Role Of Vav1 Research Articles

P164 MRT-6160, a VAV1-directed molecular glue degrader, inhibits disease progression and inflammation in a T-cell transfer model of Colitis

Abstract Background VAV1 is an immune-restricted guanine nucleotide exchange factor critical for T-cell receptor (TCR) and B-cell receptor (BCR) signalling. The role of VAV1 in T-cells has been demonstrated in knockout mice, which exhibit decreased effector functions and resistance to autoimmune disease models, and in CRISPR-based screens, where VAV1 has been highlighted as a top hit among positive regulators of T-cell function. Until now, VAV1 has remained undruggable by conventional small molecules. MRT-6160 is a first-in-class molecular glue degrader that specifically targets VAV1 for proteasomal degradation. Given the role of VAV1 in TCR and BCR signalling, MRT-6160-mediated degradation could be an effective therapeutic approach to treating autoimmune disease. Methods We first tested the impact of MRT-6160-mediated human (h)VAV1 degradation on primary human cells using in vitro stimulation-based assays. Oral bioavailability of MRT-6160 and in vivo degradation of murine (m)VAV1 was then examined in mice. We tested the efficacy of MRT-6160 using a T-cell adoptive transfer model of colitis treating recipients of pathogenic CD45RBhigh CD4+ T-cells (8 per group) with anti-TNF (25 mg/kg, Q3D) or MRT-6160 (1 mg/kg, QD) for 41 days following cell transfer. Disease activity index (DAI), comprising stool consistency and weight loss, was assessed every 3 days. At study termination, mesenteric lymph nodes (mLN) were profiled by flow cytometry and colon tissues were assessed for cytokine expression and histopathology. Results Degradation of hVAV1 by MRT-6160 inhibited T-cell activation, proliferation, and cytokine production in a dose-dependent manner. Orally administered MRT-6160 degraded mVAV1 and inhibited colitis disease progression by 85%, reducing average end point DAI scores more than a standard of care control (vehicle, p<0.0001; anti-TNF, p=0.0107). Compared to vehicle treated groups, MRT-6160 treated mice had fewer IL-17A+ (12.6 and 5.3% respectively) and TNF+ (37.6 and 17% respectively) CD4+ T cells in mLN tissue. Colon weight:length ratio was significantly reduced in MRT-6160-treated mice (42.5 mg/cm) compared to vehicle (70.3 mg/cm) and anti-TNF (61.2 mg/cm). Finally, pro-inflammatory cytokine expression in the colon mucosa was reduced in MRT-6160-treated mice compared to vehicle (IL-6: 1305.8 and 682.3 fg/mg; TNF: 175.5 and 66.9 ng/mg; respectively), as well as histopathological evidence of disease. Conclusion MRT-6160 demonstrates strong activity in a preclinical model of colitis reducing DAI, effector cytokine production, and colon tissue damage. These data suggest that MRT-6160-mediated degradation of VAV1 may have therapeutic benefit in IBD patients and warrants further clinical development.

Role of Vav1, a hematopoietic signal transduction molecule, as an adaptor protein in health and disease

The growth and differentiation of normal cells are controlled by protein-tyrosine kinases, which serve as receptors for a wide variety of external signals. Small protein modules called Src homology 2 (SH2) and SH3 domains mediate protein-protein interactions in signaling pathways that are triggered by protein tyrosine kinases. The SH2 domain, a protein module of around 100 amino acids, is present in tyrosine kinase targets within the cell. SH2 domains are recruited to activated and autophosphorylated growth factor receptors by directly recognizing tyrosine phosphorylation sites. Growth factor receptors and other phosphoproteins have short phosphotyrosine (pTyr)-containing sequences that are bound by SH2 domains. The SH3 domain, a distinct element of approximately 50 residues that recognizes proline-rich and hydrophobic-amino-acid-containing regions, is frequently found in SH2-containing proteins. Tyrosine kinases can be coupled to downstream targets with SH3-binding sites by proteins with SH2 and SH3 domains acting as adaptors. These intricate and precise biochemical signaling pathways result in the regulation of gene expression, cytoskeletal architecture, and cell metabolism. The role of SH2/SH3 proteins in T cell signaling will be discussed. A special focus will be on the role of the hematopoietic signal transducer with SH2/SH3 domains, Vav1, in health and cancer.

