Guanine Nucleotide Exchange Factor Vav1 Research Articles

Abstract 382: Oxidized LDL Signaling “Primes” GPVI-Mediated Platelet Activation in a CD36-Dependent Manner

CD36 acts as an important participant in the prothrombotic state associated with chronic inflammatory diseases such as atherosclerosis and diabetes by serving as a signaling relay point for danger-associated molecular patterns (DAMPs), including oxidized low-density lipoproteins (oxLDL) and advanced glycated proteins. Although CD36-mediated signaling pathways have been well characterized in macrophages, less is known about CD36-mediated signaling in platelets. Our group identified important roles for specific Src family kinases (SFK) Lyn and Fyn, and for the guanine nucleotide exchange factors Vav1 and Vav3. Since platelet activation by the collagen receptor GPVI also involves downstream activation of SFKs, we now hypothesize that oxLDL signaling via CD36 primes platelets for hyperactivity by activating components of the GPVI signaling pathway. To test this hypothesis, we treated fluorescently labeled murine platelets with either native LDL (nLDL) or oxLDL and exposed them to immobilized collagen under defined shear flow in a microfluidic flow chamber. A significant increase (~1.2 fold) in platelet accumulation was observed with oxLDL treatment. No increase in accumulation was observed in oxLDL-treated platelets from cd36 null mice, suggesting that this phenomenon was CD36-dependent. Furthermore, addition of tirofiban ablated the oxLDL-induced increase in platelet accumulation, suggesting that the increase was likely due to enhanced platelet-platelet contacts (i.e. enhanced platelet activation). This supports previous data from our laboratory showing that treatment of murine platelets with oxLDL alone resulted in activation of αIIbβ3 integrin. Additionally, oxLDL-treated platelets from vav1/vav3 double null mice phenocopied cd36 null platelets and displayed no increase in platelet accumulation compared to nLDL-treated platelets, strengthening the functional link between CD36 and GPVI signaling pathways. These data suggest a functional link between CD36 and GPVI signaling pathways in platelets, which may contribute to platelet hyperactivity in athero-inflammatory diseases.

Tiam1/Rac1 signals contribute to the proliferation and chemoresistance, but not motility, of chronic lymphocytic leukemia cells

AbstractSignals from the tumor microenvironment promote the migration, survival, and proliferation of chronic lymphocytic leukemia (CLL) cells. Rho GTPases control various signaling pathways downstream of microenvironmental cues. Here, we analyze the function of Rac1 in the motility and proliferation of CLL cells. We found decreased transcription of the Rac guanine nucleotide exchange factors Tiam1 and Vav1 in unstimulated peripheral blood CLL cells with almost complete loss of Tiam1 but increased transcription of the potential Rac antagonist RhoH. Consistently, stimulation of CLL cells with the chemokine CXCL12 induced RhoA but not Rac1 activation, whereas chemokine-induced CLL cell motility was Rac1-independent. Coculture of CLL cells with activated T cells induced their activation and subsequent proliferation. Here, Tiam1 expression was induced in the malignant cells in line with increased Ki-67 and c-Myc expression. Rac1 or Tiam1 knockdown using siRNA or treatment with the Tiam1/Rac inhibitor NSC-23766 attenuated c-Myc transcription. Furthermore, treatment of CLL cells with NSC-23766 reduced their proliferation. Rac inhibition also antagonized the chemoresistance of activated CLL cells toward fludarabine. Collectively, our data suggest a dynamic regulation of Rac1 function in the CLL microenvironment. Rac inhibition could be of clinical use by selectively interfering with CLL cell proliferation and chemoresistance.

