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• W1972234127 abstract "Article16 November 1998free access Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2 Kornel E. Schuebel Kornel E. Schuebel Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, NY, 11794-7025 USA Search for more papers by this author Nieves Movilla Nieves Movilla Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, NY, 11794-7025 USA Search for more papers by this author José Luis Rosa José Luis Rosa Present address: Unidad de Bioquímica (4178), Facultad de Medicina, Campus de Bellvitge, Universidad de Barcelona, C/ Feixa Llarga s/n, 08907 Hospitalet (Barcelona), Spain Search for more papers by this author Xosé R. Bustelo Corresponding Author Xosé R. Bustelo Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, NY, 11794-7025 USA Search for more papers by this author Kornel E. Schuebel Kornel E. Schuebel Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, NY, 11794-7025 USA Search for more papers by this author Nieves Movilla Nieves Movilla Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, NY, 11794-7025 USA Search for more papers by this author José Luis Rosa José Luis Rosa Present address: Unidad de Bioquímica (4178), Facultad de Medicina, Campus de Bellvitge, Universidad de Barcelona, C/ Feixa Llarga s/n, 08907 Hospitalet (Barcelona), Spain Search for more papers by this author Xosé R. Bustelo Corresponding Author Xosé R. Bustelo Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, NY, 11794-7025 USA Search for more papers by this author Author Information Kornel E. Schuebel1, Nieves Movilla1, José Luis Rosa2 and Xosé R. Bustelo 1 1Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, NY, 11794-7025 USA 2Present address: Unidad de Bioquímica (4178), Facultad de Medicina, Campus de Bellvitge, Universidad de Barcelona, C/ Feixa Llarga s/n, 08907 Hospitalet (Barcelona), Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:6608-6621https://doi.org/10.1093/emboj/17.22.6608 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info We show here that Vav-2, a member of the Vav family of oncoproteins, acts as a guanosine nucleotide exchange factor (GEF) for RhoG and RhoA-like GTPases in a phosphotyrosine-dependent manner. Moreover, we show that Vav-2 oncogenic activation correlates with the acquisition of phosphorylation-independent exchange activity. In vivo, wild-type Vav-2 is activated oncogenically by tyrosine kinases, an effect enhanced further by co-expression of RhoA. Likewise, the Vav-2 oncoprotein synergizes with RhoA and RhoB proteins in cellular transformation. Transient transfection assays in NIH-3T3 cells show that phosphorylated wild-type Vav-2 and the Vav-2 oncoprotein induce cytoskeletal changes resembling those observed by the activation of the RhoG pathway. In contrast, the constitutive expression of the Vav-2 oncoprotein in rodent fibroblasts leads to major alterations in cell morphology and to highly enlarged cells in which karyokinesis and cytokinesis frequently are uncoupled. These results identify a regulated GEF for the RhoA subfamily, provide a biochemical explanation for vav family oncogenicity, and establish a new signaling model in which specific Vav-like proteins couple tyrosine kinase signals with the activation of distinct subsets of the Rho/Rac family of GTPases. Introduction The GTP-binding proteins of the Rho/Rac family participate in coordinated cellular responses to extracellular stimuli (for a review see Van Aelst and D‘Souza-Chorey, 1997; Hall, 1998). Their action is essential to promote the formation of cytoskeletal structures, the activation of kinase cascades and the induction of nuclear responses required for both developmental and proliferative decisions (Van Aelst and D'Souza-Chorey, 1997; Hall, 1998). Members of this family can be grouped into three classes according to amino acid sequence similarities. The first subfamily is composed of four Rac proteins (Rac-1, Rac-2, Rac-3 and RhoG). Some of these proteins promote the activation of protein kinases such as PAK, c-Jun N-terminal kinase (JNK) and p38MAPK. They are also involved in the activation of other independent pathways regulating membrane ruffling and cell proliferation (Van Aelst and D‘Souza-Chorey, 1997; Hall, 1998). The second subfamily, Rho, includes RhoA, RhoB, RhoC, RhoD, RhoE and