Function Of Vav1 Research Articles

Lysine Acetylation Reshapes the Downstream Signaling Landscape of Vav1 in Lymphocytes.

Vav1 works both as a catalytic Rho GTPase activator and an adaptor molecule. These functions, which are critical for T cell development and antigenic responses, are tyrosine phosphorylation-dependent. However, it is not known whether other posttranslational modifications can contribute to the regulation of the biological activity of this protein. Here, we show that Vav1 becomes acetylated on lysine residues in a stimulation- and SH2 domain-dependent manner. Using a collection of both acetylation- and deacetylation-mimicking mutants, we show that the acetylation of four lysine residues (Lys222, Lys252, Lys587, and Lys716) leads to the downmodulation of the adaptor function of Vav1 that triggers the stimulation of the nuclear factor of activated T cells (NFAT). These sites belong to two functional subclasses according to mechanistic criteria. We have also unveiled additional acetylation sites potentially involved in either the stimulation (Lys782) or the downmodulation (Lys335, Lys374) of specific Vav1-dependent downstream responses. Collectively, these results indicate that Nε-lysine acetylation can play variegated roles in the regulation of Vav1 signaling. Unlike the case of the tyrosine phosphorylation step, this new regulatory layer is not conserved in other Vav family paralogs.

Vav1, a Rho GTPase Guanine Exchange Factor (GEF) Is Required to Integrate gp130 Cytokine Signaling for Successful Engraftment of Murine Hematopoietic Stem Cells Following Irradiation

Successful engraftment of hematopoietic stem and progenitor cells (HSPCs) during bone marrow transplantation requires appropriate homing and retention of transplanted cells in the bone marrow (BM) and the activation of a proliferative program in response to signals from the hematopoietic microenvironment (HM). Molecular pathways regulating migration, homing and retention of HSPCs in the BM are integrated by RhoGTPasesincluding Rac and CDC42, however the complex cues that drive the proliferative response of these cells following transplantation are less clear.We have previously described the hematopoietic phenotype of adult mice lacking Vav1, a multi-domain, hematopoietic-specific GEF for Rac and CDC42. Deletion of Vav1 does not affect steady state hematopoiesis in adult mice, but severely compromises the engraftment potential of HSPCs. In the absence of Vav1, signal transduction from SDF1α is impaired in HSPCs, leading to abnormal localization and reduced retention of these cells in the HM, a phenotype similar to deficiency of Rac (Sanchez-Aguilera et al. PNAS, 2011). Here, we define an unexpected role for Vav1 in mediating post-irradiation proliferative responses of HSPCs.Surprisingly, we observed that deletion of Vav1 does not affect HSPC migration during ontogeny, a process largely mediated by SDF1α. The number of immunophenotypically and functionally defined HSPCs in Vav1-/- E13.5 fetal liver (FL) was comparable to WT (Table 1). Similarly, no difference was detected in HSPCs in the peripheral blood (PB) of E18.5 embryos or in the BM of newborn mice. However, similar to adult cells, Vav1-/- fetal HSCPs showed severely defective engraftment in lethally irradiated recipients (see Table 2). In marked contrast, both adult and fetal Vav1-/- HSCPs could engraft non-irradiated (Kit W/Wv Rag2- γc-) recipients, achieving successful correction of the macrocytic anemia and B cell leukopenia phenotype of recipient mice (Table 2, in red). Reduced proliferation of Vav1-/- HSPCs was also observed in vitro upon co-culture with primary irradiated stromal cells (Table 3). No differences among genotypes were detected when using non-irradiated stromal cells. These data suggest a distinct role for Vav1 in mediating responses of HSPCs to the HM after irradiation.To further clarify this phenotype, we investigated the role of individual soluble factors on proliferative responses of Vav1 HSPCs. Given their known role in expansion and proliferation of hematopoietic progenitors we focused on gp130 cytokines. We found that both IL-6 and IL-11, prominent members of this cytokine family, were increased in the BM of irradiated WT recipients, compared to both non-irradiated WT recipients (3x and 2x increase) and Kit W/Wv Rag2- γc- (2x and 3x increase) age- and sex-matched animals. To validate the potential role of IL-6 and IL-11 in Vav1 function, we stimulated HSPCs with both cytokines and observed that they induced phosphorylation of Vav1 and activation of Rac, but not CDC42. Using CFU assays, liquid culture experiments and BrdU analysis we confirmed that deletion of Vav1 abolishes the proliferative responses elicited by IL-6 and IL-11 on HSPCs in vitro.In summary, we show that Vav1 acts in HSPCs to mitigate responses to pro-inflammatory cytokines present in the HM during engraftment following irradiation. Manipulating the gp130-Vav-Rac axis in HSPCs could represent a strategy to enhance engraftment of normal cells in conditioned recipients. [Display omitted] [Display omitted] [Display omitted] DisclosuresWilliams:bluebird bio: Research Funding; Novartis: Consultancy; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Vav1 Regulates Mesenchymal Stem Cell Differentiation Decision Between Adipocyte and Chondrocyte via Sirt1.

Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in the bone marrow. MSCs have the potential to differentiate to adipocytes, chondrocytes, and other types of cells. In this study, we investigated the molecular mechanism that controls MSC cell fate decisions for differentiation. We found that Vav1, a guanine nucleotide exchange factor for Rho GTPase, was highly expressed in MSCs. Interestingly, loss of Vav1 in MSCs led to spontaneous adipogenic but impaired chondrogenic differentiation, and accordingly Vav1 null mice displayed an increase in fat content and a decrease in cartilage. Conversely, ectopic expression of Vav1 in MSCs reversed this phenotype, and led to enhanced MSC differentiation into chondrocyte but retarded adipogenesis. Mechanistically, loss of Vav1 reduced the level of Sirt1, which was responsible for an increase of acetylated PPARγ. As acetylation activates PPARγ, it increased C/EBPα expression and promoted adipogenesis. On the other hand, loss of Vav1 resulted in an increase of acetylated Sox9, a target of Sirt1. As acetylation represses Sox9 activity, it led to a dramatic reduction of collagen 2α1, a key regulator in chondrocyte differentiation. Finally, we found that Vav1 regulates Sirt1 in MSCs through Creb. Together this study reveals a novel function of Vav1 in regulating MSC cell fate decisions for differentiation through Sirt1. Sirt1 deacetylates PPARγ and Sox9, two key mediators that control adipocyte and chondrocyte differentiation. The acetylation status of PPARγ and Sox9 has opposite effects on its activity, thereby controlling cell fate decision. Stem Cells 2016;34:1934-1946.

VAV-1 acts in a single interneuron to inhibit motor circuit activity in Caenorhabditis elegans.

The complex molecular and cellular mechanisms underlying neuronal control of animal movement are not well understood. Locomotion of Caenorhabditis elegans is mediated by a neuronal circuit that produces coordinated sinusoidal movement. Here we utilize this simple, yet elegant, behaviour to show that VAV-1, a conserved guanine nucleotide exchange factor for Rho-family GTPases, negatively regulates motor circuit activity and the rate of locomotion. While vav-1 is expressed in a small subset of neurons, we find that VAV-1 function is required in a single interneuron, ALA, to regulate motor neuron circuit activity. Furthermore, we show by genetic and optogenetic manipulation of ALA that VAV-1 is required for the excitation and activation of this neuron. We find that ALA signalling inhibits command interneuron activity by abrogating excitatory signalling in the command interneurons, which is responsible for promoting motor neuron circuit activity. Together, our data describe a novel neuromodulatory role for VAV-1-dependent signalling in the regulation of motor circuit activity and locomotion.

