Visualization of Negative Signaling in B Cells by Quantitative Confocal Microscopy

Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcgammaRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcgammaRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcgammaRII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were co-recruited to the BCR, regardless of the presence or absence of FcgammaRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcgammaRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcgammaRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.     

Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex

The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here, we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.     

Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors

Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.     

Integrin-dependent tyrosine phosphorylation and growth regulation by Vav

The proto-oncogene product p95Vav (Vav) undergoes rapid phosphorylation on tyrosine following stimulation of the T or B cell antigen receptor, and in response to a variety of other cell surface stimuli. Vav contains, among other, a guanine nucleotide exchange factor domain with homology to the Rho/Rac/CDC42 exchange protein Db1. It has been recently shown that Vav is functionally linked to small GTPases of the Rho family, suggesting that it is an activator of Rho GTPases and may participate in regulation of cytoskeletal organization. The present study shows that cell adhesion to fibronectin triggers rapid phosphorylation of Vav on tyrosine in Vav-transfected CHO cells and in Jurkat T cells. Vav phosphorylation is strongly dependent on adhesion and is mediated by beta 1 integrins. Furthermore, Vav overexpression enhances the adhesion-dependent increase in the rate and extent of phosphorylation on focal adhesion kinase and paxillin, and the formation of stress fibers and lamellipodia. In addition, there is a marked increase in the amount of Vav localized to the triton-insoluble fraction following 1 h of incubation on FN. Finally, Vav increases the growth rate of the cells in an adhesion-dependent manner. Our results strongly implicate Vav as a mediator of integrin signal transduction.     

Loss of Vav2 proto-oncogene causes tachycardia and cardiovascular disease in mice

The Vav family is a group of signal transduction molecules that activate Rho/Rac GTPases during cell signaling. Experiments using knockout mice have indicated that the three Vav proteins present in mammals (Vav1, Vav2, and Vav3) are essential for proper signaling responses in hematopoietic cells. However, Vav2 and Vav3 are also highly expressed in nonhematopoietic tissues, suggesting that they may have additional functions outside blood cells. Here, we report that this is the case for Vav2, because the disruption of its locus in mice causes tachycardia, hypertension, and defects in the heart, arterial walls, and kidneys. We also provide physiological and pharmacological evidence demonstrating that the hypertensive condition of Vav2-deficient mice is due to a chronic stimulation of the renin/angiotensin II and sympathetic nervous systems. Together, these results indicate that Vav2 plays crucial roles in the maintenance of cardiovascular homeostasis in mice.     

Signaling through CD5 Activates a Pathway Involving Phosphatidylinositol 3-Kinase, Vav, and Rac1 in Human Mature T Lymphocytes

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell–B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca²⁺ levels, and subsequently in the activation of Ca²⁺/calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CD5-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated Rac1 mutant (Rac1 · N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. A role for Vav in the CD5-induced signaling pathway is further supported by the findings that the expression of a dominant negative Vav mutant (Vav-C) completely abolishes the response to CD5 costimulation while the expression of a constitutively active Vav mutant [Vav(Δ1–65)] makes the CD5 costimulation signal superfluous. Wortmannin is unable to block the Vav(Δ1–65)- or Rac1 · V12-induced signals, indicating that both Vav and Rac1 function downstream from PI 3-kinase. Vav and Rac1 both act upstream from the CD5-induced activation of CaM kinase IV, since KN-62, an inhibitor of CaM kinases, and a dominant negative CaM kinase IV mutant block the Vav(Δ1–65)-and Rac1 · V12-mediated signals. We propose a model for the CD5-induced signaling pathway in which the PI 3-kinase lipid products, together with tyrosine phosphorylation, activate Vav, resulting in the activation of Rac1 by the Vav-mediated exchange of GDP for GTP.     

The granulocyte receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) directly associates with Vav to promote phagocytosis of human pathogens

The human granulocyte-specific receptor carcinoembryonic antigen-related cell adhesion molecule (CEACAM)3 is critically involved in the opsonin-independent recognition of several bacterial pathogens. CEACAM3-mediated phagocytosis depends on the integrity of an ITAM-like sequence within the cytoplasmic domain of CEACAM3 and is characterized by rapid stimulation of the GTPase Rac. By performing a functional screen with CEACAM3-expressing cells, we found that overexpression of a dominant-negative form of the guanine nucleotide exchange factor Vav, but not the dominant-negative versions SWAP70, Dock2, or ELMO1 interfered with CEACAM3-initiated phagocytosis. Moreover, small interfering RNA-mediated silencing of Vav reduced uptake and abrogated the stimulation of Rac in response to bacterial CEACAM3 engagement. In Vav1/Vav2-deficient cells, CEACAM3-mediated internalization was only observed after re-expression of Vav. Vav colocalized with CEACAM3 upon bacterial infection, coimmunoprecipitated in a complex with CEACAM3, and the Vav Src homology 2 domain directly associated with phosphorylated Tyr(230) of CEACAM3. In primary human granulocytes, TAT-mediated transduction of dominant-negative Vav, but not SWAP70, severely impaired the uptake of CEACAM3-binding bacteria. These data support the view that, different from canonical ITAM signaling, the CEACAM3 ITAM-like sequence short-wires bacterial recognition and Rac stimulation via a direct association with Vav to promote rapid phagocytosis and elimination of CEACAM-binding human pathogens.     

