed cell migration (Fernandez-Espartero, 2014).

Regulation of Vav signalling downstream of RTKs can participate not only in development or normal physiology but also in tumorigenesis (Billadeau, 2002; Lazer, 2009). Vav1 is mis-expressed in a high percentage of pancreatic ductular adenocarcinomas and lung cancer patients (Fernandez-Zapico, 2005; Lazer, 2009). Thus, understanding the mechanisms by which Vav controls cellular processes downstream of RTKs is likely to be relevant for both developmental and tumor biology (Fernandez-Espartero, 2014).

The guanine exchange factor Vav controls axon growth and guidance during Drosophila development

The Vav proteins are guanine exchange factors (GEFs) that trigger the activation of the Rho GTPases in general and the Rac family in particular. While the role of the mammalian vav genes has been extensively studied in the hematopoietic system and the immune response, there is little information regarding the role of vav outside of these systems. This study reports that the single Drosophila vav homolog is ubiquitously expressed during development, although it is enriched along the embryonic ventral midline and in the larval eye discs and brain. The role that vav plays during development was analyzed by generating Drosophila null mutant alleles. The results indicate that vav is required during embryogenesis to prevent longitudinal axons from crossing the midline. Later on, during larval development, vav is required within the axons to regulate photoreceptor axon targeting to the optic lobe. Finally, it was demonstrated that adult vav mutant escapers, which exhibit coordination problems, display axon growth defects in the ellipsoid body, a brain area associated with locomotion control. In addition, this study showed that vav interacts with other GEFs known to act downstream of guidance receptors. Thus, it is proposed that vav acts in coordination with other GEFs to regulate axon growth and guidance during development by linking guidance signals to the cytoskeleton via the modulation of Rac activity (Malartre, 2010).

Vav members are key regulators of the Rho GTPases and the Rac proteins in particular. However, although many studies have implicated the Rac proteins in controlling several aspects of axon growth and guidance during development, understanding of vav function in these processes is far more primitive. This is quite surprising given that all vav members are expressed in neural tissues in mammals. Recently, analysis of postnatal vav2/vav3 -deficient mice has revealed abnormal retinogeniculate projections (Cowan, 2005). This study demonstrates that in Drosophila vav is required for axon growth and guidance at embryonic, larval, and pupal stages. Hence, these data strengthen the role of vav in multiple aspects of axogenesis during development (Malartre, 2010).

During the formation of the embryonic central nervous system of Drosophila, the neurons send out axons that project either ipsilaterally or contralaterally to form the complex axonal lattice. A small number of neurons project ipsilaterally as they receive repulsive signals from the midline glia and never cross the midline, while most neurons project contralaterally, cross the midline, and form the commissures. In vav2/vav3 mutant mouse brains, ipsilateral but not contralateral projections are affected (Cowan, 2005). These results are consistent with the current data showing that Drosophila vav is required to regulate proper ipsilateral axon projection, as in vav mutant embryos the most medial longitudinal axons occasionally cross the midline when they should not. These fascicles are particularly sensitive to perturbations in axon guidance mechanism and cross the midline whenever repulsive signaling is altered, suggesting that Vav might participate in the regulation of repulsive signaling from the midline (Malartre, 2010).

This study also demonstrated that vav is required during subsequent larval development in regulating photoreceptor axon targeting to the optic lobe. This is again consistent with the finding that vav2/vav3 mutant mice display abnormal projections of axons connecting the retinal cells to the brain (Cowan, 2005), suggesting that the role of vav in mediating axon guidance decisions is conserved between species. Interestingly, vav function in photoreceptors (R cells) seems to be more important than in the embryonic CNS. Indeed, this study found that in 100% of the larvae, R cell axons projected aberrantly to the lamina and the medulla target regions, while only 14% of vav mutant embryos displayed guidance defects. Finally, it was shown that later, during metamorphosis, vav is required once more for the correct formation of the ellipsoid body, one of the central brain structures. The ellipsoid body, in a majority of vav mutant adult brains, does not close properly and remains ventrally opened, most likely reflecting defects in the growth of the axons forming the ellipsoid body rather than guidance errors. Interestingly, the ellipsoid body has been involved in the control of locomotion, and vav mutant adults display strong locomotion defects. Opened ellipsoid bodies have also been found in ciboulot mutants . However these mutants do not display locomotion defects, suggesting that a disruption of the ellipsoid body alone is not sufficient to produce the locomotion phenotype observed in vav mutants. This implies that vav might also be required to regulate other aspects of axogenesis, in addition to the ones identified in this study (Malartre, 2010).

In summary, these results show that vav is required reiteratively throughout life to regulate different axogenesis events, including axon growth and guidance (Malartre, 2010).

During larval development, R cell axon targeting to the optic lobe is controlled, on the one hand, by some genes that are acting within the axons themselves, and on the other hand, by some genes that are sending signals to the axons from the glia to guide them. MARCM experiments clearly demonstrate a role for vav within the R cell axons to regulate their projections. This is also the case in mammals, where Vav2 is highly expressed in the growth cones of cultured neurons where it is required to control guidance (Malartre, 2010).

Interestingly, in Drosophila, the GEF Trio has been shown to activate Rac, which in turn activates Pak, which is recruited to the membrane by Dock. These proteins participate in a signal transduction pathway that plays an essential role during photoreceptor axon guidance. Vav also acts via Rac in photoreceptors, and vav and trio interact genetically. Thus, in this context, it is tempting to speculate that like Trio, Vav could also contribute to the precise spatial control of Pak activity. In this scenario, the combination of signals via Vav, Trio, and Dock would allow growth cones to integrate multiple guidance signals (Malartre, 2010).

Vav function in the axons could be to regulate the intracellular trafficking of guidance receptors through the activation of Rac. In mammals, for instance, vav2 has been proposed to be required in axons downstream of ephrin signaling for proper axon guidance. In this case, when ephrins bind their Eph receptors, Vav becomes transiently activated upon phosphorylation and promotes local Rac-dependent endocytosis of the ephrin/Eph complex, a key event in axonal repulsion. In Drosophila however, mutations in Eph surprisingly show no obvious axon guidance defects in the photoreceptor axons targeting to the optic lobe nor in the embryonic CNS. This suggests that in Drosophila, vav would need to be acting downstream of other guidance signals besides Eph (Malartre, 2010).

In conclusion, it is proposed that Vav, after being activated by signaling receptors, could be required to stimulate Rac proteins to participate in the regulation of axon growth and guidance during development (Malartre, 2010).