TTF proteins. Of these, RhoA has been characterized extensively and shown to be involved in cell transformation, formation of stress fibers and focal adhesions, and in the stimulation of protein kinases such as PKN and p160Rock (Van Aelst and D'Souza-Chorey, 1997). Finally, the third subfamily is composed of TC10 and the two isoforms of the Cdc42 protein. In this subfamily, Cdc42 was shown to be involved in the activation of JNK, PAK and p38MAPK as well as in the formation of filopodia in the plasma membrane (Van Aelst and D'Souza-Chorey, 1997; Hall, 1998). The activation and deactivation cycle of most Rho proteins is regulated by the differential binding of guanosine nucleotides (for a review see Boguski and McCormick, 1993). In quiescent cells, these GTPases are in an inactive state maintained by the presence of bound GDP molecules. Stimulation of cells via a number of extracellular stimuli leads to the exchange of GDP by GTP molecules, a transition that allows the release of inhibitory molecules from the GTPases (GDP dissociation inhibitors), the translocation of the GTP-binding proteins to the plasma membrane and the acquisition of a tertiary conformation optimal for the binding of their effector molecules (Boguski and McCormick, 1993). The exchange of guanosine nucleotides on these GTPases is catalyzed by guanosine nucleotide exchange factors (GEFs) (Boguski and McCormick, 1993). To date, two different families of Rho GEFs have been identified that differ in the structure of their catalytic domains. The first group is composed of Rho GDP dissociation stimulators (GDS), a family of proteins distantly related to the Cdc25 homology regions present in Ras GEFs (Boguski and McCormick, 1993). GDSs work at stoichiometric concentrations and have a rather broad catalytic specificity, being active on prenylated K-Ras, Rho and Rap proteins (Boguski and McCormick, 1993). The second subset of Rho activators comprises an extensive number of enzymes containing Dbl-homology (DH) domains with catalytic activity exclusively directed towards Rho/Rac GTPases (for a review see Cerione and Zheng, 1996). The majority of these GEFs are highly transforming when overexpressed either as wild-type or truncated proteins (Cerione and Zheng, 1996), a property that highlights their importance as regulators of mitogenic processes. Although Rho GEFs have been characterized extensively both biochemically and oncogenically, little information is available regarding the mechanism by which they become activated during signal transduction. To date, the best example for the participation of a DH-containing protein in receptor-mediated cell signaling is the product of the vav proto-oncogene, a protein preferentially expressed in the hematopoietic system (for a review see Bustelo, 1996). In addition to the DH and pleckstrin homology (PH) regions commonly found in Rho/Rac GEFs, Vav contains other structural motifs, including a calponin-homology (CH) region, an acidic domain (AD), a zinc finger butterfly motif, two SH3 regions and one SH2 domain (Bustelo, 1996). Vav becomes tyrosine phosphorylated during the signaling of many receptors with intrinsic or associated protein tyrosine activity (Bustelo, 1996), and binds to a number of signaling molecules via its SH2 and SH3 domains (Bustelo, 1996). Recently, biochemical experiments have demonstrated that the phosphorylation of Vav on tyrosine residues leads to the activation of its GDP/GTP exchange activity towards Rac-1 (Crespo et al., 1997). In good agreement with such observations, it has been shown that several elements of the Rac-1 pathway, including Rac-1 itself and JNK, are activated by wild-type Vav protein upon co-transfection with protein tyrosine kinases (Crespo et al., 1997; Teramoto et al., 1997). More recently, Han et al. (1998) have shown that the activity of phosphorylated Vav can be enhanced further by the binding to the Vav PH domain of products of the phosphatidylinositol 3-kinase. Vav appears, therefore, to be an essential mediator of mitogenic and antigenic signals, providing a direct connection between membrane-derived signals and the activation of the Rac-1 pathway. Interestingly, Vav appears to be a member of a new family of signal transduction molecules highly conserved during evolution. Thus, a vav-related gene was identified recently in