VAV1 controls inflammation in Experimental Autoimmune Encephalomyelitis (EAE)

Vav1, a guanine nucleotide exchange factors for the Rho family of GTPases, plays an important role in T-cell and B-cell development, activation, tissue homing and effector functions. Vav1 was found within a rat QTL that regulates the MS animal model EAE, and overlaps with several QTLs for immune-mediated diseases. Analysis of a human cohort showed association of certain haplotypes of the gene with MS. In this work we attempt to further characterize the function of Vav1 by using congenic rats that express different levels of Vav1 and are differentially susceptible to EAE.

Vav1 as a Central Regulator of Invadopodia Assembly

Invadopodia are protrusive structures used by tumor cells for degradation of the extracellular matrix to promote invasion [1]. Invadopodia formation and function are regulated by cytoskeletal-remodeling pathways and the oncogenic kinase Src. The guanine nucleotide exchange factor Vav1, which is an activator of Rho family GTPases, is ectopically expressed in many pancreatic cancers, where it promotes tumor cell survival and migration [2, 3]. We have now determined that Vav1 is also a potent regulator of matrix degradation by pancreatic tumor cells as depletion of Vav1 by siRNA-mediated knockdown inhibits the formation of invadopodia. This requires the exchange function of Vav1 toward the GTPase Cdc42, which is required for invadopodia assembly [4, 5]. In addition, we have determined that Src-mediated phosphorylation and activation of Vav1 are both required for, and, unexpectedly, sufficient for, invadopodia formation. Expression of Vav1 Y174F, which mimics its activated state, is a potent inducer of invadopodia formation through Cdc42, even in the absence of Src activation and phosphorylation of other Src substrates, such as cortactin. Thus, these data identify a novel mechanism by which Vav1 can enhance the tumorigenicity and invasive potential of cancer cells. These data suggest that Vav1 promotes the matrix-degrading processes underlying tumor cell migration and further, under conditions of ectopic Vav1 expression, that Vav1 is a central regulator and major driver of invasive matrix remodeling by pancreatic tumor cells.

Vav1 regulates MHCII expression in murine resting and activated B cells

Vav1 is a guanine nucleotide exchange factor (GEF) for Rho GTPases, which is exclusively expressed in cells of the hematopoietic system. In addition to its well-documented GEF activity, it was suggested to have other functions due to the presence of multiple domains and nuclear localization signals in its protein structure. Although GEF-dependent and GEF-independent functions of vav have been implicated in T-cell development and T-cell receptor signaling, the role of vav1 in antigen-presenting cells is poorly understood. We found that vav1 is an important regulator of MHCII expression and transport. Microarray analysis of unstimulated bone marrow-derived macrophages revealed a novel role of vav1 in transcriptional regulation of the MHCII locus, possibly by indirect means. Primary immune cells from vav1-deficient mice had a significantly lower constitutive surface expression of MHCII with the strongest impact observed on splenic and peritoneal B cells. Impaired MHCII expression resulted in a diminished capacity for T-cell activation. Using 6-thio-GTP, a specific inhibitor of the GEF function of vav1, we were able to show that the GEF activity is required for MHCII upregulation in B cells after stimulation with LPS. Furthermore, our data show that vav1 not only affects transcription of the MHCII locus but also is an important regulator of MHCII protein transport to the cell surface.

Vav1 Fine Tunes p53 Control of Apoptosis versus Proliferation in Breast Cancer

Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.