The Drosophila genome contains 22 GEFs. At least nine of them are expressed in the CNS, five of which are thought to be Rac activators. Why are there several Rac GEFs acting in the nervous system? A possible explanation is that the different GEFs might be activated in response to distinct guidance cues, thus triggering Rac-dependent specific cellular responses. For instance, beside its function in longitudinal axon growth, Trio has been involved in promoting commissure formation through its interaction with the attractive Netrin receptor Frazzled. Furthermore, another GEF, Sos, has been proposed to mediate Rac activation downstream of the Robo receptor to control axon repulsion at the midline. In this scenario, Vav, Sos, and Trio could coexist and be activated in response to different guidance molecules to control distinct aspects of axon guidance during the formation of the CNS (Malartre, 2010).

In another scenario, different set of GEFs could also act redundantly to activate Rac proteins to a certain level, or at precise time points or in specific subcellular locations, allowing a unique cellular response. In fact, this study has shown that the loss of both vav and sos function enhances dramatically the individual midline guidance phenotypes, suggesting that vav and sos can act redundantly in a common pathway. Similarly, the phenotype of the vav;trio double mutant in the nervous system, both at the midline and along the longitudinal axons, is more severe than the single mutants. In addition, while mutations in either vav or trio do not show any obvious defects outside the nervous system despite their widespread expression, elimination of both results in gross morphological defects. This indicates that both genes can act redundantly in vivo in different tissues and suggests that vav and trio are the main regulators of Rac activity (Malartre, 2010).

A final explanation for the existence of different rac GEFs is that they could preferentially activate a particular Rac. There are three highly related rac genes in Drosophila, rac1, rac2, and mtl, and it has been suggested that Rac1 and Rac2 are preferred substrates of Trio. By performing a similar epistasis analysis, this study has shown that in photoreceptor cells Vav activates preferentially Rac1 and Rac2. The fact that vav and trio show similar substrate specificities could explain why these two genes were found to be redundant during embryogenesis (Malartre, 2010).

In conclusion, although the vav family has been mainly implicated in the hematopoietic system and immune response, new roles are beginning to emerge for these genes. The fact that vav is required for axon growth and guidance at different stages of development suggests that it could be playing a multiplicity of functions in response to diverse signals. The existence of various protein-protein interaction domains in Vav represents a means of integrating Rac activities. These results also suggest that vav function must overlap with that of other Rac modulators. Having isolated mutations in the Drosophila vav gene will help elucidate not only the role of this GEF during neural development but also the molecular mechanisms underlying general remodeling of the embryonic and adult nervous systems (Malartre, 2010).

Antagonistic roles of Rac and Rho in organizing the germ cell microenvironment

The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments. However, how microenvironments are shaped is poorly understood. In testes of Drosophila, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells. Germ cells signal via the Egf class ligand Spitz (Spi), and evidence is provided that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppresses the germ cell enclosure defects of the conditional spi allele. It is proposed that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. These novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage (Sarkar, 2007).

In the male gonad of Drosophila, germ cells are surrounded by somatic cells that define their cellular microenvironmen. Germline stem cells (GSCs) are attached to a cluster of nondividing cells at the apical tip, called hub cells, and associated with cyst progenitor cells (CPCs) that act as stem cells for the somatic support cell lineage. Two CPCs extend their cytoplasm around one GSC, toward the hub, and toward each other such that each GSC appears to be completely enclosed in its cellular microenvironment. GSCc and CPCs generate differentiating daughters, called gonialblasts and cyst cells, respectively. The gonialblasts undergo transit amplification divisions to produce 16 spermatogonia, which become spermatocytes, grow in size, undergo the meiotic divisions, and differentiate into sperm. Two cyst cells grow cytoplasmic extensions around one gonialblast to form the germ cell cellular microenvironment that controls various aspects of germ cell differentiation (Sarkar, 2007).

Germ cells associated with somatic cells mutant for the Map-Kinase Raf fail to differentiate and accumulated as early-stage germ cells instead. A similar accumulation of early-stage germ cells was observed in Egfr^ts mutant testes shifted to nonpermissive temperature, and in testes from animals mutant for Stem cell tumor (Stet; Rhomboid2), a protease that cleaves Egfr ligands. However, stet mutant germ cells in addition fail to associate with somatic support cells, suggesting that the Egfr pathway is required for setting up the critical cellular microenvironment (Sarkar, 2007).

Loss of spi results in a failure of germ cells to differentiate, similar to the effects of loss of stet or the Egfr. Wild-type testes are long (~2 mm) tubular structures that contain germ cells in a spatio-temporal order along the apical-to-basal axis. Early germ cells (GSCs, gonialblasts, and spermatogonia) are small and have small, densely packed nuclei in DAPI-stained preparations. Spermatocytes are located basal to the spermatogonia, and differentiating spermatids fill the distal part of the testis (Sarkar, 2007).

Animals carrying a temperature-sensitive allele of spi, spi^77-20, die when raised at 29°C. However, spi^77-20 animals raised at a slightly permissive temperature (27°C) survive and have tiny testes. Most of these testes (40 of 50) contain only small cells, as seen at the tip of wild-type testes, and do not have spermatocytes or differentiating spermatids. Staining with molecular markers revealed that the testes contains increased numbers of GSCs, gonialblasts, and spermatogonia compared to wild-type. The remaining testes (10 of 50) have high numbers of early germ cells and a few spermatocytes, but no differentiating spermatids (Sarkar, 2007).

Testes from spi^77-20 animals raised at an intermediate permissive temperature (25°C) are longer than testes from animals raised at 27°C, but significantly shorter (500 μm-1.5 mm) than wild-type testes. A substantial part of the testes is occupied by tumor-like aggregates of early-stage germ cells. However, spermatocytes and differentiating spermatids are also present (Sarkar, 2007).

spi activity is both sufficient and required within the germ cells. Expression of a cleaved version of Spi (sSpi) in germ cells but not in somatic support cells of spi^77-20 testes restores the wild-type phenotype, and germ cell clones mutant for spi accumulate at early stages based on phase-contrast microscopy and DAPI-stained preparations) (Sarkar, 2007).

spi was also required for somatic support cells to associate with and enclose the germ cells. Germ cell clones mutant for the conditional spi^77-20 allele from animals raised at 27°C either do not associate with somatic support cells or associated with only one somatic support cell (4 of 20 clones), based on staining with soma-specific antibodies, such as the transcription factor Traffic Jam (Tj). Tj labels the nuclei of somatic support cells that are normally associated with early-stage germ cells (Sarkar, 2007).

