Vav Proto-oncogene Research Articles

The fusion oncogene VAV1-MYO1F triggers aberrant T-cell receptor signaling in vivo and drives peripheral T-cell lymphoma in mice.

VAV1-MYO1F is a recently identified gain-of-function fusion protein of the proto-oncogene Vav guanine nucleotide exchange factor 1 (VAV1) that is recurrently detected in T-cell non-Hodgkin's lymphoma (T-NHL) patients. However, the pathophysiological functions of VAV1-MYO1F in lymphomagenesis are insufficiently defined. Therefore, we generated transgenic mouse models to conditionally express VAV1-MYO1F in T-cells in vivo. We demonstrate that VAV1-MYO1F triggers cell autonomous activation of T-cell signaling with an activation of the ERK, JNK, and AKT pathways. VAV1-MYO1F expression induces a T-cell activation phenotype with high surface expression of CD25, ICOS, CD44, PD-1, and decreased CD62L as well as aberrant T-cell differentiation, proliferation, and neoplastic transformation. Consequently, the VAV1-MYO1F expressing T-cells induce a malignant T lymphoproliferative disease with 100% penetrance in vivo that mimics key aspects of human peripheral T-cell lymphoma. These results demonstrate that the human T-cell oncogene VAV1-MYO1F is sufficient to trigger oncogenic T-cell signaling and neoplastic transformation, and moreover, it provides a new clinically relevant mouse model to explore the pathogenesis of and treatment concepts for human T-cell lymphoma.

Transcriptome-based screening of intracellular pathways and angiogenesis related genes at different stages of thiram induced tibial lesions in broiler chickens

BackgroundThe Tibial dyschondroplasia (TD) in fast-growing chickens is mainly caused by improper blood circulation. The exact mechanism underlying angiogenesis and vascularization in tibial growth plate of broiler chickens remains unclear. Therefore, this research attempts to study genes involved in the regulation of angiogenesis in chicken red blood cells. Twenty-four broiler chickens were allotted into a control and thiram (Tetramethyl thiuram disulfide) group. Blood samples were collected on day 2, 6 (8- and 14-days old chickens) and 15 (23 days old chickens).ResultsHistopathology and hematoxylin and eosin (H&E) results showed that angiogenesis decreased on the 6th day of the experiment but started to recover on the 15th day of the experiment. Immunohistochemistry (IHC) results confirmed the expressions of integrin alpha-v precursor (ITGAV) and clusterin precursor (CLU). Transcriptome sequencing analysis evaluated 293 differentially expressed genes (DEGs), of which 103 up-regulated genes and 190 down-regulated genes were enriched in the pathways of neuroactive ligand receptor interaction, mitogen-activated protein kinase (MAPK), ribosome, regulation of actin cytoskeleton, focal adhesion, natural killer cell mediated cytotoxicity and the notch signalling pathways. DEGs (n = 20) related to angiogenesis of chicken erythrocytes in the enriched pathways were thromboxane A2 receptor (TBXA2R), interleukin-1 receptor type 1 precursor (IL1R1), ribosomal protein L17 (RPL17), integrin beta-3 precursor (ITGB3), ITGAV, integrin beta-2 precursor (ITGB2), ras-related C3 botulinum toxin substrate 2 (RAC2), integrin alpha-2 (ITGA2), IQ motif containing GTPase activating protein 2 (IQGAP2), ARF GTPase-activating protein (GIT1), proto-oncogene vav (VAV1), integrin alpha-IIb-like (ITGA5), ras-related protein Rap-1b precursor (RAP1B), tyrosine protein kinase Fyn-like (FYN), tyrosine-protein phosphatase non-receptor type 11 (PTPN11), protein patched homolog 1 (PTCH1), nuclear receptor corepressor 2 (NCOR2) and mastermind like protein 3 (MAML3) selected for further confirmation with qPCR. However, commonly DEGs were sarcoplasmic/endoplasmic reticulum calcium ATPase 3 (ATP2A3), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), centriole cilia and spindle-associated protein (CCSAP), coagulation factor XIII A chain protein (F13A1), shroom 2 isoform X6 (SHROOM2), ras GTPase-activating protein 3 (RASA3) and CLU.ConclusionWe have found potential therapeutic genes concerned to erythrocytes and blood regulation, which regulated the angiogenesis in thiram induced TD chickens. This study also revealed the potential functions of erythrocytes.Graphical abstract1. Tibial dyschondroplasia (TD) in chickens were more on day 6, which started recovering on day 15. 2. The enriched pathway observed in TD chickens on day 6 was ribosome pathway, on day 15 were regulation of actin cytoskeleton and focal adhesion pathway. 3. The genes involved in the ribosome pathways was ribosomal protein L17 (RPL17). regulation of actin cytoskeleton pathway were Ras-related C3 botulinum toxin substrate 2 (RAC2), Ras-related protein Rap-1b precursor (RAP1B), ARF GTPase-activating protein (GIT1), IQ motif containing GTPase activating protein 2 (IQGAP2), Integrin alpha-v precursor (ITGAV), Integrin alpha-2 (ITGA2), Integrin beta-2 precursor (ITGB2), Integrin beta-3 precursor (ITGB3), Integrin alpha-IIb-like (ITGA5). Focal adhesion Proto-oncogene vav (Vav-like), Tyrosine-protein kinase Fyn-like (FYN).

