Article |
Address correspondence to Joan S. Brugge, Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: (617) 432-3974. Fax: (617) 432-3969. email: Joan_Brugge{at}hms.harvard.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: integrins; cell adhesion; Rho GTPases; chemotaxis; phagocytosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Vav family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases (Rho, RhoG, Rac, and Cdc42) by catalyzing the exchange of GDP for GTP. The three mammalian Vav proteins (Vav1, -2, -3) differ in their tissue distribution: Vav1 is expressed predominantly in hematopoietic cells, whereas Vav2 and Vav3 are more broadly expressed. Each Vav protein shares the same set of structural domains (calponin homology, Dbl homology, pleckstrin homology, cysteine-rich, and Src homology 2 and 3 [SH2, SH3] domains) and display overall 5060% sequence homology (Bustelo, 2000). Studies of lymphocytes from mice lacking vav1, vav2, vav3, or combinations of these genes indicate that whereas certain functions of Vav proteins are redundant, individual Vav proteins also serve specialized functions in lymphocytes (Bustelo, 2000; Fujikawa et al., 2003). It has not been established which targets of individual Vav proteins mediate the specific functions in lymphocytes nor is it known which specific Rho family GTPases serve as direct substrates for each Vav family member in vivo.
The function of Vav proteins has been best characterized downstream of immune response receptors (IRRs). Targeted disruption of Vav1 results in severe impairment of T lymphocyte function in response to antigen receptor stimulation (Bustelo, 2000) and impaired FcR-induced degranulation and cytokine production in mast cells (Manetz et al., 2001). In particular, T cell receptor (TCR)-induced IL-2 production, proliferation, and calcium mobilization are impaired in vav1/ T lymphocytes (Bustelo, 2000). Interestingly, actin cytoskeletal rearrangements such as TCR capping and actin patch formation are defective in vav1/ lymphocytes (Fischer et al., 1998; Holsinger et al., 1998), suggesting Vav1 is specifically required to couple the TCR to the actin cytoskeleton. A double deficiency in Vav1/2 or Vav1/3 causes distinct phenotypic effects in lymphocytes, with Vav2 loss causing more severe effects in B cells and Vav3 loss further enhancing T cell defects (Doody et al., 2001; Tedford et al., 2001; Fujikawa et al., 2003). Deletion of all Vav family members prevents production of all mature B and T cells (Fujikawa et al., 2003). Therefore, Vav proteins serve critical functions downstream of IRR signaling in lymphocytes. However, the role of Vav proteins downstream of receptors other than IRRs has not been extensively examined.
The exchange activity of Vav proteins is regulated by phosphorylation of regulatory tyrosine residues in the amino terminus (Bustelo, 2000). In addition to IRRs, stimulation of receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors (GPCRs) induces tyrosine phosphorylation of Vav1, the best characterized family member (Bustelo, 2000). Thus, Vav proteins can couple to diverse signaling receptors and, due to their exchange activity and adaptor domains, are ideally suited to transduce signals to Rho GTPases and the actin cytoskeleton. Previous studies have implicated a kinase cascade involving the tyrosine kinases Src and Syk in IIbß3 integrin-induced Vav1 phosphorylation and cytoskeletal rearrangements (Miranti et al., 1998; Obergfell et al., 2002). Inducible Vav1 phosphorylation in response to ß1 and ß2 integrin ligation has also been reported in myeloid cells (Gotoh et al., 1997), T cells (Yron et al., 1999), and platelets (Cichowski et al., 1996). In addition, overexpression studies have implicated Vav proteins in integrin-dependent cell spreading (Marignani and Carpenter, 2001; del Pozo et al., 2003). Thus, Vav proteins can be activated by integrins in multiple hematopoietic cell types and may regulate intracellular signaling pathways important for proper integrin function.