Vav transformation requires activation of multiple GTPases and regulation of gene expression

Although Vav can act as a guanine nucleotide exchange factor for RhoA, Rac1, and Cdc42, its transforming activity has been ascribed primarily to its ability to activate Rac1. However, because activated Vav, but not Rac-specific guanine nucleotide exchange factors, exhibits very potent focus-forming transforming activity when assayed in NIH 3T3 cells, Vav transforming activity must also involve activation of Rac-independent pathways. In this study, we determined the involvement of other Rho family proteins and their signaling pathways in Vav transformation. We found that RhoA, Rac1, and Cdc42 functions are all required for Vav transforming activity. Furthermore, we determined that Vav activation of nuclear factor-kappaB and the Jun NH2-terminal kinase mitogen-activated protein kinase (MAPK) is necessary for full transformation by Vav, whereas p38 MAPK does not seem to play an important role. We also determined that Vav is a weak activator of Elk-1 via a Ras- and MAPK/extracellular signal-regulated kinase kinase-dependent pathway, and this activity was essential for Vav transformation. Thus, we conclude that full Vav transforming activation is mediated by the activation of multiple small GTPases and their subsequent activation of signaling pathways that regulate changes in gene expression. Because Vav is activated by the epidermal growth factor receptor and other tyrosine kinases involved in cancer development, defining the role of aberrant Vav signaling may identify activities of receptor tyrosine kinases important for human oncogenesis.     

Collagen phagocytosis is regulated by the guanine nucleotide exchange factor Vav2

Collagen phagocytosis is a crucial alpha2beta1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5-20 min) but not Cdc42 or RhoA activity. Analysis of collagen bead-associated proteins showed enrichment with Vav2, which correlated temporally with increased Rac1 activity. Knockdown of Vav2 prevented Rac activation, recruitment of Rac1 to collagen bead binding sites, and collagen bead binding, but knockdown of Sos-1 or beta-Pix had no effect on Rac activation or collagen binding. Vav2 was associated with the nucleotide-free Rac1 mutant (G15ARac1) after collagen binding. Collagen bead binding promoted phosphorylation of Vav2, which temporally correlated with Rac1 activation and which required Src kinase activity. Blockage of Src activity prevented collagen bead-induced Rac activation and collagen bead binding. Collectively these data indicate that Vav2 regulates the Rac1 activity associated with the binding step of collagen phagocytosis.     

ZAP-70 is essential for the T cell antigen receptor-induced plasma membrane targeting of SOS and Vav in T cells

Translocation of the SOS and Vav GDP/GTP exchange factors proximal to Ras and Rac GTPases localized in the plasma membrane glycolipid-enriched microdomains is a pivotal step required for T cell antigen receptor-induced T cell activation. Here we demonstrate that the T cell antigen receptor zeta-chain-associated ZAP-70 kinase and T cell antigen receptor zeta-chain immunoreceptor tyrosine-based activation motifs are essential for the membrane recruitment of SOS and Vav. Plasma membrane targeting of SOS or Vav begins with the assembly of ZAP-70 with Grb-2 and SOS. The subsequent tyrosine phosphorylation of LAT (linker for activation of T cell) by ZAP-70 leads to a shift in equilibrium from the ZAP-70.Grb-2.SOS(Vav) complex to the (Vav)SOS.Grb-2.LAT complex. This shift results in the targeting of SOS and Vav into glycolipid-enriched microdomains and initiation of the Ras and Rac signaling cascades involved in T cell activation, proliferation, and cytokine production.     

Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.     

Tyrosine phosphorylation of Vav stimulates IL-6 production in mast cells by a Rac/c-Jun N-terminal kinase-dependent pathway.

This study investigates whether the guanine nucleotide exchange activity of Vav is linked to cytokine production in mast cells. Overexpression of Vav in the RBL-2H3 mast cell line resulted in the constitutive tyrosine phosphorylation and activation of Vav. We analyzed the functional effect of Vav overexpression on cytokine production. IL-2 and IL-6 mRNA levels were dramatically increased in Vav-overexpressing cells and correlated with increased NF-AT activity. Little or no effect was observed on the mRNA levels of IL-3, IL-4, GM-CSF, TNF-alpha, and TGF-beta. FcepsilonRI engagement did not further enhance IL-2 and IL-6 mRNA levels and only slightly enhanced NF-AT activity, but dramatically increased the mRNA levels of other tested cytokines. To understand the signal transduction required, we focused primarily on IL-6 induction by measuring mitogen-activated protein kinase activity and analyzing the effects of mutant or dominant negative forms of Vav, Rac1, and c-Jun N-terminal kinase-1 (JNK1). Vav overexpression resulted in the constitutive activation of JNK1 with little or no effect on p38 mitogen-activated protein kinase and ERK2. This was dependent on Vav-mediated activation of Rac1 as a Dbl domain-mutated Vav, inactive Rac N17, and inactive JNK1 down-regulated the Vav-induced JNK1 or IL-6 responses. Vav expression, but not expression of domain-mutated Vav, increased IL-6 secretion from nonimmortalized bone marrow-derived mast cells upon FcepsilonRI engagement. We conclude that Vav phosphorylation contributes to IL-6 induction in mast cells.     

Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells.

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1-/-) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.     

Vav2 is an activator of Cdc42, Rac1, and RhoA

Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity in their Dbl homology domains, it has been reported that they are active GEFs with distinct substrate specificities toward Rho family members. Whereas Vav displayed GEF activity for Rac1, Cdc42, RhoA, and RhoG, Vav2 was reported to exhibit GEF activity for RhoA, RhoB, and RhoG but not for Rac1 or Cdc42. Consistent with their distinct substrate targets, it was found that constitutively activated versions of Vav and Vav2 caused distinct transformed phenotypes when expressed in NIH 3T3 cells. In contrast to the previous findings, we found that Vav2 can act as a potent GEF for Cdc42, Rac1, and RhoA in vitro. Furthermore, we found that NH(2)-terminally truncated and activated Vav and Vav2 caused indistinguishable transforming actions in NIH 3T3 cells that required Cdc42, Rac1, and RhoA function. In addition, like Vav and Rac1, we found that Vav2 activated the Jun NH(2)-terminal kinase cascade and also caused the formation of lamellipodia and membrane ruffles in NIH 3T3 cells. Finally, Vav2-transformed NIH 3T3 cells showed up-regulated levels of Rac-GTP. We conclude that Vav2 and Vav share overlapping downstream targets and are activators of multiple Rho family proteins. Therefore, Vav2 may mediate the same cellular consequences in nonhematopoietic cells as Vav does in hematopoietic cells.     

Mechanistic analysis of the amplification and diversification events induced by Vav proteins in B-lymphocytes

Vav proteins participate in the assembly of a multibranched signal transduction pathway in lymphocytes, including the stimulation of the phosphatidylinositol 3-kinase/protein kinase B and the phospholipase C-gamma/Ras GDP-releasing protein/Ras/Erk routes. In the present work, we used a genetic approach in chicken DT40 B-cell lines to investigate additional elements of the Vav route, the synergisms existing among the different Vav signaling branches, and the activities exerted by wild-type and oncogenic Vav proteins in B-lymphocytes. We show here that the Vav pathway is ramified in B-lymphocytes in additional diacylglycerol-dependent signaling branches such as those involving protein kinase C, protein kinase D, and phospholipase D. By using side-by-side comparisons of the activation levels of those signal transduction pathways in inhibitor-treated and knockout DT40 cells, we show that B-cells have different requirements regarding Vav proteins for the activation of antigen receptor downstream elements. Furthermore, we have detected interpathway cross-talk at the level of the most proximal elements but not among the most distal effector molecules of the Vav route. Finally, we show that the oncogenic versions of Vav1 and RhoA can activate alternative routes that could contribute to signal amplification and diversification events in transformed lymphocytes.     

Vav proteins in neutrophils are required for FcgammaR-mediated signaling to Rac GTPases and nicotinamide adenine dinucleotide phosphate oxidase component p40(phox)

Phagocytes generate reactive oxygen species, the regulation of which is important in eliminating ingested microbes while limiting tissue damage. Clustering of FcgammaRs results in the activation of Vav proteins, Rho/Rac guanine nucleotide exchange factors, and results in robust superoxide generation through the NADPH oxidase. In this study, studies in neutrophils isolated from mice deficient in Vav or Rac isoforms demonstrate a critical role for Vav3 in Rac2-dependent activation of the NADPH oxidase following FcgammaR clustering. However, studies in cytokine-primed cells revealed a strict requirement for Vav1 and Vav3 and Rac1 and Rac2 in the FcgammaR-mediated oxidative burst. In comparison, Vav was not essential for PMA or G protein-coupled receptor-mediated superoxide generation. The FcgammaR-mediated oxidative burst defect in Vav-deficient cells was linked to aberrant Rac activation as well as Rac- and actin-polymerization-independent, but PI3K-dependent, phosphorylation of the NADPH oxidase component p40(phox). In macrophages, Vav regulation of Rac GTPases was required specifically in FcgammaR-mediated activation of the oxidative burst, but not in phagocytosis. Thus, Vav proteins specifically couple FcgammaR signaling to NADPH oxidase function through a Rac-dependent as well as an unexpected Rac-independent signal that is proximal to NADPH oxidase activation and does not require actin polymerization.     

Vav2 is required for cell spreading.

Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.