Self-binding peptides: Binding-upon-folding versus folding-upon-binding

Self-binding peptide (SBP) represents a novel biomolecular phenomenon spanning between folding and binding. It is a structurally independent, short peptide segment within a monomeric protein and fulfills biological function by dynamically binding to/unbinding from its target domain in the same monomer. Here, four representative SBP systems, including mouse proto-oncogene Vav, human retinoic acid receptor RARγ, fruit fly scaffold module INAD and crypto 14-3-3 protein Cp14b, are investigated systematically by using atomistic molecular dynamics (MD) simulations and post binding energetics analyses. The native bound structure, artificial unbound state and isolated peptide segment of SBP moieties in the four systems were constructed, analyzed and compared in detail. It is revealed that the SBP interaction with their targets is almost a binding phenomenon at single-molecule level, but presence of a polypeptide linker between the SBP and target can promote the binding efficiency since the linker restriction largely increases the probability of SBP–target encounters in a statistical physics point of view. In this respect, unlike classical peptide-mediated interactions where the intrinsically disordered peptides are folded into an ordered structure upon binding to their protein partners (folding-upon-binding), we herein propose SBPs as a new and reversed biological event that is naturally a folding phenomenon but exhibits a typical binding behavior (binding-upon-folding).

A systems medicine approach reveals disordered immune system and lipid metabolism in multiple sclerosis patients.

Identification of autoimmune processes and introduction of new autoantigens involved in the pathogenesis of multiple sclerosis (MS) can be helpful in the design of new drugs to prevent unresponsiveness and side effects in patients. To find significant changes, we evaluated the autoantibody repertoires in newly diagnosed relapsing-remitting MS patients (NDP) and those receiving disease-modifying therapy (RP). Through a random peptide phage library, a panel of NDP- and RP-specific peptides was identified, producing two protein data sets visualized using Gephi, based on protein--protein interactions in the STRING database. The top modules of NDP and RP networks were assessed using Enrichr. Based on the findings, a set of proteins, including ATP binding cassette subfamily C member 1 (ABCC1), neurogenic locus notch homologue protein 1 (NOTCH1), hepatocyte growth factor receptor (MET), RAF proto-oncogene serine/threonine-protein kinase (RAF1) and proto-oncogene vav (VAV1) was found in NDP and was involved in over-represented terms correlated with cell-mediated immunity and cancer. In contrast, transcription factor RelB (RELB), histone acetyltransferase p300 (EP300), acetyl-CoA carboxylase 2 (ACACB), adiponectin (ADIPOQ) and phosphoenolpyruvate carboxykinase 2 mitochondrial (PCK2) had major contributions to viral infections and lipid metabolism as significant events in RP. According to these findings, further research is required to demonstrate the pathogenic roles of such proteins and autoantibodies targeting them in MS and to develop therapeutic agents which can ameliorate disease severity.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

Hematopoietic stem cell (HSC) gene therapy for two patients with adenosine deaminase (ADA) deficiency without cytoreductive conditioning; a suggestion for the optimal protocol for HSC gene therapy for ADA deficiency