Germ cell enclosure can be investigated by labeling testes with molecular markers such as antibodies against the membrane-bound β-catenin Armadillo (Arm) that labels the cell membranes of somatic support cells as they surround the germ cells. In wild-type testes, each GSC, gonialblast, and cluster of developing germ cells is associated with and surrounded by two somatic support cells. In testes from spi^77-20 animals raised at 27°C, Tj-positive cells did not form cytoplasmic extensions around the germ cells. Similar results were obtained with other markers, including a cytoplasmic UAS-Green Fluorescent Protein (UAS-GFP) expressed in somatic support cells under control of a soma-specific Gal4-driver. In control testes, GFP is detected in the cell bodies of the somatic cells surrounding the germ cells. In contrast, in spi^77-20 testes from animals grown at 27°C, GFP is detected in balls, most likely small round cell bodies of somatic support cells. Occasionally, cytoplasmic extensions emerged from somatic support cells, but they remained short and did not enclose the germ cells (Sarkar, 2007).

The lack of cytoplasmic extensions from Tj-positive cells in spi^77-20 mutant testes is similar to the phenotype observed in stet mutants. This suggests that the Egf class ligand Spi, expressed in germ cells and processed by Stet, stimulates the Egfr on somatic support cells, inducing them to send out cytoplasmic extensions to enclose the neighboring germ cells (Sarkar, 2007).

Association of germ cells with somatic support cells is sensitive to the level of Spi. Germ cell clones from spi^77-20 animals raised at 25°C and germ cell clones from animals mutant for the spi² allele often associated with more than two somatic support cells (Sarkar, 2007).

The growth of cytoplasmic extensions around the germ cells is also sensitive to the level of Spi. When spi^77-20 animals are raised at 25°C, many Tj-positive cells form cytoplasmic extensions directed toward and/or around the germ cells. However, not every germ cell cluster appear to be associated with and/or surrounded by somatic support cells. Furthermore, many of the Tj-positive cells forme cytoplasmic extensions toward each other, suggesting that multiple somatic support cells may surround one tumor-like aggregate of germ cells. Similar abnormal associations of somatic support cells with germ cells are also seen in Egfr^ts mutants shifted to nonpermissive temperature. One possible explanation for the different phenotypes of loss compared to reduction of Egfr signaling is that different levels of Egfr stimulation may affect different cellular properties of somatic support cells, such as cell adhesiveness and/or growth (Sarkar, 2007).

To identify novel players in germ cell enclosure, the sensitized background of the spi^77-20 allele was used to search for genetic modifiers. It was found that impaired activity of the small monomeric GTPase (small GTPase) Rac1 enhances the spi^77-20 testes phenotype. Activity of Rac1 was impaired by two strategies-either by removing one copy of the rac1 gene or by expressing a dominant-negative version of Rac1 (dnRac1) in somatic support cells of testes from spi^77-20 animals raised at 25°C. In either case, the enhanced testes are shorter than testes from spi^77-20 animals raised at 25°C. In 12 of 20 enhanced testes, Tj-positive cells do not enclose the germ cells, and early-stage germ cells accumulate (Sarkar, 2007).

Reducing activity of Vav, a guanine nucleotide exchange factor for Rac-type small GTPases, from somatic support cells by antisense expression also enhances the spi^77-20 testes phenotype from animals raised at 25°C. 11 of 20 enhanced testes were tiny and contained mostly early-stage germ cells that were not surrounded by somatic support cells. The enhanced phenotypes caused by impairing Rac or Vav raises the possibility that Rac1 and Vav act downstream of the Egfr in somatic support cells and that Vav plays a role in regulating somatic support cell-shape changes associated with germ cell enclosure (Sarkar, 2007).

In mammalian cells, autophosphorylation of specific Vav-binding motifs within the cytoplamic tail of the Egfr allows for binding and phosphorylation of mammalian Vav2 (Tamas, 2003). Phosphorylation of Drosophila Vav has been shown to depend on Egfr stimulation in both mammalian and Drosophila cultured cells, and Drosophila Vav bound to mammalian Egfr (Bishop, 2000; Dekel, 2000; Sarkar, 2007 and references therein).

Consistent with a role for Drosophila Vav in Egfr signaling in testes, Vav protein immunoprecipitates from testis extracts with an antibody against the Egfr. Vav does not immunoprecipitate from testis extracts that had been pretreated with phosphatase, suggesting that the interaction between Vav and the Egfr is phosphorylation dependent. The immunoprecipitated Vav band comigrates with a band detected by antibodies against phospho-tyrosine, suggesting that Vav is phosphorylated when in a complex with the Egfr (Sarkar, 2007).

In the classical view of the Drosophila Egfr pathway, only one docking protein-Downstream receptor kinase (Drk)-binds to the stimulated Egfr and activates a MAP-Kinase cascade for transcription of target genes. However, genetic and biochemical data suggest that the Egfr pathway is branched at the level of docking proteins and that the adaptor protein Vav binds to the Egfr to activate the small GTPase Rac1. These data suggest that Rac regulates cell-shape changes associated with germ cell enclosure, and studies on Raf suggested that it regulates the transcription of target genes. However, the possibility of crosstalk between the two branches cannot be excluded: Vav may contribute to transcriptional regulation and Map-Kinases may contribute to germ cell enclosure. A possible crosstalk is consistent with findings that in cultured Drosophila cells (Hornstein, 2003), Vav can contribute to Erk phosphorylation (Sarkar, 2007).

Surprisingly, impairing activity of the Rho-type small GTPase Rho1 has the opposite effect to impairing Rac1. Testes from spi^77-20 animals raised at 27°C that express dominant-negative Rho1 (dnRho1) in somatic support cells are long and appear almost wild-type. In contrast to somatic support cells in spi^77-20 testes from animals raised at 27°C without dnRho1 expression, the somatic support cells expressing dnRho1 enclose the germ cells. The same dominant suppression is observed in spi^77-20, rho1/+ testes, indicating that expression of dnRho1 reflects loss of Rho1 activity (Sarkar, 2007).

These data raise the possibility that Rac and Rho have antagonistic effects on germ cell enclosure. Rac appears to be required for somatic support cells to grow cytoplasmic extensions around the germ cells, and Rho appears to suppress this growth. Antagonistic roles for Rac and Rho have also been reported in cultured mammalian cells, where Rac and Rho regulate cell-shape changes and growth via different effects on the actin cytoskeleton. Prominent readouts for small GTPase activities on the actin cytoskeleton are the appearances of ruffles and lamellipodia in the cell membranes (Sarkar, 2007).

