In this study, we describe the identification and functional characterization of VAV-3
, a new member of the VAV
gene family of guanosine nucleotide exchange factors. The product of the human VAV-3
gene is a 98-kDa protein which shares a high degree of homology with the products of the VAV
genes. Experiments conducted with both endogenous and ectopically expressed Vav-3 demonstrate its participation in the signal transduction processes. Moreover, the analysis of the enzyme specificity of Vav-3 indicates that it catalyzes preferentially the exchange of guanosine nucleotides on RhoA and RhoG and, to a lower extent, on the Rac-1 GTPase. Vav-3 binds also physically to those GTPases when they are in the nucleotide-free state, a result previously shown for most Rho and Ras GEFs (12
). By analogy to those proteins, we can infer that Vav-3 will catalyze GDP-GTP exchange by stabilizing the nucleotide-free state of its substrates.
The role of Vav-3 as an activator of Rho GTPases is further substantiated by transient transfections in rodent fibroblasts. We have shown that truncated Vav-3 proteins (Δ1–144 and Δ1–144+Δ607–847) are capable of inducing a rapid reorganization of the actin cytoskeleton, leading to membrane ruffling, lamellipodia, and the formation of thin bundles of stress fibers in NIH 3T3 cells. In COS-1 cells, the expression of Vav-3 leads also to the formation of extensive membrane ruffling, a phenotype similar to that observed upon overexpression of the other Vav family proteins, Rac-1Q61L
, and RhoGQ61L
). Given the similarity of the morphologies induced by Rac-1 and RhoG, it is not possible at present to discriminate whether such lamellipodia and membrane ruffling formation represents the activation of Rac-1, RhoG, or both. However, since Vav-3 works only at substoichiometric concentrations on RhoA and RhoG, we favor the idea that those responses are mediated mainly through the stimulation of RhoG.
The expression of truncated versions of Vav-3 induces also marked alterations in the process of cell division, leading to the generation of binucleated cells in about 35% of all Vav-3-expressing cells. This phenotype is shared with Vav-2, but it is not observed upon expression of Vav and other Rho GEFs such as Lbc. The rapid induction of this phenotype (28 h) suggests that this response is the consequence of the direct unregulated activity of Vav-3 and Vav-2 rather than to an epistatic event occurring upon the long-term constitutive activation of the Vav-2/Vav-3 pathways. Interestingly, the examination of Vav-3-expressing cells shows at least two different types of alterations. In the majority of cases, the two new nuclei separate effectively into the daughter cells but the septum fails to form, leading to lack of partition of the cells. In a minority of cases, it has been observed that one of the nuclei of binucleated cells undergoes mitosis, while the second one remains resting. Division of those cells leads to the generation of a new cell inheriting one of the new nucleus, while the second daughter cell inherits the other newly formed nucleus and the undivided one (19
). Thus, multinucleation of Vav-3-expressing cells appears to be derived from alterations in both the cytokinetic machinery and from the asynchronous DNA synthesis in the nuclei of already binucleated cells. A recent report has shown that a RhoA downstream target, citron, can induce polynucleation in HeLa cells due to abnormal cytokinesis (18
). It is tempting to speculate that the biological effects of Vav-3 and Vav-2 could be mediated, at least in part, by activation of this RhoA-regulated serine and threonine kinase.
Despite the similar activity of Vav-3 and Vav-2 both in vitro and in vivo, we were unable to detect any transforming activity of this new gene in standard focus formation assays. The explanation for this result is not straightforward to us. Due to the lack of transformation of the initial VAV-3
cDNA obtained by PCR (see Materials and Methods), we recloned the full length VAV-3
cDNA using standard screening procedures to avoid the possibility of a recurrent mutation in all our PCR clones. However, the library-derived clones showed the same primary structure and still lacked transforming activity, even when truncations that hyperactivated the oncogenicity of Vav and Vav-2 (23
) were generated in the VAV-3
was also inactive when linearized plasmids were used in the transfections, a method that traditionally enhances the transforming activity of oncogene-harboring plasmids by up to 200-fold, including those for vav
). Species differences can also be ruled out, because the human VAV
gene is as transforming as its rodent counterpart (5
). It is possible, therefore, that the lack of VAV-3
oncogenic activity could derive from a different behavior of this gene in vivo, such as distinct subcellular localization or to lower levels of activity of Vav-3 in vivo. More work will be necessary to fully understand the lack of transformation potential of this new Vav family member.