Vav1 Down-Modulates Akt2 Expression in Cells from Pancreatic Ductal Adenocarcinoma: Nuclear Vav1 as a Potential Regulator of Akt Related Malignancy in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive tumor malignancy worldwide, mainly due to uncontrolled metastasis. Among the numerous molecules deregulated in PDAC, different members of the Akt pathways are of great importance because they are involved in tumor cell proliferation, migration, and invasion. We have recently demonstrated that Vav1, ectopically expressed in solid tumors, is capable of down-modulating expression and/or activation of specific Akt isoforms in breast cancer cells. By using pancreatic cell lines expressing different basal levels of Vav1, we demonstrated here that Vav1 down-regulates the expression of Akt2, known to correlate with tumor metastases and resistance to therapy. In particular, while the silencing of Vav1 is sufficient to induce Akt2, its up-modulation reduces Akt2 levels only when Vav1 accumulates inside the nucleus of PDAC cells. Moreover, in PDAC tissues, we revealed that high nuclear levels of Vav1 correlate with low Akt2 expression. Although we cannot demonstrate the mechanisms involved, our results provide new insights into the role of Vav1 in PDAC and, as targeting specific members of the Akt family is a promising therapeutic chance in solid tumors, they suggest that Vav1, by down-modulating Akt2, has potential as a molecular target in PDAC.

A Natural Variant of the Signaling Molecule Vav1 Enhances Susceptibility to Myasthenia Gravis and Influences the T Cell Receptor Repertoire.

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1R63W). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1R63W) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4+ T cells, as well as an increased frequency of antigen-specific CD4+ T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes.

Novel role of Vav1-Rac1 pathway in actin cytoskeleton regulation in interleukin-13-induced minimal change-like nephropathy.

Our established interleukin-13 (IL-13) overexpression rat model of minimal change-like nephropathy provided a platform to study the molecular signalling pathways in T-helper 2 (Th2) cytokine associated minimal change nephrotic syndrome (MCNS). We hypothesized that IL-13 may act directly on podocytes, causing podocyte foot process effacement and hence proteinuria in our rat model of minimal change-like nephropathy. The present study aimed firstly to delineate the glomerular 'gene signature' associated with IL-13-mediated dysregulation of podocyte-related proteins, and subsequently to investigate the role of the differentially regulated genes (DEGs) in IL-13-mediated podocyte injury. Glomerular transcriptional profile of IL-13-overexpressed rats showed characteristic features of podocyte injury with 87% of podocyte-related genes being significantly down-regulated. Gene expression of Vav1 was shown to be highly up-regulated in the glomeruli of IL-13-overexpressed rats and pathway analysis of the DEGs suggested a possible novel role of Vav1in podocyte cytoskeleton remodelling. Immunofluorescence examination demonstrated glomerular expression of Vav1in rats which co-localized with synaptopodin, confirming podocyte expression. However, positive staining for the phosphorylated form of Vav1 (p-Vav1) was only seen in IL-13-overexpressed rats. Moreover, invitro IL-13 stimulation of human podocytes resulted in phosphorylation of Vav1. This was associated with Rac1 activation and actin cytoskeleton rearrangement, which was abrogated in Vav1 knockdown podocytes. In conclusion, we have demonstrated the role of Vav1-Rac1 pathway characterized by phosphorylation of Vav1, activation of Rac1 and the subsequent actin cytoskeleton rearrangement in IL-13-induced podocyte injury, possibly explaining the podocyte foot process effacement seen in our IL-13 overexpression rat model.

A Conserved GEF for Rho-Family GTPases Acts in an EGF Signaling Pathway to Promote Sleep-like Quiescence in Caenorhabditis elegans.