To address a potential role of Rac and Rho in shape changes of somatic support cells, dominant-negative Rac or Rho were expressed in somatic support cells of otherwise wild-type testes, and transmission electron microscopy (TEM) was used to investigate changes in the membranes of somatic support cells surrounding single germ cells and spermatogonia at the apical tip of the testes. Germ cells and somatic support cells can be identified based on their different shapes and density of staining in TEM. In wild-type, the somatic support cells surrounding single germ cells and spermatogonia exhibit wavy plasma membranes, possibly analogous to membrane ruffles accompanying cellular growth and rearrangements of the actin cytoskeleton in cultured cells (Sarkar, 2007).

Somatic support cells expressing dnRac1 have much smoother plasma membranes than do wild-type somatic support cells. Conversely, somatic support cells expressing dnRho1 have lamellipodia-like extensions in their membranes. Lamellipodia-like extensions were not detected in somatic support cell membranes in serial sections of wild-type testes or in testes expressing dnRac1. In mammalian cells, formation of lamellipodia depends on Rac-type small GTPases. The presence of lamellipodia-like extensions in somatic support cells expressing dnRho1 suggests that Rac may become hyperactive in the absence of Rho. Based on these TEM data, it is hypothesized that, just as their mammalian counterparts do in cultured cells, Drosophila small GTPases may act on the cytoskeleton of somatic support cells to mediate cell-shape changes and growth of cellular extensions and that the effects of Rac and Rho are antagonistic (Sarkar, 2007).

This model predicts that expression of a constitutively active Egfr ligand in somatic support cells might compromise the differential in smGTPase activities. Indeed, forced expression of cleaved ligand in somatic support cells, but not in germ cells, closely mimics the effect of dnRho expression: the somatic support cells formed lamellipodia-like structures in their membranes (Sarkar, 2007).

This research on the Drosophila gonad provides a striking example how one cell type in tissue communicates with another cell type to induce and direct the formation of a proper cellular microenvironment: a signal from one cell induces subcellular changes throughout the body of another cells. This mechanism underlying the formation of a cellular microenvironment may be conserved across species (Sarkar, 2007).

Phylogenetic conservation of the regulatory and functional properties of the Vav oncoprotein family

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, a characterization of the regulatory, catalytic, and functional properties was performed 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. It was also shown 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 (Couceiro, 2005).

Rho/Rac GTPase pathways originated in yeast to regulate functions related to stress responses and F-actin dynamics. Since then, they have adapted to the new functional needs of more complex organisms, such as embryonic development, the maintenance of physiological circuits, or the engagement of immune responses. This has led to the development of signaling elements that allowed the insertion of these GTPases into new biological pathways. A good example for this progressive acquisition of signaling elements is the Vav oncoprotein family, a group of Rho/Rac GEFs of animal metazoans that have originated to facilitate the connection of Rho/Rac proteins to receptors with intrinsic or associated tyrosine kinase activity. The evolution of these proteins was progressive, both in terms of total gene family number and protein domain structure. Thus, the Vav family has single representatives in protostomes and early chordates but, upon genome duplication events occurring during evolution, diversified later on to give rise to the three known Vav proteins of vertebrates (Vav, Vav2, and Vav3). Vav proteins acquired new structural features during those transitions, such as the insertion of a proline rich region (missing in the Vav protein from C. elegans) and an additional SH3 domain (missing in the Vav proteins of all protostome species). In addition, upon the triplication of the ancestral vav gene, they diversified functionally. As a consequence, the three mammalian Vav proteins share a core of basic pathways (i.e., activation of GTPases, modulation of F-actin dynamics) but differ in their ability to engage other signaling responses (i.e., the activation of the nuclear factor of stimulated T-cells) (Couceiro, 2005).

The availability of Vav family proteins from a wide range of species has afforded the opportunity to take a phylogenetic perspective of the regulation and function of these proteins. In this regard, the characterization of the single Vav family protein of Drosophila indicates that the regulatory mechanisms controlling the catalytic activity of its mammalian counterparts have been set up early in evolution. Using a mutagenesis approach, this study demonstrated that the two known structural interactions for regulating the phosphorylation-dependent catalytic activity of Vav proteins are also at work in Drosophila. Moreover, it was observed that DmVav activates the same spectrum of GTPases as mammalian Vav. As a consequence, DmVav induces biological responses quite similar to its mammalian counterparts when expressed in mammalian cells, including oncogenesis, changes in the cell cytoskeleton, and enhanced cell motility. These results indicate that the foundations for the regulatory and catalytic properties of this protein group were established before the split between protostomes and deuterostomes (Couceiro, 2005).

The similarity of the regulatory properties of DmVav and mouse Vav protein can be extended to most of the other structural domains. On one hand, this study has shown that the mutation of key residues of the DH, PH, and ZF region results in the total abrogation of the biological activity of all Vav proteins tested, both in terms of transforming activity and cytoskeletal change. On the other hand, it was demonstrated that the SH2 regions do not play an essential role in the biological activity of Vav oncoproteins. This is probably due to the fact that the N-terminally deleted oncoproteins show a constitutive, phosphorylation-independent exchange activity. Due to this, they do not rely necessarily in the imperative interaction with upstream tyrosine kinases for activation. This is in agreement with the extensive work with mammalian Vav proteins indicating that the SH2 domains are only essential for the activity of the wild type forms of these exchange factors. In this regard, the lower transformation observed in the SH2 mutants has been attributed not to lack of phosphorylation but, rather, to a deficient translocation to the plasma membrane. Indeed, if such defect is bypassed by the attachment of membrane localization signals to the Vav C-terminus, the DH-PH-ZF domains of mammalian Vav proteins show even higher transforming activities than the normal, N-terminal deleted oncoproteins that contain the SH3-SH2-SH3 cassette (Couceiro, 2005).

Unexpectedly, the mutagenesis experiments have revealed that such functional conservation cannot be extended to the SH3 regions. Thus, unlike mammalian Vav proteins, DmVav does not elicit cytoskeletal change when its SH3 region is inactivated by point mutation. Likewise, the transforming activity of this mutant is also severely reduced. This differential effect cannot be attributed to the presence of a second SH3 region in mammalian Vav proteins, because mouse Vav proteins lacking both SH3 regions can still promote cell transformation and cytoskeletal change. Despite intense efforts aimed at characterizing the function of the SH3 regions of mammalian Vav proteins, their specific role within the cell remains still obscure. On one hand, it has been shown that this SH3 can bind to a number of proline-rich region containing proteins such as hnRNP-K, Cbl-b and zyxin. On the other hand, it has been postulated that it plays a role in ensuring the proper and efficient subcellular localization of the protein since its missing function can be fully replaced by the introduction of ectopic membrane localization signals at the C-terminus of the Vav ZF region. It is likely that this last function could be conserved in DmVav, because its SH3 mutant cannot be ever detected at the plasma membrane. In any case, these results suggest that, in contrast to the CH, Ac, DH, PH, ZF, and SH2 regions, the regulatory plan for the SH3 regions (both in terms of number of domains and function) have been set up after the protostome/deuterostome split. In this regard, it must be recalled that the most N-terminal SH3 region of Vav proteins has been acquired at the level of C. intestinalis, a urochordate species that is considered the most immediate ancestor of the vertebrate lineage (Couceiro, 2005).