The study of Vav-3 has also allowed us to gain a better understanding on the intramolecular interactions that regulate the activity of this GDP-GTP exchange factor. Thus, we have shown that the biochemical activity of wild-type Vav-3 is strictly dependent on tyrosine phosphorylation. Since these assays are done with purified proteins, this result indicates that this posttranslational modification induces an intramolecular effect that activates the latent biochemical activity of Vav. In this regard, we have shown that this effect is mediated, at least in part, by an increase in the binding avidity of Vav-3 toward its substrates. However, since Vav-3 activity is always associated with both the physical interaction to the GTPases and catalysis of GDP-GTP exchange, it is impossible at this point to determine whether phosphorylation plays only a role in the former process or whether, in addition, it also participates in regulating the catalytic activity of Vav-3 towards the bound substrate. Mutagenesis experiments capable of dissociating these two functional points in the Vav-3–Rho relationship will help to answer this important question. By using an N-terminally truncated Vav-3 protein (Δ1–144), we have also demonstrated that these Vav-3 mutant proteins are constitutively active due to the lack of dependency of tyrosine phosphorylation for the activation of Rho proteins. This is not due to higher levels of tyrosine phosphorylation, because this truncated protein displays comparable levels of phosphorylation that the wild type protein when purified from Sf9 cells and after phosphorylation by Hck. In good agreement with these in vitro observations, we have shown that Vav-3 proteins lacking the N terminus can induce effective morphological change and cell multinucleation even in the absence of the C-terminal SH3-SH2-SH3 domains, a region essential for the tyrosine phosphorylation of Vav-3 and for its interaction with protein tyrosine kinases and other cytoplasmic phosphoproteins. This constitutive activation appears to be another intramolecular effect mediated by the missing CH region, since it can be induced in vitro by using highly purified preparations of Vav-3. Taken together, these results are consistent with the idea that the main function of the Vav-3 SH3-SH2-SH3 is to mediate the phosphorylation of the wild-type protein, thereby eliminating the inhibitory function of the CH region on other structural domains of Vav-3 (Fig. C).
Since the DH, PH, and ZF domains appear to drive all the biological and biochemical activities of Vav-3, we decided to investigate the individual contributions of the DH, PH, and ZF regions for both the enzyme and biological activities of Vav-3. Not surprisingly, we found that a point mutation affecting the DH region (L211Q) inhibits the biological activity of Vav-3. This finding is in good agreement with our previous results with Vav showing that the mutation in the same residue (L213Q) disrupts its ability to promote JNK-1 activation and Rac-1 GDP-GTP exchange in vivo (6
). This inactivation is not due to a major alteration in the folding of Vav-3 because this mutant protein works well in other functions such as the EGF-dependent binding to the EGF-R and Shc.
Further analysis of the DH-PH-ZF region has shown that the integrity of the Vav-3 ZF domain is also essential for the function of this protein. Indeed, we have shown that a point mutation affecting a conserved cysteine residue (C527S) of the Vav-3 ZF totally eliminates the activity of this protein in vivo and in vitro. Several independent observations indicate that this inhibition reflects a truly functional role of the Vav-3 ZF. First, we have shown that this C527S mutant does participate in signal transduction pathways, becoming phosphorylated on tyrosine residues after treatment of quiescent COS-1 cells with EGF. Second, this mutant protein can also interact physically with EGF-R and the p48/p52Shc isoforms in a ligand-dependent manner. These two properties are similar to the ones seen for the intact Vav-3 protein when expressed in the same cell background. Third, we have demonstrated that different Vav-3 deletion mutants lacking the entire ZF region are also inactive in vitro. Thus, the inactivation of the Vav-3 C527S mutant protein cannot be attributed to artifactual causes, such as the formation of new intramolecular covalent bonds between cysteines of the disrupted ZF and cysteine residues located elsewhere in the molecule.