In this paper, we have examined the role of Vav proteins in ß2 integrin- and GPCR-dependent biological functions of neutrophils using mice deficient in both Vav1 and Vav3. We show that Vav proteins are required for multiple ß2 integrin-induced functions, including sustained adhesion, spreading, and complement-mediated phagocytosis, but are not essential for initial agonist-induced inside-out activation. ß2 integrin-induced activation of Cdc42, Rac1, and RhoA is defective in the absence of Vav1 and Vav3. In addition, Pyk2 and paxillin phosphorylation, Akt activation, and phosphorylation of the Rho family GTPase effectors PAK and myosin light chain (MLC) are impaired in the absence of Vav proteins. The requirement for Vav is specific for integrin-dependent events, as GPCR-induced signaling pathways and chemotaxis are largely unaffected in Vav-deficient neutrophils. Thus, our work has defined a novel role for Vav proteins as critical regulators of integrin function in neutrophils.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
To investigate inside-out activation, we examined the distribution of ß2 integrins in suspended neutrophils stimulated with fMLP. First, Mß2 was constitutively clustered in unstimulated WT neutrophils and was not notably altered by fMLP stimulation. In contrast,
Lß2 was uniformly distributed in unstimulated WT neutrophils; however, we were able to detect clustering in response to fMLP in only a very small percentage of cells (unpublished data). Therefore, chemoattractant-induced, ligand-independent ß2 integrin clustering does not seem to significantly contribute to neutrophil adhesion under the conditions examined in this work.
Neutrophils rolling on selectins in vitro and in vivo rapidly arrest in response to proinflammatory stimuli by binding to ICAMs such as ICAM-1 via inside-out activation of ß2 integrins. To address whether the phenotype of Vav1/3ko neutrophils was attributable to reduced initial adhesion due to defective inside-out ß2 integrin activation, we compared the ability of WT and Vav1/3ko neutrophils to arrest on coimmobilized P-selectin/ICAM-1 in response to LTB4 under low shear (1 dyne/cm2) in vitro. LTB4-mediated neutrophil arrest on ICAM-1 requires ß2 integrin function, as ß2/ neutrophils did not arrest in response to this stimulus (unpublished data). Vav1/3ko neutrophils rapidly arrested on ICAM-1 in response to LTB4 indistinguishably from WT neutrophils, suggesting that initial inside-out integrin activation was not defective (Fig. 3 A). However, visual inspection of the video data indicated that the arrested Vav1/3ko neutrophils frequently lifted from the surface if the flow rate was increased (unpublished data). These results suggest that Vav proteins are not essential for initial inside-out integrin activation induced by agonists but are required for sustained adhesion of neutrophils in vitro. To rule out any intrinsic defects in ß2 integrin ligand binding activity, neutrophil adhesion was induced by manganese, which binds to the extracellular metal ion-dependent adhesion site on integrins and induces the active, high affinity conformation in the absence of additional stimuli. Comparable levels of WT and Vav1/3ko neutrophils adhered to immobilized fibrinogen, C3bi and ICAM-1 in response to manganese treatment (Fig. 3 B), indicating that Vav deficiency does not affect the intrinsic ability of ß2 integrins to bind ligand.