Abstract 3854: Identification of crucial residues at the rim of the binding cleft of the C1 domain of Vav1 that determine its ligand sensitivity

Abstract Vav1 is a guanine exchange factor (GEF) for the Rho family of GTPases. It plays a pivotal role in T-cell maturation and development, cytoskeleton organization, and oncogenic transformation. The GEF activity of Vav1 is regulated by several factors, including interaction between its catalytic DH domain and its C1 domain. Its C1 domain shows homology with “typical” C1 domains that are sensitive to the second messenger diacylglycerol (DAG) and phorbol esters (PEs), but it is classified as “atypical” based on its unresponsiveness to these ligands. However, crystallographic analysis has shown that, unlike atypical C1 domains (e.g. Raf1) which possess a distorted structure, the Vav1 C1 retains the geometry of the binding cavity. We hypothesized that residues in the vicinity of the binding pocket might interfere with ligand binding. Sequence alignment with typical C1 domains revealed six unique residues situated along the rim of the putative binding cleft in Vav1 C1: Glu9, Glu10, Pro11, Trp22, Thr24, Tyr26.To probe the role of these residues on DAG/PE sensitivity, we first mutated these sites in the potent PE-sensitive C1b domain of PKCΔ to that of the corresponding sites of Vav1 C1, and analyzed the potency of the mutants for PEs. In vitro binding assays showed that 5 of 6 single-site-mutations (except Trp22) caused significant but limited (10-15 fold) reduction in the binding affinity to phorbol 12,13-dibutyrate (PDBU). Introduction of multiple mutations further decreased the affinity, in a cumulative fashion, leading to no detectable binding in the quintuple mutant. Correspondingly, in vivo confocal microscopy revealed that double and triple GFP-tagged mutants showed much slower and weaker plasma membrane translocation in response to PE than did WT C1bΔ, whereas the quintuple mutation was completely unresponsive. Thus, the ligand-insensitivity of Vav1 C1 reflects the combined effects of Glu9, Glu10, Pro11, Thr24, Tyr26 rather than the effect of a single specific residue. Conversely, introducing “reverse” mutations (corresponding to the residues of PKCΔ C1b) into Vav1 C1 generate binding activity. The quintuple (PKCΔ-like) mutation restored the phorbol-ester-sensitivity of Vav1 C1 to the level of the potent PKCΔ C1b both in vitro and in vivo. In addition, the quintuple mutation conferred PE-sensitivity to the full length Vav1, as revealed by translocation studies. Computer modeling suggests that the presence of these residues confers on Vav1 C1 a hydrophilic surface at the tip of the binding cavity (as opposed to the rather lipophilic surface of PKCΔ C1b), thus impeding interactions with the membrane bilayer and hindering the formation of the ternary binding complex of ligand, receptor (C1) and membrane lipid. We speculate that targeting those unique hydrophilic residues with specific DAG/PE analogs may provide a rationale for selectively manipulating Vav1 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3854. doi:10.1158/1538-7445.AM2011-3854

Vav1 Regulates Perivascular Homing, Bone Marrow Retention and Engraftment of Hematopoietic Stem Cells Via SDF1a Signaling

