Nuclear Vav3 is required for polycomb repression complex-1 activity in B-cell lymphoblastic leukemogenesis.

Acute B-cell lymphoblastic leukemia (B-ALL) results from oligo-clonal evolution of B-cell progenitors endowed with initiating and propagating leukemia properties. The activation of both the Rac guanine nucleotide exchange factor (Rac GEF) Vav3 and Rac GTPases is required for leukemogenesis mediated by the oncogenic fusion protein BCR-ABL. Vav3 expression becomes predominantly nuclear upon expression of BCR-ABL signature. In the nucleus, Vav3 interacts with BCR-ABL, Rac, and the polycomb repression complex (PRC) proteins Bmi1, Ring1b and Ezh2. The GEF activity of Vav3 is required for the proliferation, Bmi1-dependent B-cell progenitor self-renewal, nuclear Rac activation, protein interaction with Bmi1, mono-ubiquitination of H2A(K119) (H2AK119Ub) and repression of PRC-1 (PRC1) downstream target loci, of leukemic B-cell progenitors. Vav3 deficiency results in de-repression of negative regulators of cell proliferation and repression of oncogenic transcriptional factors. Mechanistically, we show that Vav3 prevents the Phlpp2-sensitive and Akt (S473)-dependent phosphorylation of Bmi1 on the regulatory residue S314 that, in turn, promotes the transcriptional factor reprogramming of leukemic B-cell progenitors. These results highlight the importance of non-canonical nuclear Rho GTPase signaling in leukemogenesis.

A transcriptional cross-talk between RhoA and c-Myc inhibits the RhoA/Rock-dependent cytoskeleton.

The GTPase RhoA participates in a number of cellular processes, including cytoskeletal organization, mitogenesis and tumorigenesis. We have previously shown that the transforming activity of an oncogenic version of RhoA (Q63L mutant) was highly dependent on the transcriptional factor c-Myc. In contrast to these positive effects in the RhoA route, we show here that c-Myc affects negatively the F-actin cytoskeleton induced by RhoA(Q63L) and its downstream effector, the serine/threonine kinase Rock. This effect entails the activation of a transcriptional program that requires synergistic interactions with RhoA-derived signals and that includes the upregulation of the GTPase Cdc42 and its downstream element Pak1 as well as the repression of specific integrin subunits. The negative effects of c-Myc in the F-actin cytoskeleton are eliminated by the establishment of cell-to-cell contacts, an effect associated with the rescue of Pak1 and integrin levels at the post-transcriptional and transcriptional levels, respectively. These results reveal the presence of a hitherto unknown signaling feed-back loop between RhoA and c--Myc oncogenes that can contribute to maintain fluid cytoskeletal dynamics in cancer cells.

Identification of the Rock-dependent transcriptome in rodent fibroblasts

Rock proteins are Rho GTPase-dependent serine/ threonine kinases with crucial roles in F-actin dynamics and cell transformation. By analogy with other protein kinase families, it can be assumed that Rock proteins act, at least in part, through the regulation of gene expression events. However, with the exception of some singular transcriptional targets recently identified, the actual impact of these kinases on the overall cell transcriptome remains unknown. To address this issue, we have used a microarray approach to compare the transcriptomes of exponentially growing NIH3T3 cells that had been untreated or treated with Y27632, a well known specific inhibitor for Rock kinase activity. We show here that the Rock pathway promotes a weak impact on the fibroblast transcriptome, since its inhibition only results in changes in the expression of 2.3% of all the genes surveyed in the microarrays. Most Y27632-dependent genes are downregulated at moderate levels, indicating that the Rock pathway predominantly induces the upregulation of transcriptionally active genes. Although functionally diverse, a common functional leitmotiv of Y27632-dependent genes is the implication of their protein products in cytoskeletal-dependent processes. Taken together, these results indicate that Rock proteins can modify cytoskeletal dynamics by acting at post-transcriptional and transcriptional levels. In addition, they suggest that the main target of these serine/threonine kinases is the phosphoproteome and not the transcriptome (AU)

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Transcriptomal profiling of the cellular transformation induced by Rho subfamily GTPases.

We have used microarray technology to identify the transcriptional targets of Rho subfamily guanosine 5'-triphosphate (GTP)ases in NIH3T3 cells. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are upregulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells.

Involvement of the Rho/Rac family member RhoG in caveolar endocytosis.