Caenorhabditis elegans during the characterization of the genome of this nematode. The C.elegans Vav-like protein lacks the two C-terminal SH3 domains but maintains all the other structural domains present in the mammalian counterpart (Bustelo, 1996). In addition, a new protein (Vav-2) with the same arrangement of structural domains as Vav has been identified both in human and mouse (Henske et al., 1995; Schuebel et al., 1996). In spite of their structural similarity, Vav and Vav-2 differ in several biological properties. For example, Vav-2 displays a ubiquitous pattern of expression during both embryonic and adult mouse stages (Schuebel et al., 1996). Moreover, Vav-2 requires a more extensive deletion of the N-terminus than Vav to become oncogenically active (Schuebel et al., 1996). Perhaps more importantly, it has been shown that the expression of the vav and vav-2 oncogenes leads to different types of morphological transformation in rodent fibroblasts (Schuebel et al., 1996), a result that suggests that they may work through similar, but not identical, signal transduction pathways. Thus, the discovery of new Vav-like proteins has given further relevance to the role of this protein family in cell signaling and, in addition, has raised new functional questions such as those regarding the type(s) of mechanism(s) of activation and the functional redundancy of the different Vav family members. In order to characterize the function of this new member of the Vav family in more detail, we have investigated the biochemical and biological properties of both the wild-type and oncogenic forms of Vav-2. Using GDP/GTP exchange reactions, we show here that wild-type Vav-2 is a phosphorylation-dependent GDP/GTP exchange factor that targets a subset of GTP-binding proteins overlapping, but not identical to, those engaged by Vav. Moreover, we demonstrate that Vav-2 transformation is mediated by the expression of a constitutively active protein that leads to morphological changes different from those induced by the Vav oncoprotein. Hence, these results identify a phosphorylation-dependent RhoA subfamily GEF, establish a general mechanism by which Vav family members achieve full oncogenic activity, and suggest a new signaling pathway in which membrane receptors will turn on distinct GTPase pathways via the stimulation of different members of the Vav family. Results The Vav-2 oncoprotein induces morphological changes in rodent fibroblasts We have shown previously that the expression of vav and vav-2 oncogenes in NIH-3T3 cells leads to the generation of foci of different morphology. Thus, expression of both the human and mouse vav oncogene induces the generation of dense, non-refractile cells that pile up to form mountain-like foci (Coppola et al., 1991; Schuebel et al., 1996). Instead, the expression of the vav-2 oncogene leads to the formation of flat foci composed of monolayers of very enlarged and multinucleated cells that are accompanied by clumps of small, rounded, highly refractile cells (Schuebel et al., 1996). To verify that these differences are stable in time, several foci of vav-2-transformed cells were randomly picked, expanded and purified further by cloning in soft agar. After this step, individual colonies of vav-2-transformed cells were expanded to obtain stable vav-2-transformed cell lines. The microscopic examination of these cells showed that they had conserved the morphological change previously detected in the cells within the vav-2 foci. These cell lines are composed of enlarged and multinucleated cells (Figure 1B, C and F–H), small flat cells, and small and refractile rounded cells whose presence becomes more apparent in confluent cultures (Figure 1B and C). A similar morphological change was observed in cells expressing the dbl oncogene (data not shown, Eva and Aaronson, 1985). This phenotype is conserved during multiple passages, although the giant cells are significantly reduced in number after prolonged culture (Figure 1C). Unlike vav-2- and dbl-expressing cells, vav-transformed NIH-3T3 cells display a very low proportion of multinucleated giant cells and the majority of the culture is composed of small flat cells showing frequent membrane ruffling (Figure 1I) that can reach very high cell densities in confluent cultures (Figure 1D). As a negative control, NIH-3T3 