AbstractAbstract 400Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depends on extracellular signals present in specialized microenvironments in the bone marrow (BM). Osteoblasts, endothelium, and nestin-positive mesenchymal cells (N+MC), potentially contribute to the hematopoietic microenvironment (HM) and HSPC “niche” through membrane and secreted molecules such as stem cell factor, CXCL12/SDF1α, and integrin ligands. The crucial intracellular pathways that coordinate these signals, and thus regulate HSPC localization and interaction with components of their niche, remain poorly understood. We have implicated Rho GTPases Rac1 and Rac2 in the regulation of HSPC trafficking and engraftment (Gu et al. Science, 2003; Cancelas et al. Nat Med, 2005). Rho GTPases are activated by c-kit, CXCR4 and β1 integrin membrane receptors via largely undefined guanine nucleotide exchange factors (GEF). We hypothesized that the hematopoietic-specific GEF Vav1 regulates HSPC engraftment and retention by controlling Rho GTPase activation in response to signals from the HM and studied the hematopoietic phenotype and function of Vav1-/- HSPC mice. Analysis of the frequency of circulating progenitors in Vav1-/- mice revealed unexpected alterations in HSPC trafficking, including significantly decreased numbers of circulating CFC, absence of circadian fluctuation, and impaired progenitor mobilization induced by AMD3100 (AMD3100 mobilized 925+/−200 vs. 420+/−300 CFC/ml in WT vs Vav1-/-, P<0.01). Total leukocyte counts were similar in both genotypes after AMD3100 treatment, showing that this change was specific for primitive cells. In addition, GCSF-induced mobilization was also defective in Vav1-/- mice (640+/−40 CFC/ml vs 105+/−40 CFC/ml in WT vs Vav1-/-; P<0.01). These processes depend on SDF1α signalling, suggesting that in the absence of Vav1, HSPC were insensitive to induced changes in SDF1α levels or activity in the BM. Confirming that Vav1 was specifically required for SDF1α-mediated chemotactic responses, Vav1-/- HSPC demonstrated defective linear migration in a SDF1α gradient in vitro (net path length 53+/−10μm vs 27+/−4μm in WT vs Vav1-/- cells, P<0.01); however, Vav1 was not required for baseline or SDF1α -induced β1 integrin-mediated adhesion. These changes were associated with a lack ofagonist-dependent increase in Rac/Cdc42 activation after treatment with SDF-1α and an unexpectedly high baseline Rac/Cdc42 activity level. The downstream effector PAK-1 showed the same abnormal activation profile. To investigate the role of this pathway in in vivo engraftment and retention in the HM, we performed repopulation assays and determined the localization of transplanted Vav1-/- HSPC in the BM by intravital microscopy. Deletion of Vav1 was associated with nearly complete loss of HSPC engraftment in short-term (50% donor WT chimerism vs 0% Vav1-/- at 1 month) and long-term (56% vs 0%, WT vs Vav1-/- at 4 months) assays. Microscopically, Vav1-/- HSPC localized normally with respect to osteoblasts and the endosteal matrix.(Col2.3-GFP transgenic recipients), but showed significantly impaired localization to the proximity of SDF1α-secreting, N+MC (nestin-GFP+ recipients), 1 hr after transplant. At this early timepoint, only 6.25% of Vav1-/- HSPC, compared with 45.8% of WT HSPC, were located at less than 30 μm from a nestin+ cell. Considering only HSPC with nestin+ cells in their proximity, the mean distance of HSPC to the nearest nestin+ cell was significantly (p=0.005) shorter for WT vs Vav1-/- HSPC. This early abnormal localization was associated with significantly decreased retention of Vav1-/- HSPC (44-60% relative to WT) at 48 hours post-transplant in the BM cavities of both nestin-GFP and Col2.3-GFP recipient mice; this alteration in numbers of HSPC was not related to alterations in HSPC proliferative status or survival. In addition to previously characterized roles in immune responses, Vav1 functions as a critical molecule in HSPC, where it is specifically required for Rac/Cdc42 activation, SDF1α-dependent perivascular post-transplant homing, BM retention and engraftment. Our data define a pathway differentially regulating HSPC positioning with respect to N+MC, and support the concept that localization in the proximity of this perivascular niche component is important in the initial stages of post-transplant HSPC homing and is required for their early retention. Disclosures:Sanchez-Aguilera:Children's Hospital Boston: Employment.

Tyrosine Residues at the Carboxyl Terminus of Vav1 Play an Important Role in Regulation of Its Biological Activity