While biochemical and tissue culture experiments have pinpointed the connection of DmVav with Rac1 and F-actin dynamics, the gain-of-function studies carried out in Drosophila embryos has afforded the opportunity to check the effect of the catalytic activity of DmVav in a more physiological context. Using transgenic flies expressing the constitutively active form of DmVav in specific tissues of the Drosophila embryo, it was demonstrated that the ectopic activation of this GEF results in developmental problems remarkably similar to those previously observed for Rac1 mutants. Those included defects in embryonic dorsal closure, myoblast fusion, axon growth and guidance, tracheal cell development, and the migration of different populations of cells. The similarity of phenotypes is consistent with the idea that DmVav and DmRac1 act in common pathways. These results are probably a reflection of the actual role of DmVav in those cells, since this protein has a ubiquitous expression in most of the tissues used in these studies (Couceiro, 2005).

Interestingly, genetic studies have also indicated that DmVav may not be able to induce the activation of all the specific downstream elements of the Rac1 route, as evidenced by the lack of proper activation of the JNK-Dpp pathway in specific cells of Drosophila embryos. Although these observations may seem counterintuitive in principle, recent results have shown that it is not a rare signaling event in GEF/Rho-Rac GTPases relationships. For instance, DmTrio, a Rac1-specific GEF widely expressed in Drosophila, is only required for Rac1 function in axon growth and guidance but not for epithelial morphogenesis or myoblasts fusion. In mammalian cells, it has been shown that the FGD1 GEF triggers JNK activation while having no effect on Pak1. Conversely, the GEFs Tiam1 and Dbl induce the activation of Pak1 but not of JNK. There are several functional scenarios to explain such signaling selectivity. It can be argued that GEFs may act at subcellular localizations that can be fully compatible with Rac1 activation but not accessible to specific downstream elements. However, experimental evidence does not support this possibility, since the subcellular localizations of Trio, Tiam1, and Dbl are very similar, at least when their oncogenic variants are expressed in mammalian cells. It is also possible that the stimulation of specific signaling pathways may require the presence of intermediary adaptors recruited by the GEF that facilitate the physical proximity between the GTP-bound GTPase and the primary effector. If that is the case, the specificity of the effector molecules would be determined by the spectrum of adaptor molecules that the GEF can bind to. This possibility has been confirmed already for some GEFs for the Rho/Rac family. Thus, it has been reported that the N-terminal region of the Tiam1 GEF can bind to either spinophilin or IB2/JIP2, two proteins that facilitate the connection of the activated GTPase with p70S6 and p38 kinases, respectively. Moreover, it has been postulated that the effective activation of Pak1 by Rac1 during T-cell signaling requires the simultaneous association of Vav with Rac1 and Nck, an adaptor protein that can bind to that Rac1 effector. Further genetic and biochemical work in this area will be needed to elucidate the group of Rac1 effectors stimulated by DmVav and the basis for such signaling specificity. Based on these results, it will also be interesting to use cells from the available vav, vav2, and vav3 knockout mice to check the Rho/Rac downstream elements that are specifically affected by the catalytic activity of Vav proteins (Couceiro, 2005).

The observation that the catalytic regulation of the Vav family has been established before the split between protostomes and deuterostomes poises interesting questions regarding the evolutionary time-point at which such functional plan may have been set up originally. Based on previous sequencing data from unicellular and multi-cellular organisms, it was assumed that Vav proteins were totally restricted to animal metazoans. However, a recent report has indicated the presence of tyrosine kinase-related pathways in choanoflagellates (i.e., Monosiga brevicollis)], a group of unicellular and colonial flagellates that resemble cells found only in metazoa. Recent characterization of EST clones from those protozoa resulted in the isolation of five tyrosine kinases distantly related to the Src/Abl, Tie/Tec, and the FGF-receptor families. More importantly to the curren case, they appear to express also vav-related cDNA sequences. Thus, the ancestor for vav family genes could be located much earlier in the phylogenetic tree than previously anticipated. If this is the case, the isolation of this distant family ancestor will be an invaluable tool to track down the molecular evolution of this group of signal transduction molecules (Couceiro, 2005).

DroVav, the Drosophila melanogaster homologue of the mammalian Vav proteins, serves as a signal transducer protein in the Rac and DER pathways

Mammalian Vav signal transducer proteins couple receptor tyrosine kinase signals to the activation of the Rho/Rac GTPases, leading to cell differentiation and/or proliferation. The unique and complex structure of mammalian Vav proteins is preserved in the Drosophila homologue, Vav. Drosophila Vav functions as a guanine-nucleotide exchange factor (GEF) for DRac. Drosophila cells overexpressing wild-type (wt) Vav exhibit a normal morphology. However, overexpression of a truncated Vav mutant (that functions as an oncogene when expressed in NIH3T3 cells) results in striking changes in the actin cytoskeleton, resembling those usually visible following Rac activation. Dominant-negative Rac abrogates these morphological changes, suggesting that the effect of the truncated Vav mutant is mediated by activation of Rac. In Drosophila cells, stimulation of the Drosophila EGF receptor (DER) increases tyrosine phosphorylation of Vav, which in turn associates with tyrosine-phosphorylated DER. In addition, the following results imply that Vav participates in downstream DER signalling, such as ERK phosphorylation: (1) overexpression of Vav induces ERK phosphorylation; and (2) 'knockout' of Vav by RNA interference blocks ERK phosphorylation induced by DER stimulation. Unlike mammalian Vav proteins, Drosophila Vav was not found to induce Jnk phosphorylation under the experimental circumstances tested in fly cells. These results establish the role of Vav as a signal transducer that participates in receptor tyrosine kinase pathways and functions as a GEF for the small RhoGTPase, Rac (Hornstein, 2003).