cells show a fibroblast-like morphology (Figure 1E) and undergo cell growth arrest after reaching confluency (Figure 1A). As expected, the morphology of both vav- and vav-2-transformed cells is also quite different from that induced by other unrelated exchange factors, such as Ras GRF, a Ras- and R-Ras-specific GDP/GTP exchange factor (Figure 1J). The morphologies of vav- and vav-2-transformed cells were observed in six independently cloned cell lines (data not shown). Figure 1.Morphology of vav-2- and vav-transformed cells. Confluent (A–D) or subconfluent (E–J) cultures of parental NIH-3T3 cells (A and E) and established cell lines expressing the mouse vav-2 oncogene at early (B and F–H) or after 10 passages (∼1 month of culture; C), the mouse vav oncogene (D and I) or the farnesylated Cdc25 domain of the rat Ras GRF protein (J) were observed under the microscope using Nomarski optics. The scale bars for (A–D) and (E–J) are below (D) and (H), respectively. Download figure Download PowerPoint The morphological transformation induced in NIH-3T3 cells by Vav family proteins results in marked changes in the cytoskeleton. Thus, while exponentially growing NIH-3T3 cells show the presence of thin bundles of F-actin in the form of stress fibers (Figure 2A), the expression of the vav oncogene leads to a general disruption of stress fibers and to a preferential localization of actin molecules in peripheral membrane structures (Figure 2B). In contrast, expression of the Vav-2 oncoprotein in the same cell background induces the formation of abundant stress fibers in the giant cells (Figure 2C–E). These stress fibers display a parallel distribution in the majority of vav-2-transformed cells (Figure 2C and D) although, occasionally, adopt a radial configuration (Figure 2E). The co-staining of these cells with anti-vinculin antibodies revealed that many of the vav-2-transformed cells contain thick and long focal adhesion plaques that generally co-localize with the distal tips of the actin fibers (Figure 2F–H). In contrast to the enlarged cells, the morphology of the vav-2-transformed cells of normal size was more heterogenous, including the presence of flat cells with stress fiber distributions similar to those discussed above as well as flat or rounded cells showing a total absence of stress fibers and focal adhesions (Figure 2C and F, and E and H). Under these culture conditions, no filopodia were observed in either vav- or vav-2-transformed cells (Figures 1 and 2). The change in the cytoskeleton in vav- and vav-2-transformed cells appears to be restricted to the actin network, as other structures such as tubulin microfilaments showed no alteration in those cells (Figure 2I–K). Taken together, these results indicate that the constitutive expression of Vav and Vav-2 oncoproteins induces different morphological changes in rodent fibroblasts, suggesting that their signaling pathways are not identical. Figure 2.Cytoskeletal organization of vav-2-transformed cells. NIH-3T3 cells (A and I), vav- (B) and vav-2- (C–H, J and K) transformed cells were submitted to incubations with FITC-labeled phalloidin (A and B–E), or antibodies to either vinculin (F–H) or α-tubulin (I–K), as indicated in Materials and methods. (C and F), (D and G) and (E and H) represent the same cell co-stained with phalloidin and anti-vinculin antibodies. Download figure Download PowerPoint Vav-2 is a phosphorylation-dependent guanosine nucleotide exchange factor for the RhoA subfamily The different morphology of vav- and vav-2-transformed cells led us to investigate the catalytic specificity of Vav-2 towards GTP-binding proteins of the Rho family. To this end, we first generated a baculovirus capable of expressing the full-length mouse Vav-2 protein after infection of Spodoptera frugiperda (Sf9) cells. To facilitate the recovery of the protein from the total cellular extracts, we included a stretch of polyhistidine residues at the N-terminus of the protein to allow its purification by chromatography onto nickel beads. This method allowed the efficient purification of full-length Vav-2 protein free of other protein contaminants as determined by Coomassie Blue staining of SDS–polyacrylamide gels (Figure 3A). Since the activity of Vav is dependent on tyrosine phosphorylation (Crespo et al., 1997), the purified Vav-2 protein was then incubated with the protein tyrosine kinase Lck in the presence of ATP, a treatment that leads to optimal Vav-2 phosphorylation as determined by in vitro kinase assays in the presence of [γ-32P]ATP (Figure 3B). Next, we purified several representative members of the Rho family as GST fusion proteins to be used as substrates by using a standard bacterial expression system (Figure 3C). After purification, the activity of these proteins was demonstrated by testing their ability to hydrolyze [α-32P]GTP into [α-32P]GDP (Figure 3D). Figure 3.