The guanine nucleotide exchange factor (GEF) Vav1 is an essential signal transducer protein in the hematopoietic system, where it is expressed physiologically. It is also involved in several human malignancies. Tyrosine phosphorylation at the Vav1 amino terminus plays a central role in regulating its activity; however, the role of carboxyl terminal tyrosine residues is unknown. We found that mutation of either Tyr-826 (Y826F) or Tyr-841 (Y841F) to phenylalanine led to loss of Vav1 GEF activity. When these Vav1 mutants were ectopically expressed in pancreatic cancer cells lacking Vav1, they failed to induce growth in agar, indicating loss of transforming potential. Furthermore, although Y841F had no effect on Vav1-stimulated nuclear factor of activated T cells (NFAT) activity, Y826F doubled NFAT activity when compared with Vav1, suggesting that Tyr-826 mediates an autoinhibitory effect on NFAT activity. SH2 profiling revealed that Shc, Csk, Abl, and Sap associate with Tyr-826, whereas SH2-B, Src, Brk, GTPase-activating protein, and phospholipase C-gamma associate with Tyr-841. Although the mutations in the Tyr-826 and Tyr-841 did not affect the binding of the carboxyl SH3 of Vav1 to other proteins, binding to several of the proteins identified by the SH2 profiling was lost. Of interest is Csk, which associates with wild-type Vav1 and Y841F, yet it fails to associate with Y826F, suggesting that loss of binding between Y826F and Csk might relieve an autoinhibitory effect, leading to increased NFAT. Our data indicate that GEF activity is critical for the function of Vav1 as a transforming protein but not for NFAT stimulation. The association of Vav1 with other proteins, detected by SH2 profiling, might affect other Vav1-dependent activities, such as NFAT stimulation.

Vav1 couples the T cell receptor to cAMP response element activation via a PKC-dependent pathway

The transcription factor cAMP-responsive element binding protein (CREB) is a regulator of the expression of several genes important for lymphocyte activation and proliferation. However, the proximal signaling events leading to activation of CREB in T cells upon antigen receptor stimulation remain unknown. Here we identify a role for Vav1 in the activation of the cAMP response element (CRE), the binding site for CREB. T cell receptor (TCR)/CD28 — induced costimulation of Jurkat T cells expressing Vav1 but not a GEF-deficient mutant showed increased CRE activation (7.2 ± 2.4 fold over control), whereas Vav1 downregulation by siRNA reduced activation of CRE by 2.6 ± 1.3 fold. Inhibition of PKC and MEK but not p38 could reduce Vav1-mediated CRE activation, suggesting that Vav1 transmits TCR and CD28 signals to activation of CRE via PKC and ERK signaling pathways. As a consequence, downregulation of Vav1 impaired the expression of several CRE-containing genes like cyclin D1, INFγ and IL-2, whereas overexpression of Vav1 enhanced CRE-dependent gene expression. Furthermore, cAMP-induced CRE-dependent transcription and gene expression was also modulated by Vav1, but did not require activation of PKC and the GEF function of Vav1. Our data provide insights into the signal transduction events regulating CRE-mediated gene expression in T cells, which affects T cell development, proliferation and activation. We identify Vav1 as an essential component of TCR-induced CRE activation and gene expression, which underlines the central role for Vav1 as key player for TCR signal transduction and gene expression.

The Use of Knockout Mice Reveals a Synergistic Role of the Vav1 and Rasgrf2 Gene Deficiencies in Lymphomagenesis and Metastasis

BackgroundVav1 and RasGRF2 are GDP/GTP exchange factors for Ras superfamily GTPases with roles in the development and/or effector functions of T–lymphocytes.Methodology/Principal FindingsGiven that the phenotype of Vav1 –/–, Rasgrf2 –/– and Vav1 –/–;Rasgrf2 –/– mice has been studied so far in young animals, we decided to explore the long–term consequences of the inactivation of those loci in the immune system. Unexpectedly, our studies revealed that the inactivation of the Vav1 proto–oncogene favors the formation of lymphoblastic lymphoma–like tumors in aging mice. Those tumors, that can be found either localized exclusively inside the thymus or widely disseminated in hematopoietic and non–hematopoietic tissues, are composed of CD3+ lymphoblasts that display heterogeneous combinations of CD4 and CD8 surface markers. Interestingly, the additional deletion of the Rasgrf2 gene induces a shortening in the latency period for the development of those tumors, an increase in the percentage of disseminated tumors outside the thymus and, as a result, higher mortality rates.Conclusions/SignificanceThese data reveal unexpected negative roles for Vav1 and RasGRF2 in different stages of T–cell lymphoma progression. They also suggest that the inactivation of Vav1 function may represent an inadequate strategy to treat T–cell lymphomas, especially those associated with low levels of Rasgrf2 gene expression.