We show here that the GTPase RhoG is involved in caveolar trafficking. Wild-type RhoG moves sequentially to the plasma membrane, intracellular vesicles, and the Golgi apparatus along markers of this endocytic pathway. Such translocation is associated with changes in RhoG GDP/GTP levels and is highly dependent on lipid raft integrity and on the function of the GTPase dynamin2. In addition, the constitutively active RhoG(Q61L) mutant is preferentially located in endocytic vesicles that can be decorated with markers of the caveola-derived endocytic pathway. RhoG(Q61L), but not the analogous Rac1 mutant protein, affects caveola internalization and the subsequent delivery of endocytic vesicles to the Golgi apparatus. The expression of RhoG/Rac1 chimeric proteins and RhoG(Q61L) effector mutants in cells induces alterations in the internalization of caveolae and severe changes in vesicle structure, respectively. However, the knockdown of endogenous rhoG transcripts using small interfering RNAs does not affect significantly the trafficking of caveola-derived vesicles, suggesting that RhoG function is dispensable for this endocytic process or, alternatively, that its function is compensated by other molecules. Taken together, these observations assign a novel function to RhoG and suggest that caveolar trafficking, as previously shown for other endocytic routes, is modulated by GTPases of the Ras superfamily.

Knocked out by Rho/Rac T-cell biology.

The Rho/Rac family is a group o fRas-related proteins with demonstrated roles in the regulation of proliferation and cytoskeletal structures in a number of cell lineages. Despite this, the actual role of these proteins in T-cells could not be addressed in vivo due to the lack of adequate animal models. Recently, the use of knockout and transgenic animals for Rac1, Rac2, and RhoA has provided a genetic proof of the importance of Rho/Rac protein in different aspects of T-cell signaling. These animals have also allowed us to get better views about the influence of these GTPases proteins on the maturation decisions of immature lymphocytes and on the signaling strategies these GTPases utilize to favor the generation of coherent and robust immune responses.

Vav proteins, adaptors and cell signaling.

The Vav family is a group of signal transduction molecules with oncogenic potential that play important roles in development and cell signaling. Members of this family are distributed in all animal metazoans but not in unicellular organisms. Recent genomic studies suggest that the function of Vav proteins co-evolved with tyrosine kinase pathways, probably to assure the optimal conversion of extracellular signals into biological responses coupled to the cytoskeleton and gene transcription. To date, the best-known function of Vav proteins is their role as GDP/GTP exchange factors for Rho/Rac molecules, a function strictly controlled by tyrosine phosphorylation. Recent publications indicate that this function is highly dependent on the interaction of adaptor proteins that aid in the proper phosphorylation of Vav proteins, their interaction with other signaling molecules, and in modulating the strength of their signal outputs. In addition to the function of Vav proteins as exchange factors, there is increasing evidence suggesting that Vav proteins can mediate other cellular functions independently of their exchange activities, probably by working themselves as adaptor molecules. In this review, we will give a summary of the recent advances in this field, placing special emphasis on the non-catalytic roles of Vav and its interaction with other adaptor molecules.

Rac1 mediates STAT3 activation by autocrine IL-6.

The activity of the small GTPase, Rac1, plays a role in various cellular processes including cytoskeletal rearrangement, gene transcription, and malignant transformation. In this report constitutively active Rac1 (Rac V12) is shown to stimulate the activation of STAT3, a member of the family of signal transducers and activators of transcription (STATs). The activity of Rac1 leads to STAT3 translocation to the nucleus coincident with STAT3-dependent gene expression. The expression of Vav (Delta1-187), a constitutively active guanine nucleotide exchange factor for the Rho GTPases, or activated forms of Ras or Rho family members, leads to STAT3-specific activation. The activation of STAT3 requires tyrosine phosphorylation at residue 705, but is not dependent on phosphorylation of Ser-727. Our studies indicate that Rac1 induces STAT3 activation through an indirect mechanism that involves the autocrine production and action of IL-6, a known mediator of STAT3 response. Rac V12 expression results in the induction of the IL-6 and IL-6 receptor genes and neutralizing antibodies directed against the IL-6 receptor block Rac1-induced STAT3 activation. Furthermore, inhibition of the nuclear factor-kappaB activation or disruption of IL-6-mediated signaling through the expression of IkappaBalpha S32AS36A and suppressor of cytokine signaling 3, respectively, blocks Rac1-induced STAT3 activation. These findings elucidate a mechanism dependent on the induction of an autocrine IL-6 activation loop through which Rac1 mediates STAT3 activation establishing a link between oncogenic GTPase activity and Janus kinase/STAT signaling.

How Vav proteins discriminate the GTPases Rac1 and RhoA from Cdc42.