Contribution of guanine nucleotide exchange factor Vav2 to homocysteine‐induced NLRP3 inflammasome activation in mouse podocytes (1063.6)

NADPH oxidase‐derived reactive oxygen species (ROS) have been demonstrated to mediate the activation of podocyte NLRP3 inflammasomes in response to elevated levels of homocysteine (Hcys). We have also previously provided evidence that the guanine nucleotide exchange factor Vav2 exhibits high specificity to Rac1‐mediated NADPH oxidase activation by elevated Hcys. The precise role of Vav2 in NLRP3 inflammasome activation remains unknown. The present study was designed to test whether Vav2 contributes to Hcys‐induced NLRP3 inflammasome activation through activation of NADPH oxidase. In our experiments, podocytes were transfected with either a scramble, constitutively active form of Vav2 (oncoVav2) or Vav2 shRNA plasmid directly to the nucleus via nucleofection. Confocal microscopic analysis showed Hcys treatment as well as oncoVav2 transfection alone increased colocalization between inflammasome proteins NLRP3 with ASC and NLRP3 with caspase‐1, suggesting NLRP3 inflammasome formation. Additionally, both Hcys treatment as well as oncoVav2 transfection alone resulted in increased caspase‐1 activation and IL‐1β production, signifying NLRP3 inflammasome activation. These Hcys‐induced measures of inflammasome formation and activation were all inhibited by Vav2 shRNA transfection, observed in decreased colocalization of inflammasome components, caspase‐1 activity, and IL‐1β production in podocytes. Furthermore, Vav2 shRNA transfection also prevented podocyte damage by restoring impaired VEGF secretion as well as decreased podocyte marker podocin expression seen in Hcys injured podocytes. Together, these findings suggest that NADPH oxidase activation by Vav2 overexpression by oncoVav2 was sufficient to activate NLRP3 inflammasomes, independent of Hcys treatment. However, Vav2 inhibition was able to inhibit Hcys‐induced NLRP3 inflammasome activation and podocyte injury, further substantiating the role of Vav2 in this mechanism.Grant Funding Source: Supported by NIH grants DK54927 and 1F31AG043289

Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor–independent TCR signaling hub

T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

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.

Abstract A155: The Rac and PAK inhibitor EHop-016 inhibits mammary tumor growth and metastasis in a nude mouse model.

Abstract The Rho GTPase Rac is a central regulator of cancer cell migration/invasion that has been implicated with cancer metastasis. Therefore, we designed and developed small molecule compounds that specifically target Rac in metastatic cancer cells. We recently reported the characterization of EHop-016 as a Rac inhibitor in metastatic cancer cells, where EHop-016 was shown to inhibit Rac activity with an IC50 of 1 μM. At higher concentrations (>10 μM) EHop-016 reduced cell viability and inhibited the related Rho GTPase Cdc42, but not Rho. EHop-016 decreased the activation of Rac by the oncogenic guanine nucleotide exchange factor Vav, the activity of the Rac effector p21-activated kinase (PAK), and the extension of actin cytoskeletal structures and migration of metastatic cancer cells. Next, we determined the efficacy of EHop-016 in vivo, using a mouse model of experimental metastasis. Green fluorescent protein (GFP) tagged MDA-MB-435 human metastatic cancer cells were inoculated at the mammary fat pad of nude mice and treated with 0, 5, 10, 25, or 40 mg/kg body weight (BW) EHop-016 3X a week for ∼8 weeks. Mammary tumor growth and metastases in lungs, spleens, kidneys, livers, hearts, and femurs were quantified by fluorescence image analysis. At concentrations >10 mg/kg BW, EHop-016 significantly inhibited mammary tumor growth and metastasis to distant organs. EHop-016 had no effect on mouse weight or their gross phenotype, indicating that EHop-016 did not exert toxic effects in athymic nude mice. This data demonstrate that EHop-016 and derivatives hold promise for further development as targeted anti-cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A155. Citation Format: Linette Castillo-Pichardo, Tessa Humphries-Bickley, Eliud Hernandez, Columba de la Parra, Luis Cubano, Cornelis Vlaar, Surangani F. Dharmawardhane Flanagan. The Rac and PAK inhibitor EHop-016 inhibits mammary tumor growth and metastasis in a nude mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A155.

A Specific PTPRC/CD45 Phosphorylation Event Governed by Stem Cell Chemokine CXCL12 Regulates Primitive Hematopoietic Cell Motility

CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells.