Vav Links the T Cell Antigen Receptor to the Actin Cytoskeleton and T Cell Activation Independently of Intrinsic Guanine Nucleotide Exchange Activity

BackgroundT cell receptor (TCR) engagement leads to formation of signaling microclusters and induction of rapid and dynamic changes in the actin cytoskeleton, although the exact mechanism by which the TCR initiates actin polymerization is incompletely understood. The Vav family of guanine nucleotide exchange factors (GEF) has been implicated in generation of TCR signals and immune synapse formation, however, it is currently not known if Vav's GEF activity is required in T cell activation by the TCR in general, and in actin polymerization downstream of the TCR in particular.Methodology/Principal FindingsHere, we report that Vav1 assembles into signaling microclusters at TCR contact sites and is critical for TCR-initiated actin polymerization. Surprisingly, Vav1 functions in TCR signaling and Ca++ mobilization via a mechanism that does not appear to strictly depend on the intrinsic GEF activity.Conclusions/SignificanceWe propose here a model in which Vav functions primarily as a tyrosine phosphorylated linker-protein for TCR activation of T cells. Our results indicate that, contrary to expectations based on previously published studies including from our own laboratory, pharmacological inhibition of Vav1's intrinsic GEF activity may not be an effective strategy for T cell-directed immunosuppressive therapy.

The association of Sam68 with Vav1 contributes to tumorigenesis

Vav1 functions in the hematopoietic system as a specific GDP/GTP nucleotide exchange factor regulated by tyrosine phosphorylation. An intact C-terminal SH3 domain of Vav1 (Vav1SH3C) was shown to be necessary for Vav1-induced transformation, yet the associating protein(s) necessary for this activity have not yet been identified. Using a proteomics approach, we identified Sam68 as a Vav1SH3C-associating protein. Sam68 (Src-associated in mitosis of 68 kD) belongs to the heteronuclear ribonucleoprotein particle K (hnRNP-K) homology (KH) domain family of RNA-binding proteins. The Vav1/Sam68 interaction was observed in vitro and in vivo. Mutants of Vav1SH3C previously shown to lose their transforming potential did not associate with Sam68. Co-expression of Vav1 and Sam68 in Jurkat T cells led to increased localization of Vav1 in the nucleus and changes in cell morphology. We then tested the contribution of Sam68 to known functions of Vav1, such as focus-forming in NIH3T3 fibroblasts and NFAT stimulation in T cells. Co-expression of oncogenic Vav1 with Sam68 in NIH3T3 fibroblasts resulted in a dose-dependent increase in foci, yet no further enhancement of NFAT activity was observed in Jurkat T cells, as compared to cells overexpressing only Vav1 or Sam68. Our results strongly suggest that Sam68 contributes to transformation by oncogenic Vav1.

The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cgamma1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation.

The Rho/Rac-Family Guanine Nucleotide Exchange Factor VAV-1 Regulates Rhythmic Behaviors in C. elegans

Rhythmic behaviors are a fundamental feature of all organisms. Pharyngeal pumping, the defecation cycle, and gonadal-sheath-cell contractions are three well-characterized rhythmic behaviors in the nematode C. elegans. The periodicities of the rhythms range from subsecond (pharynx) to seconds (gonadal sheath) to minutes (defecation). However, the molecular mechanisms underlying these rhythmic behaviors are not well understood. Here, we show that the C. elegans Rho/Rac-family guanine nucleotide exchange factor, VAV-1, which is homologous to the mammalian Vav proto-oncogene, has a crucial role in all three behaviors. vav-1 mutants die as larvae because VAV-1 function is required in the pharynx for synchronous contraction of the musculature. In addition, ovulation and the defecation cycle are abnormal and arrhythmic. We show that Rho/Rac-family GTPases and the signaling molecule inositol triphosphate (IP(3)) act downstream of VAV-1 signaling and that the VAV-1 pathway modulates rhythmic behaviors by dynamically regulating the concentration of intracellular Ca(2+).