The efficacy of the gene therapy for the treatment of monogenic diseases is showing evident beneficial effects. Nevertheless, risks of insertional oncogenesis have been shown in some of the clinical protocols and in different animal models. In those cases in which a low expression of the transgene could maintain the therapeutic effect of the gene therapy, the inclusion of weak promoters in self-inactivating vectors would reduce the trans-activation potential of the integrated proviruses, resulting in a safer approach for the gene therapy protocol. Here we have generated a self-inactivating lentiviral vector in which the proximal promoter of the vav protooncogene was used to drive the expression of the transgene. Compared to other lentiviral vectors in which the internal promoter was the CMV, PGK or SFFV promoter, we observed that the vav promoter mediated a weaker, though homogeneous and stable expression of the transgene, not only in vitro but also in vivo 1. The vavHS1 promoter is rich in CpG islands and therefore potentially susceptible to methylation. However, we observed that this promoter was very low methylated not only in transduced cell lines, but also in transduced human hematopoietic grafts transplanted into immunodeficient mice. To demonstrate the therapeutic efficacy of the vavHS1 promoter, we constructed a self-inactivating lentiviral vector expressing the Fanconi Anemia A gene (FANCA). In spite of the weak activity of the promoter, we observed that vav-LVs expressing the FANCA gene were capable of efficiently correcting the phenotype of cells from a FA-A patient. Taken together, our data suggest that self-inactivating vectors harboring the proximal promoter of the vav proto-oncogene will constitute efficient and safer vectors for the therapy of diseases where a high expression of the transgene is not required to achieve a therapeutic effect. Refs: 1: Almarza E, Rio P, Meza NW, et al. Characteristics of lentiviral vectors harboring the proximal promoter of the vav proto-oncogene: a weak and efficient promoter for gene therapy. Mol Ther. 2007.

Characteristics of Lentiviral Vectors Harboring the Proximal Promoter of the vav Proto-oncogene: A Weak and Efficient Promoter for Gene Therapy

Recent published data have shown the efficacy of gene therapy treatments of certain monogenic diseases. Risks of insertional oncogenesis, however, indicate the necessity of developing new vectors with weaker or cell-restricted promoters to minimize the trans-activation activity of integrated proviruses. We have inserted the proximal promoter of the vav proto-oncogene into self-inactivating lentiviral vectors (vav-LVs) and investigated the expression pattern and therapeutic efficacy of these vectors. Compared with other LVs frequently used in gene therapy, vav-LVs mediated a weak, though homogeneous and stable, expression in in vitro-cultured cells. Transplantation experiments using transduced mouse bone marrow and human CD34(+) cells confirmed the stable activity of the promoter in vivo. To investigate whether the weak activity of this promoter was compatible with a therapeutic effect, a LV expressing the Fanconi anemia A (FANCA) gene was constructed (vav-FANCA LV). Although this vector induced a low expression of FANCA, compared to the expression induced by a LV harboring the spleen focus-forming virus (SFFV) promoter, the two vectors corrected the phenotype of cells from a patient with FA-A with the same efficacy. We propose that self-inactivating vectors harboring weak promoters, such as the vav promoter, will improve the safety of gene therapy and will be of particular interest for the treatment of diseases where a high expression of the transgene is not required.

Overexpression of the VAV proto‐oncogene product is associated with B‐cell chronic lymphocytic leukaemia displaying loss on 13q

The expression of the VAV proto-oncogene in 57 patients with chronic myeloproliferative disease (CMD), B-cell acute lymphoblastic leukaemia (B-ALL) and B-cell non-Hodgkin Lymphoma (B-NHL), and 61 with B-cell chronic lymphocytic leukaemia (B-CLL) was analysed. VAV overexpression was observed in 19.5% of cases and 81% of VAV-positive tumours also displayed VAV phosphorylation. Overexpression was not observed in B-ALL or CMD, but 13% of B-NHL and 34.4% of B-CLL patients (P = 0.002) overexpressed VAV. The overexpression and phosphorylation of VAV was detected more frequently in 13q- chronic lymphocytic leukaemias (71.4%) versus other B-CLLs (23.4%, P = 0.001). Overexpression of VAV protein is a frequent event in patients with B-CLL displaying loss of 13q sequences.

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+).