Vav proteins are GDP/GTP exchange factors for Rho/Rac GTPases that are activated by tyrosine phosphorylation. These proteins activate Rac1, RhoG, and RhoA but not the highly related Cdc42 protein. At present, there is no available information to explain this substrate selectivity at the structural level. Here we show that the selection of Vav proteins substrates is achieved at two different levels. On one hand, Vav proteins utilize some residues of the beta2/beta3 region of Rho/Rac GTPases (D49 and E54) to assure the specific binding to its substrate. In addition, these exchange factors need a second structural signal located in the beta5 region of Rho/Rac proteins (residue K118) to promote proper GDP/GTP exchange. These results identify the amino acid residues that allow the discrimination of the Vav family substrates from Cdc42 and, in addition, demonstrate that the activation of specific Rho/Rac GTPases by these GEFs requires two concatenated events, binding and subsequent enzyme reaction, whose specificities are determined by two separate regions of Rho proteins.

Tyrosine phosphorylation mediates both activation and downmodulation of the biological activity of Vav.

Vav works as a GDP/GTP exchange factor for Rac GTPases, thereby facilitating the transition of these proteins from the inactive (GDP-bound) into the active (GTP-bound) state. The stimulation of Vav exchange activity during cell signaling is mediated by tyrosine phosphorylation. To understand the roles of phosphorylation in the regulation of Vav activity, we have initiated the characterization of the residues of Vav that are phosphorylated during signal transduction. Here we show that a Y-to-F mutation in one of these residues, Y174, leads to the oncogenic activation of Vav and to the enhancement of other Vav-mediated signals such as those for cytoskeletal reorganization, JNK activation, and stimulation of the nuclear factor of activated T cells. The effect induced by the Y174F mutation is further accentuated by mutations in residue Y142 or Y160. The Y174F mutation has no effect on the exchange activity of Vav in vitro but results in higher levels of phosphorylation in vivo. Using a phosphospecific antibody, we found that Y174 is phosphorylated following stimulation of mitogenic and antigenic receptors. This phosphorylation event is conserved in Vav-2 and Vav-3, the other two members of the Vav family. These results identify a previously unknown mechanism for the oncogenic activation of Vav and suggest that the activity of this exchange factor is modulated by two antagonistic phosphorylation events, one involved in Vav activation and a second one implicated in Vav inactivation.

Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors.

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Signal transduction elements of TC21, an oncogenic member of the R-Ras subfamily of GTP-binding proteins.

TC21 is a Ras-like GTPase with high oncogenic potential that is found mutated in some human tumors and overexpressed in breast cancer cell lines. We have conducted cellular and biochemical studies in order to understand the role of this protein in signal transduction and to unveil the signaling elements that participate in the TC21 pathway. Using gene transfer experiments, we demonstrate here that the TC21 oncogene can induce both cellular transformation in mouse fibroblasts and neuronal-like differentiation in rat PC12 cells. Interestingly, the proto-oncogenic version of TC21 shows also a lower, but significant, activity in both biological processes. We also demonstrate that the similarity of the cellular responses induced by TC21 and Ras derive from the utilization of overlapping pathways. Thus, the exchange of guanosine nucleotides in wild type TC21 is catalyzed by Ras exchange factors. Moreover, TC21 binds physically to c-Raf-1 in a GTP-dependent manner. Finally, overexpression of TC21G23V in NIH3T3 cells results in the activation of c-Raf-1 and the MAPK and the JNK branches of serine/threonine cascades. From these results, we conclude that TC21 promotes Ras-like responses in diverse cell types due to the use of overlapping, if not identical, signaling elements of the Ras oncogenic pathway.

Biological and regulatory properties of Vav-3, a new member of the Vav family of oncoproteins.

We report here the identification and characterization of a novel Vav family member, Vav-3. Signaling experiments demonstrate that Vav-3 participates in pathways activated by protein tyrosine kinases. Vav-3 promotes the exchange of nucleotides on RhoA, on RhoG and, to a lesser extent, on Rac-1. During this reaction, Vav-3 binds physically to the nucleotide-free states of those GTPases. These functions are stimulated by tyrosine phosphorylation in wild-type Vav-3 and become constitutively activated upon deletion of the entire calponin-homology region. Expression of truncated versions of Vav-3 leads to drastic actin relocalization and to the induction of stress fibers, lamellipodia, and membrane ruffles. Moreover, expression of Vav-3 alters cytokinesis, resulting in the formation of binucleated cells. All of these responses need only the expression of the central region of Vav-3 encompassing the Dbl homology (DH), pleckstrin homology (PH), and zinc finger (ZF) domains but do not require the presence of the C-terminal SH3-SH2-SH3 regions. Studies conducted with Vav-3 proteins containing loss-of-function mutations in the DH, PH, and ZF regions indicate that only the DH and ZF regions are essential for Vav-3 biological activity. Finally, we show that one of the functions of the Vav-3 ZF region is to work coordinately with the catalytic DH region to promote both the binding to GTP-hydrolases and their GDP-GTP nucleotide exchange. These results highlight the role of Vav-3 in signaling and cytoskeletal pathways and identify a novel functional cross-talk between the DH and ZF domains of Vav proteins that is imperative for the binding to, and activation of, Rho GTP-binding proteins.

Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2.

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.

The Vav-Rac1 pathway in cytotoxic lymphocytes regulates the generation of cell-mediated killing.

The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell-mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.

S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells.

S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.

Cbl-b, a member of the Sli-1/c-Cbl protein family, inhibits Vav-mediated c-Jun N-terminal kinase activation.

We have used the yeast two-hybrid system to identify proteins that interact with Vav, a GDP/GTP exchange factor for the Rac-1 GTPase that plays an important role in cell signaling and oncogenic transformation. This experimental approach resulted in the isolation of Cbl-b, a signal transduction molecule highly related to the mammalian c-cbl proto-oncogene product and to the C. elegans Sli-1 protein, a negative regulator of the EGF-receptor-like Let23 protein. The interaction between Vav and Cbl-b requires the entire SH3-SH2-SH3 carboxy-terminal domain of Vav and a long stretch of proline-rich sequences present in the central region of Cbl-b. Stimulation of quiescent rodent fibroblasts with either epidermal or platelet-derived growth factors induces an increased affinity of Vav for Cbl-b and results in the subsequent formation of a Vav-dependent trimeric complex with the ligand-stimulated tyrosine kinase receptors. During this process, Vav, but not Cbl-b, becomes highly phosphorylated on tyrosine residues. Overexpression of Cbl-b inhibits the signal transduction pathway of Vav that leads to the stimulation of c-Jun N-terminal kinase. By contrast, expression of truncated Cbl-b proteins and of missense mutants analogous to those found in inactive Sli-1 proteins have no detectable effect on Vav activity. These results indicate that Vav and Cbl-b act coordinately in the first steps of tyrosine protein kinase receptor-mediated signaling and suggest that members of the Sli-1/Cbl family are also negative regulators of signal transduction in mammalian cells.

Tyrosine phosphorylation of the vav proto-oncogene product links FcepsilonRI to the Rac1-JNK pathway.

Stimulation of high affinity IgE Fc receptors (FcepsilonRI) in basophils and mast cells activates the tyrosine kinases Lyn and Syk and causes the tyrosine phosphorylation of phospholipase C-gamma, resulting in the Ca2+- and protein kinase C-dependent secretion of inflammatory mediators. Concomitantly, FcepsilonRI stimulation initiates a number of signaling events resulting in the activation of mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK), which, in turn, regulate nuclear responses, including cytokine gene expression. To dissect the signaling pathway(s) linking FcepsilonRI to MAPK and JNK, we reconstructed their respective biochemical routes by expression of a chimeric interleukin-2 receptor alpha subunit (Tac)-FcepsilonRI gamma chain (Tacgamma) in COS-7 cells. Cross-linking of Tacgamma did not affect MAPK in COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tacgamma stimulation potently induced Syk and Shc tyrosine phosphorylation and MAPK activation. In contrast, Tacgamma did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav, reconstituted the Tacgamma-induced, Syk- and Rac1-dependent JNK activation; and tyrosine-phosphorylation of Vav by Syk stimulated its GEF activity for Rac1. Thus, these data strongly suggest that Vav plays a critical role linking FcepsilonRI and Syk to the Rac1-JNK pathway. Furthermore, these findings define a novel signal transduction pathway involving a multimeric cell surface receptor acting on a cytosolic tyrosine kinase, which, in turn, phosphorylates a GEF, thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade.

Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product.

The oncogenic protein Vav harbours a complex array of structural motifs, including leucine-rich, Dbl-homology, pleckstrin-homology, zinc-finger, SH2 and SH3 domains. Upon stimulation by antigens or mitogens, Vav becomes phosphorylated on key tyrosine residues and associates with other signalling proteins, including the mitogen receptors Zap-70 (ref. 6), Vap-1 (ref. 5) and Slp-76 (ref. 7). Disruption of the vav locus by homologous recombination causes severe defects in signalling by primary antigen receptors, leading to abnormal lymphocyte proliferation and lymphopenia. Despite the importance of Vav cell signalling, the function of this protein remains unknown. Here we show that tyrosine-phosphorylated Vav, but not the non-phosphorylated protein, catalyses GDP/GTP exchange on Rac-1, a protein implicated in cell proliferation and cytoskeletal organization, causing this GTPase to switch from its inactive to its active state. Transfection experiments also show that phosphorylation of Vav on tyrosine residues leads to nucleotide exchange on Rac-1 in vivo and stimulates c-Jun kinase, a downstream element in the signalling pathway involving this GTPase. Our results have identified a function for Vav and define a mechanism in which engaged membrane receptors activate its signalling pathway.