Inhibition of gastric cancer cell growth and invasion through siRNA-mediated knockdown of guanine nucleotide exchange factor Vav3

Vav3, a Rho GTPase guanine nucleotide exchange factor, is associated with tumor growth, apoptosis, invasion and metastasis, and angiogenesis. However, the role of Vav3 in gastric cancer remains unclear. In this study, Vav3 expression was blocked by specific siRNA in gastric cancer cell line MGC803. MTT was used to assay cell proliferation activity; wound healing assay and transwell assay were applied to detect cell migration and invasion ability; and qRT-PCR and Western blot were employed to detect expression levels of Vav3 as well as proliferation, migration, and invasion-related genes. The results showed that Vav3 expression in gastric cancer tissues and cell lines was significantly upregulated and was higher than that in adjacent tissues of cancer and normal gastric mucosal cell lines. Vav3 knockdown inhibited proliferation, migration, and invasion of MGC803 gastric cancer cells. The expression of P21, P27, TIMP-1, and TIMP-2 was upregulated, while proliferating cell nuclear antigen, cyclin E1, matrix metalloproteinase (MMP)-2, and MMP-7 were downregulated by Vav3 knockdown in MGC803 gastric cells. In conclusion, Vav3 is involved in the proliferation, migration, and invasion of gastric cancer cell as a tumor oncogene.

Pak and Rac GTPases promote oncogenic KIT–induced neoplasms

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21-activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT.

Vav Guanine Nucleotide Exchange Factors Regulate Atherosclerotic Lesion Development in Mice

Atherosclerosis requires migration of monocytes to the arterial intima, with subsequent differentiation into foam cells. We showed previously that the scavenger receptor CD36 contributes to the activation of Vav family guanine nucleotide exchange factors (Vavs) in aortae from hyperlipidemic apoE-null mice and that oxidatively modified low-density lipoprotein induced CD36-dependent activation of macrophage Vavs in vitro. We also discovered that CD36-dependent uptake of oxidized low-density lipoprotein and foam cell formation were reduced in Vav-deficient macrophages. We now tested the hypothesis that Vavs play a role in atherosclerotic lesion development. We showed that apoE/vav1 double-null mice fed a Western diet had significant reduction in total aortic lesion area (by en face analysis) compared with apoE-null mice, with no significant differences in body weight or plasma lipid profiles. Histological analysis of aortic sinus lesions showed fewer macrophages and foam cells in double-null mice compared with apoE-null mice, indicating impaired foam cell generation and homing of macrophages to atherosclerotic lesions. An intravital video microscopy-based adhesion assay with fluorescent (Qtracker655)-labeled monocytes showed reduced adhesion of vav1-null monocytes to hyperlipidemic carotid arteries compared with wild-type monocytes. Furthermore, fewer fluorescently labeled vav1-null monocytes accumulated in aortic sinus lesions in hyperlipidemic apoE-null mice. We also found that activation of RhoGTPase Rac and mitogen-activated protein kinase c-Jun N-terminal kinase-2 by CD36-specific oxidized phospholipids was dependent on Vavs. These results for the first time link Vavs to atherosclerotic lesion development and suggest that Vavs act as critical molecular links coupling hyperlipidemia with proatherogenic monocyte/macrophage responses.

CEACAM1 on activated NK cells inhibits NKG2D‐mediated cytolytic function and signaling

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on activated natural killer (NK) cells wherein it inhibits lysis of CEACAM1-bearing tumor cell lines. The mechanism for this is unknown. Here, we show that interleukin-2-induced expression of CEACAM1 on both mouse and primary human NK cells impairs the ability of NK gene complex group 2 member D (NKG2D) to stimulate cytolysis of CEACAM1-bearing cells. This process requires the expression of CEACAM1 on the NK cells and on the tumor cells, which is consistent with the involvement of trans-homophilic interactions between CEACAM1. Mechanistically, co-engagement of NKG2D and CEACAM1 results in a biochemical association between these two surface receptors and the recruitment of Src homology phosphatase 1 by CEACAM1 that leads to dephosphorylation of the guanine nucleotide exchange factor Vav1 and blockade of downstream signaling that is associated with the initiation of cytolysis. Thus, CEACAM1 on activated NK cells functions as an inhibitory receptor for NKG2D-mediated cytolysis, which has important implications for understanding the means by which CEACAM1 expression adversely affects tumor immunity.