(A) Purification of Vav-2 from Sf9 cells. An aliquot of a preparation of a representative Vav-2 purification were analyzed by SDS–PAGE in the presence of molecular weight markers (M) and increasing concentrations of BSA as standard for concentration. The amount of loaded proteins is given in μg. (B) Phosphorylation of wild-type Vav-2 by Lck. In vitro kinase reactions were performed with the indicated proteins in the presence of [γ-32P]ATP, separated by SDS–PAGE and submitted to autoradiographic exposure. The migration of Vav-2 and GST–Lck is indicated by an arrow and an arrowhead, respectively. (C) Purification of Rho GTP-binding proteins from E.coli cells. Aliquots (2 μg) of each purified GST–GTPase were analyzed by SDS–PAGE in the presence of molecular weight markers. (D) GTPase activity of purified Rho proteins estimated as described in Materials and methods. The mobility of the 32P-labeled GTP and GDP molecules is indicated on the left. Download figure Download PowerPoint The exchange activities of the non-phosphorylated and phosphorylated versions of Vav-2 were then determined by measuring their ability to enhance the incorporation of [35S]GTP-γS into GTPases representative of the three branches of the Rho/Rac family (Rac-1, RhoA and Cdc42). As shown in Figure 4A (left panel), the non-phosphorylated version of Vav-2 displays a low, albeit reproducible, exchange activity on RhoA. Most noticeably, phosphorylation of this protein by Lck leads to higher levels of RhoA [35S]GTP-γS binding (Figure 4A, left panel). The stimulation of Vav-2 exchange activity by tyrosine phosphorylation in vitro oscillated between 3.5- and 8-fold, depending on the batches of purified Vav-2 protein used in the assays. In contrast to this activity, all Vav-2 batches were inactive towards Rac-1 and Cdc42 (Figure 4A, left panel, and data not shown). To verify that these GTPases were active in this assay, Rac-1 and Cdc42 were submitted to exchange reactions in the presence of either the human Dbl oncoprotein or the non-phosphorylated and phosphorylated versions of mouse Vav. As shown in Figure 4A (right panel), Dbl elicited exchange activity on both RhoA and Cdc42, while phosphorylated Vav did so on Rac-1 (Figure 4A, right panel). Non-phosphorylated Vav induced no detectable nucleotide exchange in any of these GTP-binding proteins (Figure 4A, right panel). These results are in agreement with the catalytic specificity of these GEFs (Zheng et al., 1995; Crespo et al., 1997), and demonstrate that the lack of activity of Vav-2 towards Rac-1 and Cdc42 is not due to the use of inactive GTPases in these assays. Figure 4.(A) Exchange of Vav-2 on Rho/Rac proteins. Left panel: GDP-loaded GTPases were incubated for 45 min with [35S]GTP-γS in the presence of phosphorylated Vav-2 (black boxes), non-phosphorylated Vav-2 (gray boxes) or autophosphorylated GST–Lck (white boxes). Right panel: exchange of Dbl and Vav on Rho/Rac proteins. Reactions were conducted as above with either autophosphorylated Lck (white boxes), non-phosphorylated Vav (gray boxes), phosphorylated Vav (black boxes) or Dbl (shaded boxes). (B) Kinetics of Vav-2 exchange on Rho using [35S]GTP incorporation (left panel) or [3H]GDP release (right panel) assays. RhoA was pre-loaded with either cold GDP (left panel) or [3H]GDP (right panel) and submitted to exchange reactions for the indicated periods of time with autophosphorylated Lck (triangles), non-phosphorylated Vav-2 (diamonds) or phosphorylated Vav-2 (squares). (C) Activation of Vav-2 exchange activity by protein tyrosine kinases. GDP-loaded GTPases were incubated with [35S]GTP-γS in the presence of phosphorylated Vav-2 (black boxes), non-phosphorylated Vav-2 (gray boxes) or the indicated autophosphorylated protein tyrosine kinase (white boxes). After 