Regulatory elements of the vav gene drive transgene expression in hematopoietic stem cells from adult mice

Objective Previous studies have shown that the HS21/45 promoter of the vav protooncogene drives a predominant expression of exogenous transgenes in mouse hematopoietic cells, including clonogenic bone marrow (BM) progenitors. We investigated the activity of this promoter in the hematopoietic stem cell compartment of adult mice. Materials and methods Inbred Ly5.1 transgenic mice expressing a nonfunctional human CD4 marker gene (hCD4) under the control of the HS21/45 promoter were generated. BM cells from these animals were sorted based on the intensity of hCD4 expression. Fractions characterized by high, intermediate, or low/negative expression of the transgene were then assessed for their competitive repopulation ability (CRA), using unfractionated BM cells from Ly5.2 mice as a reference competitor population. Results Data showed that BM cells having a low/negative or intermediate expression of hCD4 had a very poor hematopoietic CRA. In contrast, BM cells with high hCD4 expression were characterized by a high CRA. These observations were confirmed in the short- and long-term posttransplantation of primary and secondary recipients when analyzing the lymphoid and myeloid cells of recipient mice. Conclusions Our results demonstrate for the first time that the regulatory HS21/45 sequence of the vav gene constitutes an efficient promoter for driving transgene expression in multipotent hematopoietic stem cells residing in the BM of adult mice. Thus, this promoter is proposed for the development of transgenic mice and gene therapy vectors that require restricted expression of exogenous transgenes in cells of the hematopoietic system, including primitive hematopoietic stem cells.

Protein profiling and identification of modulators regulated by the E7 oncogene in the C33A cell line by proteomics and genomics.

Human papillomaviruses (HPVs) have been recognized as the primary cause of cervical cancer. HPV 16 E7 binds to tumor suppressor retinoblastoma protein, and interferes with its function, causing release of the transcription factor E2F, which influences expression of cell cycle-related genes. This study was performed to identify the genes and proteins modulated by the HPV E7 oncogene. An HPV-negative cervical cancer cell line (C33A) was prepared to establish a stable cell line expressing E7. In order to analyze the target molecules modulated by E7 expression, we used two approaches: matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and DNA microarrays. Forty-seven spots were identified in C33A/E7 by two-dimensional electrophoresis and MALDI/TOF MS. Protein disulfide isomerase A3, integrase interactor 1 protein, growth inhibitory protein, glutathione S-transferase P, and vav proto-oncogene were down-regulated, whereas heat shock 60 kDa protein 1, Ku70 binding protein, alpha enolase, 26S proteasome subunit were up-regulated. A genomic approach using a microarray kit showed that IL-12R beta 1, cytochrome c, and tumor necrosis factor receptor II were induced by the E7 oncogene. These results suggest that E7 can evade immune surveillance by suppressing or inducing these cell signaling factors, cell cycle regulators, and chaperones.

Adaptor protein APS binds the NH2-terminal autoinhibitory domain of guanine nucleotide exchange factor Vav3 and augments its activity.

The N-terminal calponin homology (CH) domain of Vav guanine nucleotide exchange factor is thought to serve a regulatory role in the autoinhibition, however, its precise function is not entirely clear. We found that the adaptor molecule APS could bind the CH domain of Vav3, a member of the vav proto-oncogene family. The binding of Vav3 and APS was apparently stabilized by the tyrosine phosphorylation of Vav3 by Lck, and the association of APS with Vav3 in turn enhanced the Lck-mediated phosphorylation of Vav3. Focus formation assays demonstrated that APS could increase the transforming activity of proto-Vav3. Further analyses revealed that the Vav3 CH domain could bind the pleckstrin homology (PH) domain of APS and that this binding was indispensable to enhance the transforming activity of Vav3. We present here a novel stimulatory mechanism of Vav3 in which APS directly relieves the autoinhibitory CH domain and furthermore enhances its tyrosine phosphorylation by Lck.

Association of Bcr-Abl with the Proto-oncogene Vav Is Implicated in Activation of the Rac-1 Pathway

Vav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization. The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis. Previous studies by Matsuguchi et al. (Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257-265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins. Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo. Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl. Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction. Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominant negative construct. Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow. These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus. Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1.