|
Defective complement-mediated phagocytosis in Vav1/3ko neutrophils
Neutrophils are professional phagocytes that efficiently bind and clear foreign particles. Serum opsonization leads to efficient fixation of complement C3bi on the surface of bacteria and renders them susceptible to complement-mediated phagocytosis via Mß2. To examine whether Vav is required for this process, we compared the phagocytic activity of WT and Vav-deficient neutrophils using serum-opsonized fluorescent E. coli. Although there was no detectable defect in Vav1ko or Vav3ko neutrophils (not depicted), phagocytosis was severely impaired in Vav1/3ko neutrophils (Fig. 4, A and B). PMA pretreatment was able to only partially rescue this defect in that Vav1/3ko cells contained significantly fewer fluorescent bacteria per neutrophil (Fig. 4 A). The cells were imaged in the presence of trypan blue to quench any extracellular, unphagocytosed bacteria, as demonstrated by the absence of signal in cells incubated with unopsonized E. coli (Fig. 4 A). To determine whether the impaired phagocytic activity of Vav1/3ko neutrophils was due to reduced particle binding, neutrophils were incubated with serum-opsonized fluorescent E. coli in the presence of the actin polymerization inhibitor, Latrunculin B, which blocks particle ingestion (Fig. 4 A) but not binding. A similar percentage of WT and Vav1/3ko neutrophils contained bound E. coli (76% WT vs. 81% Vav1/3ko) and the amount of bound E. coli per cell was also comparable (Fig. 4 C). Thus, the defect in complement-mediated phagocytosis of Vav1/3ko neutrophils is not attributable to decreased particle binding.
|
|
Vav proteins are not required for chemotaxis
One of the first steps of directional motility is the formation of a leading edge which is stabilized by integrin attachments to the substratum. Furthermore, the creation and turnover of these integrin-based focal adhesions is a tightly coordinated process during cell migration (Webb et al., 2002). Given the defects in spreading and firm adhesion in Vav1/3ko neutrophils, we examined fMLP-dependent chemotaxis of these cells in vitro using transwell filters coated with integrin ligands. Surprisingly, we observed no defects in chemotaxis of Vav1/3ko neutrophils over a range of chemoattractant concentrations on either C3bi- or fibronectin-coated transwell filters (Fig. 6 A). To specifically examine whether Vav1/3ko neutrophils exhibited altered cell polarization, speed, or distance of migration, we monitored chemotaxis by video microscopy using Zigmond chambers (Zigmond, 1988) coated with C3bi. Similar to the transwell assays, Vav1/3ko neutrophils migrated efficiently in the direction of the gradient (Fig. 6 B; Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200404166/DC1). Furthermore, analysis of the migration tracks of these cells revealed that similar percentages of both WT and Vav1/3ko neutrophils polarized and migrated up the chemotactic gradient of fMLP (Fig. 6 C, 94% vs. 92%). Notably, Vav1/3ko neutrophils on average migrated slightly faster than WT cells and traversed a longer distance (Fig. 6 D). Similar results were obtained with LTB4 (not depicted). Thus, despite significant ß2 integrin adhesion defects, Vav1/3ko neutrophils are able to efficiently polarize and migrate in response to a chemotactic gradient.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although distinct functions have been attributed to different Vav family members in certain cell types (e.g., T and B lymphocytes), our results clearly suggest that Vav1 and Vav3 play redundant roles in regulating neutrophil functions downstream of integrins and that the contribution of Vav2 appears to be minimal. Vav2 expression represents only a small fraction of total Vav protein, its expression does not increase in Vav1/3ko neutrophils (Fig. 1 C), and neutrophils deficient in all three vav genes do not exhibit a more severe phenotype than those deficient in both vav1 and vav3, based on adhesion, spreading, and chemotaxis (unpublished data).
Vav regulation of integrin-mediated adhesion
One critical aspect of leukocyte adhesion in response to inflammatory stimuli is the induction of integrin binding activity through inside-out signaling events that convert integrins from a low to a high affinity/avidity ligand-binding state. Our evidence that Vav1/3ko neutrophils are able to undergo chemoattractant-induced arrest under flow in vitro and in circulating blood in vivo suggests that Vav is not required for initial events associated with inside-out integrin activation. However, given the complexity of this response (Ley, 2003), we cannot rule out that Vav contributes to full activation of integrins, thus affecting the strength and maintenance of adhesion.