Sleep is evolutionarily conserved and required for organism homeostasis and survival. Despite this importance, the molecular and cellular mechanisms underlying sleep are not well understood. Caenorhabditis elegans exhibits sleep-like behavioral quiescence and thus provides a valuable, simple model system for the study of cellular and molecular regulators of this process. In C. elegans, epidermal growth factor receptor (EGFR) signaling is required in the neurosecretory neuron ALA to promote sleep-like behavioral quiescence after cellular stress. We describe a novel role for VAV-1, a conserved guanine nucleotide exchange factor (GEF) for Rho-family GTPases, in regulation of sleep-like behavioral quiescence. VAV-1, in a GEF-dependent manner, acts in ALA to suppress locomotion and feeding during sleep-like behavioral quiescence in response to cellular stress. Additionally, VAV-1 activity is required for EGF-induced sleep-like quiescence and normal levels of EGFR and secretory dense core vesicles in ALA. Importantly, the role of VAV-1 in promoting cellular stress-induced behavioral quiescence is vital for organism health because VAV-1 is required for normal survival after cellular stress.

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.

Mo1785 A Key Regulatory Role of Vav1 in Controlling Septic Shock and Experimental Peritonitis via IL-6 Production in Macrophages

Mo1785 A Key Regulatory Role of Vav1 in Controlling Septic Shock and Experimental Peritonitis via IL-6 Production in Macrophages

Vav1 is a crucial molecule in monocytic/macrophagic differentiation of myeloid leukemia-derived cells

Vav1 is a critical signal transducer for both the development and function of normal hematopoietic cells, in which it regulates the acquisition of maturation-related properties, including adhesion, motility, and phagocytosis. Vav1 is also important for the agonist-induced maturation of acute promyelocytic leukemia (APL)-derived promyelocytes, in which it promotes the acquisition of a mature phenotype by playing multiple functions at both cytoplasmic and nuclear levels. We investigated the possible role of Vav1 in the differentiation of leukemic precursors to monocytes/macrophages. Tumoral promyelocytes in which Vav1 was negatively modulated were induced to differentiate into monocytes/macrophages with phorbol-12-myristate-13-acetate (PMA) and monitored for their maturation-related properties. We found that Vav1 was crucial for the phenotypical differentiation of tumoral myeloid precursors to monocytes/macrophages, in terms of CD11b expression, adhesion capability and cell morphology. Confocal analysis revealed that Vav1 may synergize with actin in modulating nuclear morphology of PMA-treated adherent cells. Our data indicate that, in tumoral promyelocytes, Vav1 is a component of lineage-specific transduction machineries that can be recruited by various differentiating agents. Since Vav1 plays a central role in the completion of the differentiation program of leukemic promyelocytes along diverse hematopoietic lineages, it can be considered a common target for developing new therapeutic strategies for the various subtypes of myeloid leukemias.

Vav1 is a crucial molecule in monocytic/macrophagic differentiation of myeloid leukemia cells

Vav1 is a critical signal transducer for both the development and function of normal hematopoietic cells, in which it regulates the acquisition of maturation-related properties, including adhesion, motility, and phagocytosis. Vav1 is also important for the agonist-induced maturation of acute promyelocytic leukemia (APL)-derived promyelo- cytes, in which it promotes the acquisition of a mature phenotype by playing multiple functions at both cytoplas- mic and nuclear levels. We investigated the possible role of Vav1 in the differentiation of leukemic precursors to mono- cytes/macrophages. Tumoral promyelocytes in which Vav1 was negatively modulated were induced to differentiate into monocytes/macrophages with phorbol-12-myristate-13-ace- tate (PMA) and monitored for their maturation-related properties. We found that Vav1 was crucial for the phenotyp- ical differentiation of tumoral myeloid precursors to mono- cytes/macrophages, in terms of CD11b expression, adhesion capability and cell morphology. Confocal analysis revealed that Vav1 may synergize with actin in modulating nuclear morphology of PMA-treated adherent cells. Our data indicate that, in tumoral promyelocytes, Vav1 is a compo- nent of lineage-specific transduction machineries that can be recruited by various differentiating agents. Since Vav1 plays a central role in the completion of the differentiation program of leukemic promyelocytes along diverse hemato- poietic lineages, it can be considered a common target for developing new therapeutic strategies for the various subtypes of myeloid leukemias.