Lineage-Specific Requirement for the PH Domain of Vav1 in the Activation of CD4 + but Not CD8 + T Cells

Vav1 is a guanine nucleotide exchange factor (GEF) for Rho-family GTPases, which is activated by tyrosine phosphorylation following TCR stimulation. Vav1-deficient mice have defects in positive and negative selection of thymocytes as well as TCR-induced proliferation in mature T cells, demonstrating a critical role for Vav1 in transducing TCR signals. Binding of phospholipids to the PH domain of Vav1 has been proposed to regulate its GEF activity in vitro. To test this model in vivo, we have generated mice carrying a point mutation in the PH domain of Vav1, and we show that they have defects in T cell development and activation. We demonstrate that the mutation affects the function of Vav1 as a GEF and perturbs PI3K-dependent pathways downstream of Vav1. Unexpectedly, the mutation selectively affects TCR-induced proliferation of CD4(+) but not CD8(+) T cells, demonstrating differences in the wiring of TCR signaling pathways between the two lineages.

CD28 costimulatory signal induces protein arginine methylation in T cells

Protein phosphorylation initiates signal transduction that triggers lymphocyte activation. However, other posttranslational modifications may contribute to this process. Here, we show that CD28 engagement induced protein arginine methyltransferase activity and methylation on arginine of several proteins, including Vav1. Methylation of Vav1 and IL-2 production were reduced by inhibiting S-adenosyl-L-homocysteine hydrolase, an enzyme that regulates cellular transmethylation. Methylated Vav1 was induced in human and mouse T cells and selectively localized in the nucleus, which suggested that this form marks a nuclear function of Vav1. Our findings uncover a signaling pathway that is controlled by CD28 that is likely to be important for T cell activation.

Role of vav1 in the lipopolysaccharide-mediated upregulation of inducible nitric oxide synthase production and nuclear factor for interleukin-6 expression activity in murine macrophages.

vav1 has been shown to play a key role in lymphocyte development and activation, but its potential importance in macrophage activation has received little attention. We have previously reported that exposure of macrophages to bacterial lipopolysaccharide (LPS) leads to increased activity of hck and other src-related tyrosine kinases and to the prompt phosphorylation of vav1 on tyrosine. In this study, we tested the role of vav1 in macrophage responses to LPS, focusing on the upregulation of nuclear factor for interleukin-6 expression (NF-IL-6) activity and inducible nitric oxide synthase (iNOS) protein accumulation in RAW-TT10 murine macrophages. We established a series of stable cell lines expressing three mutant forms of vav1 in a tetracycline-regulatable fashion: (i) a form producing a truncated protein, vavC; (ii) a form containing a point mutation in the regulatory tyrosine residue, vavYF174; and (iii) a form with an in-frame deletion of 6 amino acids required for the guanidine nucleotide exchange factor (GEF) activity of vav1 for rac family GTPases, vavGEFmt. Expression of the truncated mutant (but not the other two mutants) has been reported to interfere with T-cell activation. In contrast, we now demonstrate that expression of any of the three mutant forms of vav1 in RAW-TT10 cells consistently inhibited LPS-mediated increases in iNOS protein accumulation and NF-IL-6 activity. These data provide direct evidence for a role for vav1 in LPS-mediated macrophage activation and iNOS production and suggest that vav1 functions in part via activation of NF-IL-6. Furthermore, these findings indicate that the GEF activity of vav1 is required for its ability to mediate macrophage activation by LPS.