Impaired integrin-mediated adhesion has also been reported in Vav1-deficient T lymphocytes (Krawczyk et al., 2002; Ardouin et al., 2003). This conclusion was based on defects in static adhesion and in Lß2 clustering on the cell surface in response to TCR stimulation. Because Vav1 plays a critical role in transducing early TCR signals required for most TCR-induced biological activities, including actin polymerization and activation of many downstream effectors of in vitro (Bustelo, 2000), the specific effects of Vav deficiency on adhesion and integrin activation per se are difficult to distinguish from those required to activate early receptor-proximal events. In neutrophils, Vav is not required for fMLP or LTB4-induced chemotaxis, initial adhesion, or activation of Rac, Cdc42, Erk, or Akt. Therefore, loss of Vav does not have broad effects on early receptor-induced events in chemoattractant-stimulated neutrophils as in TCR-stimulated T cells, and our data supports the possibility that Vav specifically regulates integrin-mediated adhesion events.
Vav requirement for integrin-dependent Rho GTPase activation
Rho GTPases are key mediators of actin cytoskeletal rearrangements associated with adhesion and spreading as they nucleate events involved in formation of focal adhesions, lamellipodia, and filopodia (Ridley, 1999). However, the mechanisms responsible for integrin-mediated Rho GTPase activation are poorly defined. Here, we show that ß2 integrin-dependent activation of Rac1, Cdc42, and RhoA is defective in Vav1/3ko neutrophils, implicating Vav proteins as critical Rho family GEFs downstream of integrins. The loss of integrin-induced activation of RhoA, Rac1, and Cdc42 in Vav1/3ko neutrophils could be a consequence of direct effects of Vav-mediated GTP exchange on multiple Rho family members or from indirect Vav regulation of multiple Rho GTPases, for example, due to a cascade of Rho GTPase activation or to generalized effects of Vav deficiency on adhesion and spreading. Vav family proteins have been reported to serve as GEFs for RhoA, RhoG, Rac, and Cdc42; however, the specificity of individual Vav proteins has been difficult to establish for many reasons: lack of consistent results from in vitro experiments in different laboratories, use of distinct domains of Vav for in vitro exchange assays, and complications of interpreting experiments in cultured cells in which distinct Vav proteins have been overexpressed (Bustelo, 2000). Thus, it is not feasible to distinguish, based on previous studies, whether the phenotypic effects observed here are due to direct or indirect effects of Vav on these GTPases. Studies from Olson and coworkers demonstrated that a mutant, constitutively activated variant of Vav1 can independently activate Rho, Rac, and Cdc42 in Swiss 3T3 cells (Olson et al., 1996); however, one cannot extrapolate these findings to the endogenous Rho GTPases activated by integrins in neutrophils. There are also precedents for a hierarchical cascade linking Rho GTPases (Ridley and Hall, 1992; Nobes and Hall, 1995; Price et al., 1998), yet coupling between these proteins has not been examined in neutrophils after attachment to integrin ligands. Thus, further studies will be required to establish the basis for loss of activation of RhoA, Rac1, and Cdc42 in Vav1/3ko neutrophils.
Regardless of whether the loss of RhoA, Rac1, and Cdc42 activation is a direct or indirect result of Vav deficiency, impaired Rho GTPase activation may be the major contributing factor in the phenotypes exhibited in Vav1/3ko neutrophils. As previously discussed, Vav1/3ko neutrophils are able to make initial contacts with immobilized integrin ligands, yet these neutrophils are unable to spread. Although the precise downstream intracellular events that coordinate cell spreading are poorly understood, a role for Rac and Cdc42 has been well documented in hematopoietic and nonhematopoietic cells (Allen et al., 1997; Clark et al., 1998; D'Souza-Schorey et al., 1998). In addition, Rac1- and Rac2-deficient neutrophils exhibit defects in integrin-mediated cell spreading (Roberts et al., 1999; Glogauer et al., 2003). In contrast, Rho has been implicated in events required for integrin-mediated firm adhesion and aggregation in lymphocytes and platelets (Morii et al., 1992; Laudanna et al., 1996; Woodside et al., 1998), but not for cell spreading (Ren et al., 1999). Interestingly, Rho, but not Rac or Cdc42, has been shown to be required for complement-mediated phagocytosis in macrophages (Caron and Hall, 1998), suggesting that loss of Rho activation contributes to the defect in phagocytosis in Vav1/3ko neutrophils. Thus, failure to activate RhoA, Rac1, and Cdc42 may differentially contribute to the loss of distinct integrin-mediated events in neutrophils.