Potassium-Chloride Cotransporter 3 Interacts with Vav2 to Synchronize the Cell Volume Decrease Response with Cell Protrusion Dynamics

Loss-of-function of the potassium-chloride cotransporter 3 (KCC3) causes hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), a severe neurodegenerative disease associated with defective midline crossing of commissural axons in the brain. Conversely, KCC3 over-expression in breast, ovarian and cervical cancer is associated with enhanced tumor cell malignancy and invasiveness. We identified a highly conserved proline-rich sequence within the C-terminus of the cotransporter which when mutated leads to loss of the KCC3-dependent regulatory volume decrease (RVD) response in Xenopus Laevis oocytes. Using SH3 domain arrays, we found that this poly-proline motif is a binding site for SH3-domain containing proteins in vitro. This approach identified the guanine nucleotide exchange factor (GEF) Vav2 as a candidate partner for KCC3. KCC3/Vav2 physical interaction was confirmed using GST-pull down assays and immuno-based experiments. In cultured cervical cancer cells, KCC3 co-localized with the active form of Vav2 in swelling-induced actin-rich protruding sites and within lamellipodia of spreading and migrating cells. These data provide evidence of a molecular and functional link between the potassium-chloride co-transporters and the Rho GTPase-dependent actin remodeling machinery in RVD, cell spreading and cell protrusion dynamics, thus providing new insights into KCC3's involvement in cancer cell malignancy and in corpus callosum agenesis in HMSN/ACC.

RNA-aptamers that modulate the RhoGEF activity of Tiam1

Rho GTPases regulate the actin cytoskeleton and thereby control cell migration, cell morphology, cell motility, and other cellular functions. The gene product of the oncogene Tiam1 acts as a guanine nucleotide exchange factor (GEF) for the Rho GTPase Rac. Like other RhoGEFs, Tiam1 is involved in cancer progression, but it also counteracts invasion in different cancer cell types. Hence, further investigations are required to unravel the functions of Tiam1 in the context of cancer initiation and progression, which appear to be cell specific. Although RhoGEFs in general seem to be attractive therapeutic targets, not many inhibitors have been described, yet. Here we report the identification and characterization of inhibitory RNA aptamers that specifically target Tiam1. After 16 selection rounds three aptamers sharing a 15 nucleotides consensus motif were identified. The clones K91 and K11 inhibited the Tiam1-mediated activation of the GTPase Rac2 in vitro. The tightest binder K91 neither bound the Rho GEF Vav1 nor the Arf GEF Cytohesin-2. In the presence of Rac1, the binding of K91 to Tiam1 was impaired indicating that the binding motif on Tiam1 overlaps with the GTPase binding site. K91 and K11 are the first reported inhibitory molecules targeting the GEF function of Tiam1. Due to their specificity over related GEF proteins they may represent promising tools for further elucidation of the biological functions of Tiam1. We anticipated that these aptamers will prove useful in validating the ambiguous roles of Tiam1 in cancer biology.

The N-terminal region of the guanine nucleotide exchange factor Vav1 plays a distinguished role in T cell receptor mediated calcium signaling (P1158)

Abstract Vav1 is a guanine nucleotide exchange factor specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All the three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor mediated signal transduction. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal region of Vav1 in calponin homology domain to be essential for its interaction with Calmodulin, that leads to TCR-induced calcium mobilization. Substitution of the region of Vav1 with the homologous region of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells activation and IL-2 production. This study highlights the importance of the N-terminal region of Vav1 for calmodulin binding, and that attributes irreplaceable role of Vav1 in T cell activation and signal transduction.