Vav1 and PU.1 are recruited to the CD11b promoter in APL-derived promyelocytes: Role of Vav1 in modulating PU.1-containing complexes during ATRA-induced differentiation

Vav1 plays an important role in the all- trans retinoic acid (ATRA)-induced completion of the differentiation program of acute promyelocytic leukemia (APL)-derived cells, in which it strengthens the drug effects and is involved in the regulation of maturation-related proteins, such as the CD11b surface antigen. In both myeloid and lymphoid cells, accumulating data attribute to the multidomain protein Vav1 a functional relevance in the control of gene expression, by direct interaction with chromatin remodeling and/or transcriptional proteins. The present study provides evidence that, in the APL-derived NB4 cell line, Vav1 and the transcription factor PU.1 cooperate in regulating the ATRA-induced CD11b expression. Both chromatin immunoprecipitation (ChIP) experiments and electrophoretic mobility shift assays (EMSA) indicate that Vav1 and PU.1 are recruited to CD11b promoter. Even if the two proteins may participate in diverse protein/DNA complexes, the amounts of complexes including PU.1 seem to be dependent on the interaction of this transcription factor with tyrosine-phosphorylated Vav1. The reported data suggest that the ATRA-induced increase of Vav1 expression and tyrosine phosphorylation may be involved in recruiting PU.1 to its consensus sequence on the CD11b promoter and, ultimately, in regulating CD11b expression during the late stages of neutrophil differentiation of APL-derived promyelocytes.

S1708 A Crucial Regulatory Role of Vav1 in Chronic Intestinal Inflammation and Colitis Associated Colon Cancer

S1708 A Crucial Regulatory Role of Vav1 in Chronic Intestinal Inflammation and Colitis Associated Colon Cancer

The Role of SPECs, Small Cdc42-binding Proteins, in F-actin Accumulation at the Immunological Synapse

SPEC1 and SPEC2 are structurally similar Cdc42-binding proteins of 79 and 84 amino acid residues, respectively. We investigated the role of SPEC2 in T cell function due to its high mRNA expression in lymphocytes. Western blot analysis revealed abundant SPEC2 protein in lymphocytes, which in glutathione S-transferase-capture experiments specifically interacted with only GTP-bound Cdc42. Immunofluorescence experiments revealed that the SPEC2 protein was diffusely localized in the cytoplasm and at the cell membrane in unstimulated Jurkat T cells and Raji B cells. Recruitment of SPEC2 within Jurkat T cells to the antigen-presenting cell interface occurred following incubation with staphylococcal enterotoxin E superantigen-loaded B cells and colocalized there with F-actin and Cdc42. T cell receptor (TCR) activation studies using anti-CD3 antibody-coated polystyrene beads showed that SPEC2 was recruited to the site of bead contact, which was not observed with anti-major histocompatibility complex antibody-coated beads. Accumulation of SPEC2 following TCR engagement occurred as early as 5 min, before obvious F-actin accumulation. Biochemical studies with Jurkat T cells demonstrated that N-terminal cysteine residues in SPEC2 were palmitoylated. Overexpression studies of the related SPEC1 showed that it also was recruited to the activated TCR. Mutational analysis revealed that localization of SPEC1 to the TCR required two N-terminal cysteine residues. Furthermore, a SPEC1 Cdc42 Rac-interacting binding mutant, containing an intact N terminus but defective in Cdc42 binding, completely blocked F-actin accumulation at the activated TCR. Taken together these results suggest that SPECs may play important roles in Cdc42-mediated F-actin accumulation at the immunological synapse.