Phenotypic similarities with Syk- and SFK-deficient neutrophils
In both platelets (Obergfell et al., 2002) and neutrophils (Mocsai et al., 2002), Syk is required for adhesion-induced Vav phosphorylation, implicating Syk as an upstream regulator of Vav. Not surprisingly, Syk- and Vav-deficient neutrophils display multiple phenotypic similarities, including impaired spreading on multiple integrin ligands and reduced Pyk2 activation (Mocsai et al., 2002). Moreover, despite impaired integrin function, SFK-, Syk-, and Vav-deficient neutrophils do not exhibit defects in chemotaxis. The requirement for Syk in neutrophils is limited to integrin-dependent functions, as GPCR-mediated responses are unaffected in syk/ (Mocsai et al., 2003). This evidence, coupled with the data in this paper, suggests that Vav proteins are critical downstream mediators of integrin-Syk signaling in neutrophils.
Vav1/3ko neutrophil migration in vitro and in vivo
Neutrophils polarize in response to shallow chemotactic gradients by extending a leading edge lamellipodium that is stabilized via integrin-mediated adhesion to the substratum. The evidence that ß2 integrins are required for leukocyte migration in multiple mouse inflammatory model systems (Mizgerd et al., 1997; Walzog et al., 1999) demonstrates the importance of these receptors for optimal migration in vivo. Surprisingly, despite significant adhesion defects, Vav1/3ko neutrophils efficiently polarize and migrate on C3bi-coated glass in response to a chemotactic gradient of either fMLP or LTB4. In comparison to WT, Vav1/3ko neutrophils on average migrate slightly faster, most likely due to their decreased adhesiveness. These data, coupled with the phenotypes of syk/ and sfk/ neutrophils, indicate that optimal integrin function is not required for the amoeboid movement of neutrophils in chemotactic models in vitro. It is possible that the minimal adhesion that occurs in Vav1/3ko neutrophils is sufficient to mediate this form of chemotaxis in vitro. However, in vivo leukocytes must survey the environment by rolling on venular endothelium and arrest at sites of tissue inflammation firmly enough to withstand the constant shear force from blood flow. Intravital microscopy of the cremaster muscle indicates that Vav1/3ko leukocytes are not as stably attached as WT to the venular endothelium in response to fMLP. Despite this difference, we have observed similar levels of neutrophil extravasation to the peritoneum in WT and Vav1/3ko mice treated with thioglycollate in preliminary experiments (unpublished data). Similar results have been reported for syk/ and sfk/ mice (Mocsai et al., 2002); however Rac1- and Rac2-deficient mice display defects in chemotaxis in vitro and inflammatory recruitment in vivo (Roberts et al., 1999; Li et al., 2002; Glogauer et al., 2003). Although it is difficult to resolve this discrepancy, these studies suggest that minimal integrin activity may suffice for both in vivo and in vitro neutrophil migration. Alternatively, thioglycollate, an extremely potent inflammatory stimulus, may bypass the need for Syk and Vav signaling in neutrophils, analogous to phorbol ester stimulation in vitro. Therefore, alternative in vivo models are required to resolve this discrepancy.