45 min, the incorporation of [35S]GTP-γS onto RhoA was determined as indicated in (A). (D) Exchange activity of Vav-2 towards RhoA subfamily members. The indicated GDP-loaded GTPases were submitted to exchange reactions under the conditions indicated in (A). In (A), values represent the mean and standard deviation (SD) of three independent determinations each performed in duplicate. (B, C and D) show a representative experiment of two independent determinations, each performed in duplicate. Download figure Download PowerPoint Time course experiments showed that phosphorylated Vav-2 promotes rapid kinetics of nucleotide exchange on RhoA when compared with either non-phosphorylated Vav-2 or autophosphorylated Lck, confirming that Vav-2 activation occurs in a phosphotyrosine-dependent manner (Figure 4B, left panel). To demonstrate further that this activity represented a bona fide exchange reaction, we also analyzed the ability of Vav-2 proteins to induce the release of [3H]GDP from RhoA in the presence of cold GTP. Tyrosine-phosphorylated Vav-2 enhanced the exchange of nucleotides on RhoA at substoichiometric concentrations under these experimental conditions (Figure 4B, right panel). Taken together, these results indicate that Vav family members share a similar mechanism of activation but display distinct substrate specificity towards members of the Rho/Rac family. The finding that Vav-2 acted as a phosphorylation-dependent RhoA GEF prompted us to analyze further the specificity of Vav-2 activation. To this end, we first investigated the ability of several protein tyrosine kinases (Lck, Hck and Syk) purified from baculovirus-infected Sf9 cells to induce Vav-2 activation in vitro. As shown in Figure 4C, Vav-2 is stimulated efficiently by incubation with these protein tyrosine kinases, as determined by [35S]GTP-γS incorporation assays. Kinase experiments conducted in the presence of [γ-32P]ATP confirmed that these kinases phosphorylate Vav at comparable levels (data not shown). We also analyzed the enzyme specificity of non-phosphorylated and phosphorylated Vav-2 towards additional members of the RhoA subfamily using [35S]GTP-γS binding assays. Phosphorylated Vav-2 was found to be active on RhoA, RhoB and the more distantly related RhoG protein (Figure 4D). In these assays, we found that phosphorylated Vav was active on RhoG and Dbl lysates on all three GTPases (data not shown). These results support the notion that Vav-2 acts as a phosphorylation-dependent GEF with substrate specificity towards RhoG and RhoA subfamily members. Oncogenic activation of vav-2 leads to the production of a truncated protein with deregulated, phosphorylation-independent exchange activity The Vav-2 protein is oncogenically activated as a result of an N-terminal truncation that removes both the CH domain and the AD (Schuebel et al., 1996). To determine whether this mutation results in a constitutively active Vav-2 protein, we generated a second baculovirus capable of expressing a polyhistidine-tagged version of the Vav-2 oncoprotein in Sf9 cells. After purification from insect cells (Figure 5A), the Vav-2 oncoprotein was subjected to in vitro kinase assays in the presence or absence of GST–Lck and then tested for GDP/GTP exchange activity on bacterially expressed Rho family proteins using [35S]GTP-γS incorporation assays. Both the non-phosphorylated and phosphorylated forms of the Vav-2 oncoprotein were active preferentially on RhoA and, to a lesser extent, on RhoB and RhoG GTPases (Figure 5B, left panel). In contrast, both versions of the Vav-2 oncoprotein lacked significant activity on Rac-1 and Cdc42 (Figure 5B, right panel). The Vav-2 oncoprotein was also inactive on Rac-2 protein (data not shown). These experiments indicated, therefore, that the Vav-2 oncoprotein has the same substrate specificity as wild-type Vav-2 but, unlike this protein, its activity is independent of its phosphorylation status. We also performed [3H]GDP release experiments in order to corroborate the de-regulated activity of the Vav-2 oncoprotein. As shown in Figure 5C, the kinetics of guanosine nucleotide exchange induced on RhoA by the non-phosphorylated and phosphorylated forms of the Vav-2 oncoprotein were indistinguishable under these alternative experimental conditions. Based on these results, we conclude that the Vav-2 oncoprotein is active regardless of its phosphorylation state. Figure 5.