The P2Y2 nucleotide receptor interacts with alphav integrins to activate Go and induce cell migration

Extracellular ATP and UTP induce chemotaxis, or directed cell migration, by stimulating the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R). Previously, we found that an arginine-glycine-aspartic acid (RGD) integrin binding domain in the P2Y(2)R enables this receptor to interact selectively with alpha(v)beta(3) and alpha(V)beta(5) integrins, an interaction that is prevented by mutation of the RGD sequence to arginine-glycine-glutamic acid (RGE) (Erb, L., Liu, J., Ockerhausen, J., Kong, Q., Garrad, R. C., Griffin, K., Neal, C., Krugh, B., Santiago-Perez, L. I., Gonzalez, F. A., Gresham, H. D., Turner, J. T., and Weisman, G. A. (2001) J. Cell Biol. 153, 491-501). This RGD domain also was found to be necessary for coupling the P2Y(2)R to G(o)- but not G(q)-mediated intracellular calcium mobilization, leading us to investigate the role of P2Y(2)R interaction with integrins in nucleotide-induced chemotaxis. Here we show that mutation of the RGD sequence to RGE in the human P2Y(2)R expressed in 1321N1 astrocytoma cells completely prevented UTP-induced chemotaxis as well as activation of G(o), Rac, and Vav2, a guanine nucleotide exchange factor for Rac. UTP also increased expression of vitronectin, an extracellular matrix protein that is a ligand for alpha(v)beta(3)/beta(5) integrins, in cells expressing the wild-type but not the RGE mutant P2Y(2)R. P2Y(2)R-mediated chemotaxis, Rac and Vav2 activation, and vitronectin up-regulation were inhibited by pretreatment of the cells with anti-alpha(v)beta(5) integrin antibodies, alpha(v) integrin antisense oligonucleotides, or the G(i/o) inhibitor, pertussis toxin. Thus, the RGD-dependent interaction between the P2Y(2)R and alpha(v) integrins is necessary for the P2Y(2)R to activate G(o) and to initiate G(o)-mediated signaling events leading to chemotaxis.     

Vav GEFs are required for beta2 integrin-dependent functions of neutrophils

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple beta2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial beta2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. beta2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor-induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling beta2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis.     

Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. Beta1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on beta1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and beta1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.     

Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins

The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.     

The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1)

UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.     

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Distinct role of phosphatidylinositol 3-kinase and Rho family GTPases in Vav3-induced cell transformation, cell motility, and morphological changes

Vav3 is a member of the Vav family of guanine nucleotide exchange factors (GEFs) for the Rho family GTPases. Deleting the N-terminal calponin homology (CH) domain to generate Vav3-(5-10) or deleting both the CH and the acidic domain to generate Vav3-(6-10) results in activating the transforming potential of Vav3. Expression of either the full-length Vav3 or its truncation mutants led to activation of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), and Stat3. We investigated the requirement of these signaling molecules for Vav3-induced focus formation and found that PI3K and its downstream signaling molecules, Akt and p70 S6 kinase, are required, albeit to varying degrees. Inhibition of PI3K had a more dramatic effect than inhibition of MAPK on Vav3-(6-10)-induced focus formation. Activated PI3K enhanced the focus-forming activity of Vav3-(6-10). Wild type FAK but not Y397F mutant FAK enhanced Vav3-(6-10)-induced focus formation. Dominant negative (dn) mutant of Stat3 resulted in a 60% inhibition of the focus-forming activity of Vav3-(6-10). Moreover, Rac1, RhoA, and to a lesser extent, Cdc42, are important for Vav3-(6-10)-induced focus formation. Constitutively activated (ca) Rac synergizes with Vav3-(6-10) in focus formation. This synergy requires signaling via Rho-associated kinase (ROK) and p21-activated kinase (PAK), downstream effectors of Rac. Consistently, a ca PAK mutant enhanced, whereas a dn PAK mutant inhibited the focus-forming ability of Vav3-(6-10). Despite having potent focus-forming ability, Vav3-(6-10) has very weak colony-forming ability. This colony-forming ability of Vav3-(6-10) can be enhanced dramatically by co-expressing an activated PI3K and to some extent by co-expressing an activated PAK mutant or c-Myc. Interestingly, inhibition of PI3K and MAPK had no effect on the ability of either wild type or Vav3-(6-10) to induce cytoskeletal changes including formation of lamellipodia and filopodia in NIH 3T3 cells. Over expression of Vav3 or Vav3-(6-10) resulted in an enhancement of cell motility. This enhancement was dependent on PI3K, Rac1, and Cdc42 but not on Rho. Overall, our results show that signaling pathways of PI3K, MAPK, and Rho family GTPases are differentially required for Vav3-induced focus formation, colony formation, morphological changes, and cell motility.     

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

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

An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand

Background

Vav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity.

Results

We found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation.

Conclusion

Our results indicate that increased migration of active Vav1 expressing cells is dependent on Vav1 GEF activity and secretion of an EGF receptor ligand. In addition, activation of ERK downstream of Vav1 is dependent on autocrine EGF receptor stimulation while active Vav1 can stimulate Rac1 and PAK activation independent of ligand binding to the EGF receptor. Thus, stimulation of migration by activated Vav1 involves both EGF receptor-dependent and independent activities induced through the Rho GEF domain of Vav1.     