The receptor tyrosine kinases (RTKs) play an important role in the control of most fundamental cellular processes including the cell cycle, cell migration, cell metabolism and survival, as well as cell proliferation and differentiation. RTK stimulation leads to the deployment of signalling proteins that relay the appropriate specific signals, resulting in the desired cell fate. Many of the signal transducing proteins, including RTKs, are conserved throughout evolution. In the past few years, genetic and biochemical studies in Drosophila have revealed the identity and function of many signalling cascade molecules that are also shared by mammals. However, there are still many signalling proteins whose roles are still unknown. One such signal transducer protein, the Drosophila melanogaster homologue of mammalian Vav proteins, has been isolated and partially characterized (Dekel, 2000; Hornstein, 2003 and references therein).

Vav proteins represent a novel family of signal transducers that couple tyrosine kinase signals with the activation of the Rho/Rac GTPases and are likely to play an integral role in the regulation of cell differentiation in many tissues. The first member of the mammalian Vav family of cytoplasmic signal transducer proteins to be identified, Vav1, was isolated as an oncogene (Katzav, 1989). Removal of its amino terminus activates Vav1 as a transforming protein (Coppola, 1991; Katzav, 1991). Likewise, the corresponding molecular lesions in Vav2 and Vav3, two other members of the mammalian Vav family, render these proteins transforming (Schuebel, 1996; Movilla, 1999; Zeng, 2000). Unlike Vav1, which is exclusively expressed in hematopoietic cells, Vav2 and Vav3 are expressed in both hematopoietic cells and in many cells of nonhematopoietic origin. Numerous biochemical and overexpression experiments revealed that tyrosine phosphorylation of Vav1 in response to activation by one of several cytokines, growth factors or antigen receptors regulates its activity as a GEF for the Rho/Rac family of GTPases, RhoA, Rac1 and RhoG (Bustelo, 1992; Margolis, 1992; Crespo, 1996, Crespo, 1997; Bustelo, 2000; Turner, 2002). Activation of these GTPases leads to cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs) in T cells. Vav2 and Vav3 function in a similar but not identical fashion. While both Vav2 and Vav3 also act as GEFs, there are conflicting reports regarding which GTPases are activated by them, and whether these GTPases are distinct from those activated by Vav1. Knockout experiments revealed that in T cells, Vav1 integrates signals from lymphocyte antigen receptors and costimulatory receptors to control differentiation, proliferation and the response to activation (Fischer, 1998; Holsinger, 1998). Thus, mice deficient in Vav1 exhibit defects in numerous responses to T-cell stimulation, including capping of the T-cell receptor (TCR) postactivation, recruitment of the actin cytoskeleton to the CD3 chain of the TCR, interleukin-2 (IL-2) production and proliferation, cell cycle progression, activity of NF-AT, phosphorylation of SLP-76 and increase in Ca2+ influx. Mice deficient in Vav2 display no obvious defects in T-cell development yet exhibited some defects in B-cell function (Doody, 2001; Tedford, 2001). Mice lacking both Vav1 and Vav2 displayed major defects in B-cell function that are as dramatic as the defects in T-cell development and activation observed in Vav1-/- mice. Since there are no reports regarding Vav3-/- mice at the present time, the picture of the intricate signalling network induced by the Vav proteins is incomplete. However, it is obvious that the redundancy and complexity of the mammalian Vav proteins even in hematopoietic cells together with the possibility that they differ both in their protein-protein interactions and in their activation of various GTPases, makes it difficult to clearly interpret the results of knockout and other experiments in mammals (Hornstein, 2003 and references therein).

In Drosophila, only one Vav homologue is present (Dekel, 2000). The highly conserved and unique structure of Vav suggests that the vav genes probably evolved from one ancestral gene and that they are important regulatory molecules in flies as well as in mammals. Drosophila Vav encodes a protein whose similarity with hVav1 is 47% and with hVav2 and hVav3 is 45%. Like mammalian Vav proteins, Drosophila Vav encodes a 'calponin-homology' (CH) region, a dbl homology (DH) domain, a pleckstrin homology (PH) domain and both an Src Homology 2 (SH2) and an Src Homology 3 (SH3) domains. However, unlike mammalian Vav proteins, Drosophila Vav lacks an amino-SH3 region. Vav is the only known Drosophila Rho GEF that encodes in addition to a DH region, both SH2 and SH3 domains, attesting that it may be a uniquely versatile signal transducer. The fact that only one homologue of Vav is present in flies, combined with the unique and highly conserved structure of the protein, promises that any study of Vav in flies should be highly beneficial and instructive (Hornstein, 2003).

A hallmark of Vav signal transducer proteins is that their tyrosine residues become phosphorylated on tyrosine residues in response to EGF stimulation. Furthermore, Vav proteins are known to bind to the stimulated EGFR through their SH2 region. In mammalian cells, Drosophila Vav is tyrosine phosphorylated in response to EGFR induction; in vitro, the Drosophila Vav SH2 region is associated with tyrosine-phosphorylated EGFR (Dekel, 2000). These results combined with the encoded domain structure of Vav strongly suggest that Drosophila Vav may function as a signal transducer protein in the Drosophila EGF receptor (DER) signalling cascades. This study investigated the role of Vav in downstream signalling from the DER, its interaction with the Drosophila Rac pathway, and its ability to effect cytoskeletal changes in Drosophila cells (Hornstein, 2003).

This study demonstrates that Vav can activate Rac in vivo. Rac is involved in Drosophila in various cellular processes including cell shape, cell adhesion, gene transcription, protein trafficking and cell cycle progression, as well as numerous developmental processes. One of the best known characteristics of Rac is that it is involved in actin cytoskeletal organization. Indeed, the expression of a constitutively activated form of DRac (V12DRac) in cultured S2 cells causes marked changes in the morphology of the cells, leading to lamellipodia and microspikes. These results indicate that overexpression of oncVav (Drosophila Vav that lacks 214 residues of its amino-terminus), but not wild-type Vav, can induce a morphology similar to that obtained with V12DRac (constitutively active mutant Rac). The fact that N17DRac inhibits the changes in the morphology obtained with oncVav further substantiates the conclusion that the activation of Rac by Vav is responsible for the observed cytoskeletal reorganization. Correspondingly, an inactive hVav1 variant defective in its ability to activate Rac inhibited the ability to induce actin cytoskeletal organization (Ma, 1998), thus further supporting the tight association between Vav, Rac and actin organization. Notably, coexpression of oncVav and V12DRac leads to more profound changes in cytoskeleton organization compared to those observed in cells overexpressing each protein alone. This result could be explained by the fact that the sum of activation reached by both the endogenous Rac activated by oncVav as well as the constitutively activated Rac yields a more striking morphology. Consistent with these results with wild-type Vav, wt hVav1 does not cause any change in morphology of NIH3T3 cells (Kranewitter, 1999) and COS cells (Ma, 1998). Conversely, a remarkable change in actin organization has been observed (Michel, 2000) following overexpression of hVav1 in T cells. These conflicting results obtained with mammalian wtVav1 could stem from the use of different experimental systems, including the strong possibility that different levels of endogenous Vav2 and Vav3 exist in these systems. The cytoskeletal changes in S2 cells transfected with oncVav are compatible with a previous study demonstrating that an amino-terminus-truncated hVav1 caused depolarization of fibroblasts and triggered the bundling of actin stress fibers in NIH3T3 cells (Kranewitter, 1999). Taken together, these results support a pathway in which Drosophila Vav serves as a GEF for Rac, thereby triggering the reorganization of the actin cytoskeleton (Hornstein, 2003).