Membrane Localization and Function of Vav3 in T Cells Depend on Its Association with the Adapter SLP-76

The Vav family of guanine exchange factors plays a critical role in lymphocyte proliferation, cytoskeletal reorganization, and gene transcription upon immunoreceptor engagement. Although the role of Vav1 in T cells is well documented, the role of Vav3 is less clear. We investigated the subcellular localization of Vav3 during T cell activation. We report here that phosphorylation of Vav3 on tyrosine residue Tyr(173) is not required for T cell receptor (TCR)-induced Vav3 membrane translocation or immunological synapse (IS) recruitment, but mutation of this residue enhanced TCR-induced nuclear factor of activated T cells (NFAT) activation. However, Vav3 mutants either containing an Src homology 2 (SH2)-disabled point mutation (R697L) or lacking its SH3-SH2-SH3 domains were unable to bind SLP-76 did not translocate to the membrane or to the IS and furthermore failed to activate NFAT. Importantly, the membrane translocation of Vav3 was abrogated in Lck, ZAP-70, LAT, and SLP-76-deficient T cells, where Vav3 binding to SLP-76 was disrupted. Finally, we confirmed and underlined the critical role of Vav3 in NFAT activation by knocking down Vav3 expression in Vav1-deficient T cells. Altogether, our data show that TCR-induced association of Vav3 with SLP-76 is required for its membrane/IS localization and function.

Role of vav1- and src-related Tyrosine Kinases in Macrophage Activation by CpG DNA

Macrophage activation by CpG DNA requires toll-like receptor 9 and the adaptor protein MyD88. Gram-negative bacterial lipopolysaccharide also activates macrophages via a toll-like receptor pathway (TLR-4), but we and others have reported that lipopolysaccharide also stimulates tyrosine phosphorylation in macrophages. Herein we report that exposure of RAW 264.7 murine macrophages to CpG DNA (but not non-CpG DNA) provoked the rapid tyrosine phosphorylation of vav1. PP1, a selective inhibitor of src-related tyrosine kinases, blocked both the CpG DNA-mediated tyrosine phosphorylation of vav1 and the CpG DNA-mediated up-regulation of macrophage tumor necrosis factor secretion and inducible nitric-oxide synthase protein accumulation. Furthermore, we found that the inducible expression of any of three dominant interfering mutants of vav1 (a truncated protein, vavC; a form containing a point mutation in the regulatory tyrosine residue, vavYF174; and a form with an in-frame deletion of six amino acids required for the guanidine nucleotide exchange factor (GEF) activity of vav1 for rac family GTPases, vavGEFmt) consistently inhibited CpG DNA-mediated up-regulation of tumor necrosis factor secretion and inducible nitric-oxide synthase protein accumulation in RAW-TT10 macrophages. Finally, we determined that CpG DNA-mediated up-regulation of NF-kappaB activity (but not mitogen-activated protein kinase activation) was inhibited by preincubation with PP1 or by expression of the truncated vavC mutant. Taken together, our results indicate that the tyrosine phosphorylation of vav1 by a src-related tyrosine kinase or kinases plays an important role in the macrophage response to CpG DNA.

The hemopoietic Rho/Rac guanine nucleotide exchange factor Vav1 regulates N-formyl-methionyl-leucyl-phenylalanine-activated neutrophil functions.

Vav1 is a hemopoietic-specific Rho/Rac guanine nucleotide exchange factor that plays a prominent role in responses to multisubunit immune recognition receptors in lymphoid cells, but its contribution to regulation of neutrophil functions is unknown. Activated Rho family GTPases are critical participants in neutrophil signaling cascades initiated by binding of FMLP and other chemoattractants to their cognate G protein-coupled receptors. Therefore, we investigated whether Vav1 regulates chemoattractant-induced responses in neutrophils. We found that superoxide production elicited by FMLP in Vav1(-/-) murine neutrophils isolated from either bone marrow or from peritoneal exudates was substantially reduced compared with that of wild type. Filamentous actin generation in FMLP-stimulated Vav1(-/-) neutrophils was also markedly reduced, whereas it was normal in response to IL-8 or leukotriene B(4). FMLP induced tyrosine phosphorylation of Vav1, whereas IL-8 or leukotriene B(4) did not, correlating with the requirement for Vav1 in chemoattractant-stimulated filamentous actin generation. Neutrophil motility in vitro and neutrophil mobilization into peripheral blood in vivo elicited by FMLP were both decreased in Vav1(-/-) mice. Hence, this study defines a new role for Vav1 in regulating granulocytic leukocytes as well as linking Vav1 to specific cellular responses downstream of a seven transmembrane domain receptor.