(A) Purification of Vav-2 oncoprotein by chromatography on nickel beads. M, molecular weight markers. The amount of loaded proteins is given in μg. (B) Exchange activity of Vav-2 oncoprotein on Rho proteins. The indicated GDP-loaded GTPases were incubated for 45 min with [35S]GTP-γS in the presence of phosphorylated Vav-2 oncoprotein (black boxes), non-phosphorylated Vav-2 oncoprotein (gray boxes) or autophosphorylated GST–Lck (white boxes) and the exchange obtained under each condition determined using a filter immobilization assay. (C)[3H]GDP release assay of RhoA with phosphorylated Vav-2 oncoprotein (squares), non-phosphorylated Vav-2 oncoprotein (diamonds) and autophosphorylated GST–Lck (triangles). In (B), values represent the mean and SD of three (left panel) and four (right panel) independent determinations each performed in duplicate. (C) shows a representative experiment of two independent determinations, each performed in duplicate. (D) Immunoblot analysis using anti-phosphotyrosine (α-PTyr) or anti–polyhistidine (α-PolyHis) antibodies of total cell lysates derived from Sf9 cells infected with the indicated baculovirus. The migration of wild-type and oncogenic Vav-2 proteins is indicated by an arrow and an arrowhead, respectively. Download figure Download PowerPoint Since these experiments could not rule out the possibility that the de-regulated activity of the oncogenic version of Vav-2 was due to high levels of phosphorylation of this protein in insect cells, we investigated the levels of tyrosine phosphorylation of the wild-type and oncogenic Vav-2 proteins in baculovirus-infected Sf9 cells. As shown in Figure 5D, immunoblot analysis indicated that the basal levels of phosphorylation of the Vav-2 oncoprotein in the total cellular lysates obtained from Sf9 cells were significantly lower than those found in its wild-type counterpart, ruling out the possibility that the de-regulated activity of the Vav-2 oncoprotein in our biochemical assays is a consequence of its hyperphosphorylation in insect cells. Vav family members share a similar mechanism of oncogenic activation In contrast to Vav-2, it has been shown previously that Vav becomes activated oncogenically upon a partial deletion of the N-terminal CH domain (residues 1–67) (Coppola et al., 1990; Katzav et al., 1990). However, this oncogenic protein is not totally unregulated, as demonstrated by previous reports showing that the Vav oncoprotein requires phosphorylation for nucleotide exchange in vitro (Crespo et al., 1997). In order to investigate whether the mechanism of activation of Vav-2 could be generalized to all Vav family proteins, we compared the transforming activity of the wild-type, the originally described Vav oncogenic version (Δ1–67 deletion) (Coppola et al., 1991; Katzav et al., 1991), and a new version of Vav lacking both the CH domain and the AD (Δ1–187) (Figure 6A) using focus formation assays in rodent fibroblasts. To ensure that all proteins were expressed with similar ki" @default.
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• W1972234127 title "Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2" @default.
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• W1972234127 cites W1484050792 @default.
• W1972234127 cites W1818326785 @default.
• W1972234127 cites W1966732663 @default.
• W1972234127 cites W1979027806 @default.
• W1972234127 cites W1979542208 @default.
• W1972234127 cites W1991460507 @default.
• W1972234127 cites W1992838895 @default.
• W1972234127 cites W2001234795 @default.
• W1972234127 cites W2008217531 @default.
• W1972234127 cites W2011751979 @default.
• W1972234127 cites W2013592169 @default.
• W1972234127 cites W2052149514 @default.
• W1972234127 cites W2077792958 @default.
• W1972234127 cites W2096791952 @default.
• W1972234127 cites W2115235932 @default.
• W1972234127 cites W2121666423 @default.
• W1972234127 cites W2146055738 @default.
• W1972234127 cites W2151584690 @default.
• W1972234127 cites W2154841963 @default.
• W1972234127 cites W2160113850 @default.
• W1972234127 cites W2276489760 @default.
• W1972234127 cites W2395247613 @default.
• W1972234127 cites W58507878 @default.
• W1972234127 cites W973784641 @default.
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