Vav proteins in GPCR signaling
Binding of GPCR agonists to their cognate receptors initiates signaling cascades leading to the activation of Rho GTPases, presumably via activation of a Rho exchange factor. Specifically, Rac2 activity is required for multiple GPCR-induced events in neutrophils, including chemotaxis to fMLP and LTB4 (Roberts et al., 1999). The fact that Vav1/3ko neutrophils undergo normal chemotaxis in response to these agonists suggests that Vav1 and Vav3 are not critical mediators of GPCR signaling. In addition, GPCR-induced calcium flux, integrin up-regulation, and initial inside-out integrin activation are unaffected in Vav1/3ko neutrophils. fMLP- and LTB4-induced activation of Rho GTPases, Akt, and Erk are also unaffected, and preliminary experiments with Vav triple knockout neutrophils suggest Vav proteins are completely dispensable for fMLP- and LTB4-induced chemotaxis (unpublished data). Thus, Vav proteins are not critical mediators of many GPCR-induced signaling events in mouse neutrophils, raising the possibility that another GEF, potentially the Gß-dependent P-Rex (Welch et al., 2002), regulates Rac activation downstream of GPCRs.
Our results have defined a novel requirement for Vav GEFs as critical mediators of ß2 integrin-dependent Rho GTPase activation in neutrophils, in addition to their well-documented role downstream of IRRs. The regulation of neutrophil adhesion and phagocytosis is an important aspect of innate immunity. Thus, Vav proteins may be attractive therapeutic targets for inflammatory diseases.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of bone marrow neutrophils
Neutrophils were isolated by Percoll density centrifugation as described previously (Mocsai et al., 2003). For a detailed protocol see online supplemental material. Equal cell equivalents were analyzed for Vav expression with rabbit anti-Vav1 (C-14; Santa Cruz Biotechnology, Inc.), anti-Vav2 (2203), and anti-Vav3 (2206; Obergfell et al., 2002). Antiserum 2203 to Vav2 was produced by rabbit immunization (Covance) with a bacterially expressed GST-Vav2 fusion protein containing human Vav2 amino acids 573878. It does not cross react with endogenous or recombinant Vav1 or Vav3 by Western blotting.
Neutrophil adhesion assays
All reagents were purchased from Sigma-Aldrich, unless otherwise indicated. For C3bi coating, 10 µg/ml mouse IgM (BD Biosciences) was absorbed onto 96-well plates (Immulon) overnight at 4°C, washed with PBS (Cellgro) and coated with 3350% mouse serum. 10 µg/ml mICAM-1:Fc (R&D Systems) was bound to protein Acoated wells (Pierce Chemical Co.) according to the manufacturer's instructions. 20 µg/ml polyRGD was coated overnight at 4°C. Neutrophils in adhesion assay media (AAM) [HBSS2+/20 mM Hepes, pH 7.4, 0.1% low endotoxin HSA] were stimulated with 10 µM fMLP, 100 nM LTB4, 10 ng/ml mTNF (R&D Systems), or 100 ng/ml PMA for 30 min at 37°C, washed 2x with AAM, and adherent neutrophils quantified by LDH content using the Cytotox Kit (Promega). For flow assays, the flow apparatus used has been described previously (Luscinskas et al., 1994) with the modifications described in online supplemental material. Cells were infused onto co-immobilized P-selectin/ICAM-1 followed by 100 nM LTB4 and recorded by video microscopy. Arrested cells were defined as those that remained stationary for 6 s and the percentage was calculated by dividing the number of arrested cells by the number of interacting cells (rolling and adherent). Intravital experiments were performed in accordance with protocols approved by the University of Virginia Health Science Center institutional committee for animal use and as described previously (Ley et al., 1995). For a detailed protocol see online supplemental material.
Complement-mediated phagocytosis assays
FITC-E. coli (Molecular Probes) were opsonized with mouse serum for 30 min at 37°C. Neutrophils were treated with 100 ng/ml PMA or 500 nM Latrunculin B (Calbiochem) for 15 min and incubated with E. coli for 30 min. Extracellular fluorescence was quenched with 0.2% trypan blue and representative images were captured using a microscope (model TE300; Nikon) and 40x objective. For quantification, cells were fixed with 3.7% formalin and manually scored by fluorescence microscopy. Cells containing at least one bacterium were scored positive and >200 cells were counted. For binding activity, cells were treated with Latrunculin B and incubated with E. coli for 30 min at 37°C, washed with PBS, and fixed. Cells containing bound E. coli were scored by fluorescence microscopy. Representative images were captured with an ORCA1 CCD camera (Hamamatsu) and 60x objective and overlaid with Metamorph (Universal Imaging Corp.).