Although we do not know as yet how the ZF works with the DH domain to promote binding and catalysis of the GTPases, we believe that our in vitro results are consistent with a dual functional role for this domain. On one hand, our observations demonstrating that the isolated Vav-3 ZF can interact physically with RhoA in vitro suggest that this domain contributes to the Vav-3–GTPase interaction by establishing points of contact with specific regions of Rho proteins (Fig. C). These regions may not be conserved in all GTPases because the Vav-3 ZF binds to the Vav-3 substrates but not to Cdc42. On the other hand, the fact that the Vav-3 ZF region has a completely different specificity for the guanosine nucleotide state of the GTPases than do wild-type Vav-3 and Vav-3 (Δ1–144) is not consistent with this region being the main determinant of the stable interaction of Vav-3 with its substrates. Accordingly, we propose a model in which the ZF also makes contributions to the overall structure of the DH-PH-ZF cassette which are essential for the biochemical action of the catalytic DH domain (Fig. C). Such function may be analogous to the contribution of the PH domain of Trio to the catalytic efficiency of its DH domain (17
). Studies aimed at solving the crystal structure of Vav-3 will allow determination of the specific contributions of the ZF region to the effector functions of Vav-3.
Finally, our experiments have shown that a mutation that completely disrupts the structure of the PH domain (W493L) has no detectable consequences for biochemical and biological activity of Vav-3. In agreement with these results, recent experiments indicate that the Vav-3 W493L mutant protein is also active in the activation of another Rac-1/RhoG downstream element, JNK (19
). To date, only Vav-3 and the Lbc exchange factor (21
) appear to be completely independent of their respective PH domains for the induction of their in vivo effects. The PH domain of Dbl is also dispensable for the catalytic activity in vitro, but it is essential for its effects in vivo, presumably by facilitating the interaction of this GEF with the cytoskeleton (35
). Recent structural studies have shown that the N-terminal region of the PH domain of Sos-1 folds into the C terminus of the DH region, leading to the inhibition of the basal exchange activity of this catalytic domain (24
). This inhibitory effect appears to be eliminated by the binding of phospholipids to the Sos-1 PH region (20
). These results indicate that some PH domains may influence the activity of Rho and Rac GEFs via intramolecular interactions and through the binding to intracellular second messengers. Since our experiments have not included mutations in the N-terminal region of the Vav-3 PH domain, we cannot rule out at the present time whether the Vav-3 PH domain has regulatory functions similar to those described for Sos-1. However, two observations argue indirectly against such possibility. First, the N-terminal regions of the PH domains of Sos-1 and Vav-2 have very low amino acid sequence similarity and differ significantly in length. This suggests that the interactions that take place between the Sos-1 DH and PH domains probably cannot occur in the case of Vav-3. Second, previous nuclear magnetic resonance and crystal structure studies have shown that the residue targeted in our mutagenesis experiments (W493) establishes hydrogen bonds and water-mediated interactions with the N-terminal β1 region of the same PH region (9
). The W493L mutation created in Vav-3 is expected therefore to cause major conformational changes in the entire N-terminal region of its PH domain. In this regard, our kinetic analysis showing that the Vav-3 (W493L) protein shows catalytic activity identical to that of the wild type version further suggests that this domain does not play either positive or negative roles in the GDP-GTP exchange activity of this protein. In any case, the definitive answer to the putative negative regulatory role of the Vav-3 PH region will require extensive mutagenesis of the residues located in the N-terminal region of this structural domain. Another interesting question that needs to be addressed is whether the model reported here for the Vav-3 PH region is conserved in the other members of the family. This is an important consideration because the PH regions are, along with the most proximal SH3 domain, one of the less-conserved regions among these proteins. It will be interesting therefore to compare the functional properties of the Vav and Vav-2 PH regions and to study whether the lack of transforming activity of Vav-3 is due to structural or functional differences among Vav family members in this structural domain. Future studies with point mutants of Vav and Vav-2 and with chimeric Vav-3/Vav and Vav-3/Vav-2 proteins will help to solve these two pending questions.
In summary, we have presented a comprehensive functional characterization of the VAV-3
gene product both biochemically and biologically and have provided new data regarding mechanistic aspects of Vav-3 GDP-GTP exchange regulation. It is likely that the isolation of VAV-3
will not represent the last step on the characterization of the Vav family. Although the presence of additional Vav-related proteins in mammals remains to be further addressed, the presence of Vav-related proteins in C. elegans
has been already demonstrated (30
). It is therefore plausible that, as for many other signaling molecules involved in developmental pathways, Vav proteins will be present in other animal models such as flies and nonmammalian vertebrates. The isolation of these genes will provide in the future important tools to study genetically the signal transduction pathway activated by these proteins and to understand the different mechanisms by which tyrosine kinase receptors couple their activation with the stimulation of Rho/Rac pathways.