Vav1 and Rac control chemokine-promoted T lymphocyte adhesion mediated by the integrin alpha4beta1

The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.     

Distinct Roles for Rho Versus Rac/Cdc42 GTPases Downstream of Vav2 in Regulating Mammary Epithelial Acinar Architecture

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.     

Integrin-dependent tyrosine phosphorylation and growth regulation by Vav

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

Microtubule-dependent regulation of Rho GTPases during internalisation of Yersinia pseudotuberculosis

Internalisation of the human pathogen Yersinia pseudotuberculosis via interaction of bacterial invasin with host beta1 integrins depends on the actin cytoskeleton and involves Src family kinases, focal adhesion kinase, p130Crk-associated substrate, proline-rich tyrosine kinase 2, Rac, Arp 2/3 complex and WASP family members. We show here that Rho GTPases are regulated by the microtubule system during bacterial uptake. Interfering with microtubule organisation using nocodazole or paclitaxel suppressed uptake by HeLa cells. The nocodazole effect on microtubule depolymerisation was partially inhibited through overexpression of Rac, Cdc42, RhoG or RhoA and completely prevented by expression of Vav2. This suggests that microtubules influence Rho GTPases during invasin-mediated phagocytosis and in the absence of functional microtubules Vav2 can mimic their effect on one, or more, of the Rho family GTPases. Lastly, overexpression of p50 dynamitin partially inhibited bacterial uptake and this effect was also blocked by co-expression of Vav2, thus further implicating this guanine nucleotide exchange factor in activating Rho GTPases for internalisation during loss of microtubule function.     

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.     

Rac and phosphatidylinositol 3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3 mast cells

FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.     

Essential role of Vav family guanine nucleotide exchange factors in EphA receptor-mediated angiogenesis

Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2-/- Vav3-/- mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.     

The molecular adapter SLP-76 relays signals from platelet integrin alphaIIbbeta3 to the actin cytoskeleton

Platelet adhesion to fibrinogen through integrin alpha(IIb)beta(3) triggers actin rearrangements and cell spreading. Mice deficient in the SLP-76 adapter molecule bleed excessively, and their platelets spread poorly on fibrinogen. Here we used human platelets and a Chinese hamster ovary (CHO) cell expression system to better define the role of SLP-76 in alpha(IIb)beta(3) signaling. CHO cell adhesion to fibrinogen required alpha(IIb)beta(3) and stimulated tyrosine phosphorylation of SLP-76. SLP-76 phosphorylation required coexpression of Syk tyrosine kinase and stimulated association of SLP-76 with the adapter, Nck, and with the Rac exchange factor, Vav1. SLP-76 expression increased lamellipodia formation induced by Syk and Vav1 in adherent CHO cells (p < 0.001). Although lamellipodia formation requires Rac, SLP-76 functioned downstream of Rac by potentiating adhesion-dependent activation of PAK kinase (p < 0.001), a Rac effector that associates with Nck. In platelets, adhesion to fibrinogen stimulated the association of SLP-76 with the SLAP-130 adapter and with VASP, a SLAP-130 binding partner implicated in actin reorganization. Furthermore, SLAP-130 colocalized with VASP at the periphery of spread platelets. Thus, SLP-76 functions to relay signals from alpha(IIb)beta(3) to effectors of cytoskeletal reorganization. Therefore, deficient recruitment of specific adapters and effectors to sites of adhesion may explain the integrin phenotype of SLP-76(-/-) platelets.     

Vav2 as a Rac-GDP/GTP exchange factor responsible for the nectin-induced, c-Src- and Cdc42-mediated activation of Rac

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that form homo- and hetero-trans-dimers (trans-interactions). Nectins first form cell-cell contact and then recruit cadherins to the nectin-based cell-cell contact sites to form adherens junctions cooperatively with cadherins. In addition, the trans-interactions of nectins induce the activation of Cdc42 and Rac small G proteins, which enhances the formation of adherens junctions by forming filopodia and lamellipodia, respectively. The trans-interactions of nectins first recruit and activate c-Src at the nectin-based cell-cell contact sites. c-Src then phosphorylates and activates FRG, a Cdc42-GDP/GTP exchange factor (GEF) for Cdc42. The activation of both c-Src and Cdc42 by FRG is necessary for the activation of Rac, but the Rac-GEF responsible for this activation of Rac remains unknown. We showed here that the nectin-induced activation of Rac was inhibited by a dominant negative mutant of Vav2, a Rac-GEF. Nectins recruited and tyrosine-phosphorylated Vav2 through c-Src at the nectin-based cell-cell contact sites, whereas Cdc42 was not necessary for the nectin-induced recruitment of Vav2 or the nectin-induced, c-Src-mediated tyrosine phosphorylation of Vav2. Cdc42 activated through c-Src then enhanced the GEF activity of tyrosine-phosphorylated Vav2 on Rac1. These results indicate that Vav2 is a GEF responsible for the nectin-induced, c-Src-, and Cdc42-mediated activation of Rac.     

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

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