Drosophila Vav is tyrosine phosphorylated in response to stimulation of DER and it also associates with the stimulated receptor. This result is compatible with the known characteristics of mammalian Vav proteins. For example, when ectopically expressed in nonhematopoietic cells, Vav1 associates with the EGFR through its SH2 region and becomes tyrosine phosphorylated upon induction with EGF. A similar result was reported for the ubiquitously expressed Vav2 and Vav3 proteins. Although it is well established that the Vav proteins are tyrosine phosphorylated upon EGFR stimulation, the exact contribution of the various mammalian Vav proteins to the EGFR signalling pathway is not understood. These studies with Drosophila Vav shed some light on these events. Thus, this study demonstrates that overexpression of Vav in D2F cells leads to increased ERK phosphorylation. Moreover, its elimination by the use of dsRNAi blocks phosphorylation of ERK even following stimulation of DER. There are opposing results regarding the link between Vav and ERK activation in mammals. Overexpression of Vav1 in Jurkat T cells induced 3-4-fold activation of ERK activation (Villalba, 2000). Vav1 activates ERK when ectopically expressed in NIH3T3 or CHO cells (Khosravi-Far, 1994; Miranti, 1998). Furthermore, Vav1 coimmunoprecipitates with ERK in a human myeloma cell line stimulated with interleukin-6 (IL-6; Lee, 1997). Finally, T cells deficient in Vav1 exhibit defects in ERK phosphorylation (Costello, 1999). Conversely, the activation of Ras and ERK following stimulation of the TCR in Vav1 null Jurkat T cells appears normal. Without the complication of multiple homologues, these studies strongly suggest a role for Drosophila Vav in the ERK pathway in S2 cells (Hornstein, 2003).

Despite the existence of several studies that point to a link between hVav1 and Ras (Bustelo, 1994; Katzav, 1995; Wu, 1995), it is still unclear how Vav proteins can affect ERK. Drosophila Vav may affect the Ras/ERK pathway through its function as a GEF towards DRac. Cross talk between the Rac and Ras pathways has been shown to exist in mammals. Rho family small GTPases were found to play an important role in mediating the activation of Raf by Ras. Thus, a dominant-negative mutant of Rac can block Raf activation by Ras. Additionally, the effect of Rac can be substituted by the PAK kinase, which is a direct downstream target of Rac. Moreover, PAK directly associates with Raf-1 under both physiological and overexpressed conditions. The extent of interaction between PAK and Raf-1 is correlated with the ability of PAK to phosphorylate Raf and induce mitogen-activated protein kinase activation. These studies strongly suggest that cross talk between Rac and Ras exist and it is mediated through activation of downstream effectors of Rac, such as PAK. Furthermore, it was demonstrated that MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42. A novel Drosophila gene, DRacGAP, has been identified which behaves as a negative regulator of the GTPases, DRac1 and DCdc42. Reduced function of DRacGAP or increased expression of DRac1 in the wing imaginal disk causes effects on vein and sensory organ development and cell proliferation as a result of enhanced activity of the EGFR/Ras signalling pathway. Thus, DRac and DRas are involved in cross-talk mechanisms that modulate Drosophila development (Hornstein, 2003 and references therein).

Drosophila Vav might also activate ERK in a GEF-independent manner. For instance, Vav might stimulate the Ras/ERK pathway via PLC activation, just as Vav1 was shown to activate PLC (Costello, 1999; Reynolds, 2002). PLC contributes to the activation of Ras, probably by stimulating the activity of the diacylglycerol-dependent exchange factor, Ras GRP. It is highly conceivable that the activity of hVav1 towards PLC is mediated in a GEF-independent mode (Jordan, 2003). Whether such a mechanism is also elicited in flies remains to be determined. Drosophila Vav contains several protein-binding domains (SH2, SH3) that might participate in various pathways that result in activation of the Ras/ERK pathway. For instance, Vav may bind to the adapter molecule DShc, that was shown to be associated with the Grb2/Drk proteins leading to DRas activation. Indeed, mammalian Shc binds mammalian Vav proteins (Ramos-Morales, 1994). Collectively, the current results clearly illustrate that Vav influences both the DRac and ERK pathways. However, it is not clear yet whether it exerts its influence on ERK by an exclusive inducement of the DRac pathway and/or through a GEF-independent activity (Hornstein, 2003).

The involvement of mammalian Vav proteins, by functioning as GEFs towards Rac in the JNK signalling cascade, is well established (Hehner, 2000; Kaminuma, 2001; Moller, 2001). Moreover, Vav proteins mediate this response through their function as GEFs towards Rac. This pathway is highly conserved between mammals and flies. In Drosophila, it can transduce signals of a diversified nature, leading to changes in cell polarity and mediating immunity in the adult. It is also required for dorsal closure during embryonic development. Genetic studies focusing on these processes placed the Rho family small GTPases in the JNK signalling cascade. However, although this study demonstrated that Vav functions as a GEF towards DRac in Drosophila, Vav does not seem to be involved in the sorbitol-induced JNK activation. In accordance, no effect has been detected of Rho family small GTPases on sorbitol-induced JNK activation in S2 cells. It is therefore conceivable that in S2 cells, the sorbitol-induced activation of JNK is not mediated through activation of DRac, and therefore does not require Vav. The possibility that Vav is involved in JNK activation under other physiological pathways in Drosophila, such as dorsal closure, still exists; however, this question merits further investigation (Hornstein, 2003).

In summary, these results show that, in fly cells, Vav functions in various signalling cascades in which it can play a role as a GEF or participate as an adapter protein. A P-element insertion has recently been reported to inactivate Vav, leading to lethality of flies (Bourbon, 2000). Further genetic experiments will be required to better understand the physiological function of Vav in developmental systems (Hornstein, 2003).