Regulation of the vav proto-oncogene by LKLF

The transcriptional induction of the vav proto-oncogene coincides with the first appearance of the definitive hematopoietic stem cell in the aorta-gonad-mesonephros region. Vav promoter activity was dependent on a previously identified 23 bp DNA segment containing PU.1 and Runx1/AML-1 binding sites and on a newly identified, highly conserved, 12 bp DNA segment (Box B). The sequence of Box B was identical in the human, mouse and rat species. Mutation of the CACCC core sequence led to diminished vav promoter activity. A protein complex which bound to Box B was found in hematopoietic cells but not in cells which did not express vav. A double-stranded oligonucleotide containing a mutation of the CACCC core was less effective in electro-mobility shift assay competitions than the wild-type sequence. UV crosslinking studies identified a 37 kDa DNA binding protein which interacted with Box B in U937 cells. Antibody supershift assays identified this protein as lung Krüppel-like factor (LKLF). LKLF, expressed as a glutathione S-transferase fusion protein, was capable of binding to Box B. A dominant-negative LKLF was able to inhibit the expression of enhanced green fluorescent protein by the vav promoter and chromatin immunoprecipitations detected LKLF bound to the vav promoter in U937 but not HeLa cells. These in vitro results suggest future in vivo experiments to examine the role of LKLF, a gene required for vasculogenesis, in the induction of vav during the genesis of the definitive hematopoietic stem cell from the vascular endothelium.

GEF at work: Vav in protruding filopodia.

The Dbl family proto-oncogene vav is a nucleotide exchange factor for Rho family GTPases and is involved in triggering cytoskeletal changes contributing to the alterations of cell shape and motility, as well as in the induction of gene expression. In vitro and in vivo Vav is regulated by multiple tyrosine phosphorylation and binding to phosphatidylinositol phosphates. Although recruitment of Vav to the plasma membrane appears important for the activation of Vav function, there is little information on the precise subcellular localization of Vav in living cells. Employing live video fluorescence and immunoelectron microscopy, we show that GFP-tagged full-length Vav, and several mutants in which the N-terminal regulatory calponin homology (CH) domain has been deleted, specifically localize to the tips of filopodia. This localization was congruent with a high content of tyrosine phosphorylation in these regions. Consistent with earlier observations, mutants lacking the C-terminal SH domain region were unable to translocate to the filopodia tips. The enrichment in filopodial tips persisted despite their lateral movement but was dependent on forward growth. Upon retraction, the signal was rapidly lost, indicating that Vav undergoes a specific and transient translocation in response to actin-based, protrusive events in filopodia.

Activation of the EBV/C3d receptor (CR2, CD21) on human B lymphocyte surface triggers tyrosine phosphorylation of the 95-kDa nucleolin and its interaction with phosphatidylinositol 3 kinase.

We previously demonstrated that CR2 activation on human B lymphocyte surface triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3' (PI 3) kinase. Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1. To identify this tyrosine phosphorylated p95 component, we first purified it by affinity chromatography on anti-phosphotyrosine mAb covalently linked to Sepharose 4B, followed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa tyrosine phosphorylated band was submitted to amino acid analysis by mass spectrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, localized between aa 411--420 and 611--624. Anti-nucleolin mAb was used to confirm the antigenic properties of this p95 component. Functional studies demonstrated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domains of the p85 subunit of PI 3 kinase and of 3BP2 and Grb2, but not with Src homology 2 domains of Fyn and Gap. These properties of nucleolin were identical with those of the p95 previously described and induced by CR2 activation. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activation. These data support that tyrosine phosphorylation of nucleolin and its interaction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.

Occlusion of DH Domain Inhibits Vav

The Dbl homology (DH)-containing protein Vav acts as a guanine nucleotide exchange factor (GEF) for the Rho family of guanine triphosphatases (GTPases), and, in vitro, Vav exhibits specific GEF activity toward Rac protein. Phosphorylation of Vav in response to B cell receptor or T cell receptor stimulation precedes Rac activation. Expression of NH 2 -terminal deletion (residues 1-182) mutants of Vav leads to chronically activated Vav and increased Rac activity. Phosphorylation of Tyr 174 is important for activation of Vav, but until now, the mechanism by which Vav is activated has remained elusive. Aghazadeh et al. have used nuclear magnetic resonance (NMR) to solve the structure of the Vav DH domain in solution. By comparing the structure of the DH domain in the presence or absence of phosphate at Tyr 174 , the authors identified that, in the inactive state, Tyr 174 resides in the Vav DH domain where it occludes Vav's ability to bind and to exchange nucleotides on Rac. Upon phosphorylation of Tyr 174 , an α helix that contains Tyr 174 shifts away from the DH domain, allowing Vav to bind Rac and to exchange GDP for GTP, thus activating Rac. Within the context of the whole protein, it is suggested that the Vav pleckstrin homology (PH) domain lies juxtaposed to the Tyr 174 -occluded DH domain. Upon lipid binding by the Vav PH domain at the surface of a membrane, the Tyr 174 residue becomes available for phosphorylation, leading to Vav activation. Thus, activation of Vav occurs in a multistep process whereby proper membrane localization of Vav leads to its activation. Aghazadeh, B., Lowry, W.E., Huang, X.-Y., and Rosen, M.K. (2000) Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 102 : 625-633. [Online Journal]