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Vav1 Controls Integrin Clustering and MHC/Peptide-Specific Cell Adhesion to Antigen-Presenting Cells

Integrin-mediated adhesion is essential for the formation of stable contacts between T cells and antigen-presenting cells (APCs). We show that Vav1 controls integrin-mediated adhesion of thymocytes and T cells to ECM proteins and ICAM1 following TCR stimulation. In a peptide-specific system, Vav1 is required for T cell adhesion to peptide-loaded APCs. Intriguingly, TCR-induced cell adhesion and aggregation of integrins occurs independent of WASP. Whereas LFA-1 and actin caps colocalize in wasp −/− T cells in response to TCR stimulation, loss of WASP uncouples TCR caps from actin patches. Our data reveal a novel role for Vav1 and WASP in the regulation of TCR-induced integrin clustering and cell adhesion and show that integrin and TCR clustering are controlled by distinct pathways.

Vav2 Activates c-fos Serum Response Element and CD69 Expression but Negatively Regulates Nuclear Factor of Activated T Cells and Interleukin-2 Gene Activation in T Lymphocyte

Vav1 and Vav2 are members of the Dbl family of guanine nucleotide exchange factors for the Rho family of small GTPases. Although the role of Vav1 during lymphocyte development and activation is well characterized, the function of Vav2 is still unclear. In this study, we compared the signaling pathways regulated by Vav1 and Vav2 following engagement of the T cell receptor (TCR). We show that Vav2 is tyrosine-phosphorylated upon TCR stimulation and by co-expressed Src and Syk family kinases. Using glutathione S-transferase fusion proteins, we observed that the Src homology 2 domain of Vav2 binds tyrosine-phosphorylated proteins from TCR-stimulated Jurkat T cell lysates, including c-Cbl and SLP-76. Like Vav1, Vav2 cooperated with TCR stimulation to increase extracellular signal-regulated kinase activation and to promote c-fos serum response element transcriptional activity. Moreover, both proteins displayed a similar action in increasing the expression of the early activation marker CD69 in Jurkat T cells. However, in contrast to Vav1, Vav2 dramatically suppressed TCR signals leading to nuclear factor of activated T cells (NF-AT)-dependent transcription and induction of the interleukin-2 promoter. Vav2 appears to act upstream of the phosphatase calcineurin because a constitutively active form of calcineurin rescued the effect of Vav2 by restoring TCR-induced NF-AT activation. Interestingly, the Dbl homology and Src homology 2 domains of Vav2 were necessary for its inhibitory effect on NF-AT activation and for induction of serum response element transcriptional activity. Taken together, our results indicate that Vav1 and Vav2 exert overlapping but nonidentical functions in T cells. The negative regulatory pathway elicited by Vav2 might play an important role in regulating lymphocyte activation processes.

Activation and Association of the Tec Tyrosine Kinase with the Human Prolactin Receptor: Mapping of a Tec/Vav1-Receptor Binding Site

Stimulation of the PRL receptor (PRLr) results in the activation of the guanine nucleotide exchange factor (GEF) p95Vav1 with corresponding alterations in cytoarchitecture and cell motility. To better understand the mechanisms involved in the regulation of Vav1 activity, the role of the tyrosine kinase p70Tec was examined. Coimmunoprecipitation and in vitro kinase assays revealed that ligand stimulation of the PRLr resulted in the rapid activation of Tec and its concomitant association with the PRLr. When coexpressed in COS-1 cells, both Vav1 and Tec were found to associate with the PRLr in the presence of ligand. In the absence of receptor, a constitutive complex between Vav1 and Tec was noted. Both Vav1 and Tec, however, were capable of independent engagement of a bipartite intracellular domain of the PRLr. Deletion mapping studies confined this interaction to residues 323 to 527 of the intracellular domain of the PRLr. Furthermore, Tec enhanced the GEF activity of Vav1 as evidenced by an increase in GTP-bound Rac1. These data would suggest a pivotal function for the formation of a Tec/Vav1/PRLr complex during PRL-driven signal transduction, given the role of Vav1 in the control of cell proliferation and the regulation of Rho family-mediated cytoskeletal alterations.