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

Correction: The hemopoietic Rho/Rac guanine nucleotide exchange factor Vav1 regulates <i>N</i>-formyl-methionyl-leucyl-phenylalanine-activated neutrophil functions

Dynamin 2 Potentiates Invasive Migration of Pancreatic Tumor Cells through Stabilization of the Rac1 GEF Vav1

The large GTPase Dynamin 2 (Dyn2) is markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. Here we demonstrate an unexpected mechanism of Dyn2 action in these contexts via direct binding to the Rac1 guanine nucleotide exchange factor (GEF) Vav1. Surprisingly, disruption of the Dyn2-Vav1 interaction targets Vav1 to the lysosome for degradation via an interaction with the cytoplasmic chaperone Hsc70, resulting in a dramatic reduction of Vav1 protein stability. Importantly, a specific mutation in Vav1 near its Dyn2-binding C-terminal Src homology 3 (SH3) domain prevents Hsc70 binding, resulting in a stabilization of Vav1 levels. Dyn2 binding regulates the interaction of Vav1 with Hsc70 to control the stability and subsequent activity of this oncogenic GEF. These findings elucidate how Dyn2 activates Rac1, lamellipod protrusion, and invasive cellular migration and provide insight into how this specific Vav is ectopically expressed in pancreatic tumors.

Novel Interaction between the Co-chaperone Cdc37 and Rho GTPase Exchange Factor Vav3 Promotes Androgen Receptor Activity and Prostate Cancer Growth*

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. Vav3, a guanine nucleotide exchange factor, is up-regulated following progression to castration resistance in preclinical models and is overexpressed in a significant number of human prostate cancers. Vav3 is a novel co-activator of the AR. We sought to identify Vav3 binding partners in an effort to understand the molecular mechanisms underlying Vav3 enhancement of AR activity and to identify new therapeutic targets. The cell division cycle 37 homolog (Cdc37), a protein kinase-specific co-chaperone for Hsp90, was identified as a Vav3 interacting protein by yeast two-hybrid screening. Vav3-Cdc37 interaction was confirmed by GST pulldown and, for native proteins, by co-immunoprecipitation experiments in prostate cancer cells. Cdc37 potentiated Vav3 co-activation of AR transcriptional activity and Vav3 enhancement of AR N-terminal-C-terminal interaction, which is essential for optimal receptor transcriptional activity. Cdc37 increased prostate cancer cell proliferation selectively in Vav3-expressing cells. Cdc37 did not affect Vav3 nucleotide exchange activity, Vav3 protein levels, or subcellular localization. Disruption of Vav3-Cdc37 interaction inhibited Vav3 enhancement of AR transcriptional activity and AR N-C interaction. Diminished Vav3-Cdc37 interaction also caused decreased prostate cancer cell proliferation selectively in Vav3-expressing cells. Taken together, we identified a novel Vav3 interacting protein that enhances Vav3 co-activation of AR and prostate cancer cell proliferation. Vav3-Cdc37 interaction may provide a new therapeutic target in prostate cancer.

The N-terminal 20-Amino Acid Region of Guanine Nucleotide Exchange Factor Vav1 Plays a Distinguished Role in T Cell Receptor-mediated Calcium Signaling

Vav1 is a guanine nucleotide exchange factor (GEF) specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor (TCR)-mediated signal transduction, whereas Vav2 and Vav3 function as GEFs that overlap with Vav1 on TCR-induced cytoskeleton reorganization. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal 20 amino acids of Vav1 in the calponin homology (CH) domain to be essential for its interaction with calmodulin (CaM) that leads to TCR-induced calcium mobilization. Substitution of the 1-20 amino acids of Vav1 with those of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells (NFAT) activation and IL-2 production. This study highlights the importance of the N-terminal 20 aa of Vav1 for CaM binding, and provides new insights into the distinguished and irreplaceable role of Vav1 in T cell activation and signal transduction.