Occlusion of DH Domain Inhibits Vav

The Dbl homology (DH)-containing protein Vav acts as a guanine nucleotide exchange factor (GEF) for the Rho family of guanine triphosphatases (GTPases), and, in vitro, Vav exhibits specific GEF activity toward Rac protein. Phosphorylation of Vav in response to B cell receptor or T cell receptor stimulation precedes Rac activation. Expression of NH2-terminal deletion (residues 1-182) mutants of Vav leads to chronically activated Vav and increased Rac activity. Phosphorylation of Tyr174 is important for activation of Vav, but until now, the mechanism by which Vav is activated has remained elusive. Aghazadeh et al. have used nuclear magnetic resonance (NMR) to solve the structure of the Vav DH domain in solution. By comparing the structure of the DH domain in the presence or absence of phosphate at Tyr174, the authors identified that, in the inactive state, Tyr174 resides in the Vav DH domain where it occludes Vav's ability to bind and to exchange nucleotides on Rac. Upon phosphorylation of Tyr174, an α helix that contains Tyr174 shifts away from the DH domain, allowing Vav to bind Rac and to exchange GDP for GTP, thus activating Rac. Within the context of the whole protein, it is suggested that the Vav pleckstrin homology (PH) domain lies juxtaposed to the Tyr174-occluded DH domain. Upon lipid binding by the Vav PH domain at the surface of a membrane, the Tyr174 residue becomes available for phosphorylation, leading to Vav activation. Thus, activation of Vav occurs in a multistep process whereby proper membrane localization of Vav leads to its activation.Aghazadeh, B., Lowry, W.E., Huang, X.-Y., and Rosen, M.K. (2000) Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 102: 625-633.[Online Journal]

Structural Basis for Relief of Autoinhibition of the Dbl Homology Domain of Proto-Oncogene Vav by Tyrosine Phosphorylation

Rho-family GTPases transduce signals from receptors leading to changes in cell shape and motility, mitogenesis, and development. Proteins containing the Dbl homology (DH) domain are responsible for activating Rho GTPases by catalyzing the exchange of GDP for GTP. Receptor-initiated stimulation of Dbl protein Vav exchange activity involves tyrosine phosphorylation. We show through structure determination that the mVav1 DH domain is autoinhibited by an N-terminal extension, which lies in the GTPase interaction site. This extension contains the Tyr174 Src-family kinase recognition site, and phosphorylation or truncation of this peptide results in stimulation of GEF activity. NMR spectroscopy data show that the N-terminal peptide is released from the DH domain and becomes unstructured upon phosphorylation. Thus, tyrosine phosphorylation relieves autoinhibition by exposing the GTPase interaction surface of the DH domain, which is obligatory for Vav activation.

Lack of interferon-alpha-induced tyrosine phosphorylation of Vav proto-oncogene in patients with myelofibrosis with myeloid metaplasia.

Myelofibrosis with myeloid metaplasia (MMM) is an uncommon chronic myeloproliferative disorder characterized by clonal stem cell proliferation and reactive non-clonal proliferation of bone marrow fibroblasts with fibrosis. In the absence of curative therapy, the current management for the majority of patients is directed towards alleviation of symptoms and improvement in quality of life. A number of experimental therapies have been investigated, among which is the use of type I interferon (IFN)-alpha, whose overall results are disappointing. We recently showed that the Vav proto-oncogene product, p95Vav, which is phosphorylated under IFN-alpha treatment, associates with both chains that constitute the functional type I IFN receptor and contributes to the antiproliferative effects of IFN-alpha. The involvement of p95Vav in IFN-alpha signalling and the frequent non-responsiveness of patients to IFN-alpha led us to investigate for any functional defect(s) of Vav in response to IFN in MMM patients. Our results showed that in the majority of patients Vav is constitutively hyperphosphorylated and that IFN-alpha failed to increase substantially such a tyrosine phosphorylation of p95Vav.