Identification of the Drosophila melanogaster homologue of the mammalian signal transducer protein, Vav

Mammalian Vav signal transducer protein couples tyrosine kinase signals with the activation of the Rho/Rac GTPases, thus leading to cell differentiation and/or proliferation. The DroVav gene, the homologue of hVav in Drosophila melanogaster, was isolated and characterized in this study. DroVav encodes a protein (793 residues) whose similarity with hVav is 47% and with hVav2 and hVav3 is 45%. DroVav preserves the unique, complex structure of hVav proteins, including the 'calponin homology', dbl homology, pleckstrin homology; SH2 and SH3 domains in addition to regions that are acidic rich, proline rich and cysteine rich. DroVav is located on the X chromosome in polytene interval 18A5;18B and is expressed in all stages of development and in all tissues. In mammalian cells, DroVav is tyrosine-phosphorylated in response to epidermal growth factor receptor (EGFR) induction; in vitro, the DroVav SH2 region is associated with tyrosine-phosphorylated EGFR. Thus, DroVav probably plays a pivotal role as a signal transducer protein during fruit fly development (Dekel, 2000).

Search PubMed for articles about Drosophila Vav

Afshari, F. T., Kwok, J. C. and Fawcett, J. W. (2010). Astrocyte-produced ephrins inhibit schwann cell migration via VAV2 signaling. J Neurosci 30: 4246-4255. PubMed ID: 20335460

Bianco, A., Poukkula, M., Cliffe, A., Mathieu, J., Luque, C. M., Fulga, T. A. and Rorth, P. (2007). Two distinct modes of guidance signalling during collective migration of border cells. Nature 448: 362-365. PubMed ID: 17637670

Billadeau, D. D. (2002). Cell growth and metastasis in pancreatic cancer: is Vav the Rho'd to activation? Int J Gastrointest Cancer 31: 5-13. PubMed ID: 12622410

Bishop, A. L. and Hall, A. (2000). Rho GTPases and their effector proteins. J. Biochem. (Tokyo) 348: 241-255. PubMed ID: 10816416

Bourbon, H. M., et al. (2000). A P-insertion screen identifying novel X-linked essential genes in Drosophila. Mech. Dev. 110(1-2): 71-83. PubMed ID: 11744370

Bustelo X. R. (2000). Regulatory and signaling properties of the Vav family. Mol. Cell. Biol. 20: 1461-1477. PubMed ID: 10669724

Coppola, M. S., Bryant, S., Koda, T., Conway, D. and Barbacid, M. (1991). Mechanism of activation of the vav protooncogene. Cell Growth Differ. 2: 95-105. PubMed ID: 2069873

Costello, P. S., et al. (1999). The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK, and NF-kappaB pathways. Proc. Natl. Acad. Sci. 96: 3035-3040. PubMed ID: 10077632

Couceiro, J. R., Martin-Bermudo, M. D. and Bustelo, X. R. (2005). Phylogenetic conservation of the regulatory and functional properties of the Vav oncoprotein family. Exp Cell Res 308: 364-380. PubMed ID: 15950967

Cowan, C. W., Shao, Y. R., Sahin, M., Shamah, S. M., Lin, M. Z., Greer, P. L., Gao, S., Griffith, E. C., Brugge, J. S. and Greenberg, M. E. (2005). Vav family GEFs link activated Ephs to endocytosis and axon guidance. Neuron 46: 205-217. PubMed ID: 15848800

Crespo, P., et al. (1996). Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav. Oncogene 13(3): 455-60. PubMed ID: 8760286

Crespo, P., et al. (1997). Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385(6612): 169-72. PubMed ID: 8990121

Dekel, I., Russek, N., Jones, T., Mortin, M. A. and Katzav, S. (2000). Identification of the Drosophila melanogaster homologue of the mammalian transducer protein, Vav. FEBS Lett. 472: 99-104. PubMed ID: 10781813

Doody, G. M., et al. (2001). transduction through Vav-2 participates in humoral immune responses and B cell maturation. Nat. Immunol. 2: 542-547. PubMed ID: 11376342

Fernandez-Espartero, C. H., Ramel, D., Farago, M., Malartre, M., Luque, C. M., Limanovich, S., Katzav, S., Emery, G. and Martin-Bermudo, M. D. (2013). GTP exchange factor Vav regulates guided cell migration by coupling guidance receptor signalling to local Rac activation. J Cell Sci 126: 2285-2293. PubMed ID: 23525006

Fernandez-Zapico, M. E., Gonzalez-Paz, N. C., Weiss, E., Savoy, D. N., Molina, J. R., Fonseca, R., Smyrk, T. C., Chari, S. T., Urrutia, R. and Billadeau, D. D. (2005). Ectopic expression of VAV1 reveals an unexpected role in pancreatic cancer tumorigenesis. Cancer Cell 7: 39-49. PubMed ID: 15652748

Fischer, K. D., et al. (1998). Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor. Curr. Biol. 8: 554-562. PubMed ID: 9601639

Hehner, S. P., et al. (2000). Tyrosine-phosphorylated Vav1 as a point of integration for T-cell receptor- and CD28-mediated activation of JNK, p38, and interleukin-2 transcription. J. Biol. Chem. 275: 18160-18171. PubMed ID: 10849438

Holsinger, L. J., et al. (1998). Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction. Curr. Biol. 8: 563-572. PubMed ID: 9601640

Hornstein, I., Mortin, M. A. and Katzav, S. (2003). DroVav, the Drosophila melanogaster homologue of the mammalian Vav proteins, serves as a signal transducer protein in the Rac and DER pathways. Oncogene 22: 6774-6784. PubMed ID: 14555990

Hunter, S. G., Zhuang, G., Brantley-Sieders, D., Swat, W., Cowan, C. W. and Chen, J. (2006). Essential role of Vav family guanine nucleotide exchange factors in EphA receptor-mediated angiogenesis. Mol Cell Biol 26: 4830-4842. PubMed ID: 16782872

Jekely, G., Sung, H. H., Luque, C. M. and Rorth, P. (2005). Regulators of endocytosis maintain localized receptor tyrosine kinase signaling in guided migration. Dev Cell 9: 197-207. PubMed ID: 16054027

Jordan, M. S., Singer, A. L. and Koretzky, G. A. (2003). Adaptors as central mediators of signal transduction in immune cells. Nat. Immunol. 4: 110-116. PubMed ID: 12555096

Kaminuma, O., Deckert, M., Elly, C., Liu, Y. C. and Altman, A. (2001). Mol. Cell. Biol. 21: 3126-3136. PubMed ID:

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date revised: 22 November 2022

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