Roles of SLP-76, ADAP and Pyk2 on chemokine-stimulated T cell adhesion mediated by the integrin alpha4-beta1

Event Abstract Back to Event Roles of SLP-76, ADAP and Pyk2 on chemokine-stimulated T cell adhesion mediated by the integrin alpha4-beta1 Soledad Isern De Val1, Ana Dios1 and Joaquín Teixidó1* 1 Centro de Investigaciones Biológicas, Spain Lymphocyte extravasation at sites of tissue injury or infection depends on chemokine-activated adhesion mediated by the alpha4-beta1 and alphaL-beta2 integrins. This activation must be rapid to deliver tight cell attachment to resist the blood shear stress. The chemokine-dependent integrin activation step in lymphocytes requires an inside-out signalling that impinges on the integrin beta subunit cytoplasmic domains, a key event for transmitting conformational changes to the extracellular regions that leads to increase in integrin affinity. Talin is a recipient of this inside-out signalling that promote their binding to the beta cytoplasmic domains, finally stimulating the transition to high-affinity alpha4-beta1 and alphaL-beta2 conformations. A key inside-out molecule that regulates the activation of alpha4-beta1 is the guanine-nucleotide exchange factor Vav1, which associates with talin forming a key complex that controls chemokine-dependent, alpha4-beta1-mediated T cell adhesion. SLP-76 and Pyk2 are Vav1-binding proteins with the potential of influencing alpha4-beta1-mediated T cell adhesion in response to chemokine stimulation. We are investigating the role of SLP-76, its binding partner ADAP, and Pyk2, as possible regulators of this adhesion by inside-out mechanisms arising from chemokine stimulation. Keywords: T cells, Chemokines, leukocyte trafficking, Integrins, chemokine signaling Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Isern De Val S, Dios A and Teixidó J (2013). Roles of SLP-76, ADAP and Pyk2 on chemokine-stimulated T cell adhesion mediated by the integrin alpha4-beta1. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00944 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Joaquín Teixidó, Centro de Investigaciones Biológicas, Madrid, Spain, joaquint@cib.csic.es Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Soledad Isern De Val Ana Dios Joaquín Teixidó Google Soledad Isern De Val Ana Dios Joaquín Teixidó Google Scholar Soledad Isern De Val Ana Dios Joaquín Teixidó PubMed Soledad Isern De Val Ana Dios Joaquín Teixidó Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Molecular differences between normal breast epithelia, pure DCIS, and DCIS adjacent to invasion.

50 Background: With the use of screening mammography, the diagnosis of breast ductal carcinoma in situ (DCIS) is increasing worldwide. Currently there are no applied molecular markers to aid in predicting risk of DCIS progression to invasive breast cancer (IBC). Studies predicted that the transition from DCIS to invasive disease was associated with quantitative rather than qualitative differences in gene and protein expression. Methods: We developed monochrome imaging-based method to measure protein expression as a continuous variable in fixed tissue (Furstenau et al. BREA 2010). The insulin-like growth factor I receptor (IGF-IR), Ras oncogene –like protein 1 (Rap1), and a Rho GTPase guanine nucleotide exchange factor Vav2 implicated in the regulation of invasion in preclinical models have been chosen for protein analysis in tissue. In pilot study, we performed quantitative protein profiling on 90 samples of the breast: 17 histologically normal tissues, 16 benign lesions; 15 CIS, and 42 IBC. In addition, we analyzed 24 DCIS: 12 pure and 12 associated with IBC. Results: The expression of the IGF-IR and Rap1 was increased in pure CIS; significantly (P ≤ 0.001) increased in CIS adjacent to IBC as well as in IBC compared with non-cancerous tissue. In the majority of pure DCIS the levels of Vav2 were similar to that in normal epithelia; however, the Vav2 levels were significantly (P&lt;0.01) higher in DCIS adjacent to invasion and in IBC. Moreover, IGF-IR, Rap1 and Vav2 significantly discriminated invasive from non-invasive tissues, with receiver operating characteristic (ROC) area under curve, AUC=0.8. Interestingly, younger women (&lt; 50 years of age) were more likely to develop IBC with increased expression of the Vav2 protein (OR= 2.05; 95% CI 1.08-3.86). No significant correlation was found between IGF-IR, Rap1 expression levels and patient’s age. Conclusions: We conclude that our assessment of protein expression in breast tissue revealed previously unidentified quantitative differences in the IGF-IR, Rap1, and Vav2 protein expression in breast cancer progression series. These findings open door for studies on molecular-based prediction of individualized risk for developing invasion in early diagnosed DCIS.