Chemotaxis assays
Transwell filters were coated with C3bi or with fibronectin. Assays were performed as described previously (Mocsai et al., 2002), except migrated cells were scored by hemocytometer. For detailed information, see online supplemental material. For video microscopy, cells were plated on C3bi-coated coverslips and mounted onto Zigmond slides (Neuroprobe). The gradient was established with 10 µM fMLP for 10 min. Images were captured at 15-s intervals for >10 min with a 20x DIC objective on a microscope (model TE300; Nikon) modified with a heated stage. Migration paths, velocity, and distance were calculated with Metamorph. 50 tracks/genotype were analyzed.
Biochemistry experiments
DFP-washed cells were primed with TNF and plated on C3bi-coated dishes at 37°C. Unattached cells were removed by aspiration. Cells were lysed and lysates normalized by LDH content using the Cytotox kit and analyzed by immunoblotting against: phosphotyrosine (4G10; provided by T. Roberts, Dana Farber Cancer Institute, Boston, MA); pY881 Pyk2 and pY118 paxillin (Biosource International); pY416 Src, pS473 Akt, and pS19 MLC2 (Cell Signaling); PAK
(Santa Cruz); Pyk2 and paxillin (Transduction Laboratories); and pS198/S203 PAK
(a gift from M. Greenberg, Children's Hospital, Boston, MA). For Vav IPs, equal amounts of lysate were incubated with Vav1 (C-14), Vav2 (2203), and Vav3 (2206) antibodies for 2-3 h. For GTPase pull-down assays, neutrophils were plated on C3bi and PBD assays performed as described previously (Benard and Bokoch, 2002). Rho assays were performed as described previously (Ren et al., 1999) except cells were lysed in PBD lysis buffer. Pull-downs were blotted with Rac1 and Cdc42 (Transduction Laboratories) or RhoA (Santa Cruz Biotechnology, Inc.) antibodies. Rac2 antibody was a gift from G. Bokoch (Scripps Research Institute, La Jolla, CA). For fMLP stimulation, suspended neutrophils were treated with 10 µM fMLP followed by immediate lysis with 2x lysis buffer.
Online supplemental material
The accompanying videos to the still clips in Fig. 6 show wild-type (Video 1) and Vav1/3ko (Video 2) neutrophils migrating towards a gradient of fMLP in C3bi-coated Zigmond chambers. Analysis of adhesion of single Vav knockout neutrophils to varying concentrations of ICAM-1 is shown in Fig. S1. In addition, activation of multiple integrin signaling pathways in Vav single knockout neutrophils in response to adhesion to C3bi is shown in Fig. S2. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200404166/DC1.
![]() |
Acknowledgments |
---|
This work was supported by National Institutes of Health grants CA78773 and HL059561 (to J.S. Brugge), HL65095 and HL36028 (to T.N. Mayadas), HL54136 (to K. Ley), NRSA fellowship (to J. Wilsbacher), and Leukemia and Lymphoma Society (to S.L. Moores).
Submitted: 28 April 2004
Accepted: 1 June 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allen, W.E., G.E. Jones, J.W. Pollard, and A.J. Ridley. 1997. Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J. Cell Sci. 110:707720.
Ardouin, L., M. Bracke, A. Mathiot, S.N. Pagakis, T. Norton, N. Hogg, and V.L. Tybulewicz. 2003. Vav1 transduces TCR signals required for LFA-1 function and cell polarization at the immunological synapse. Eur. J. Immunol. 33:790797.[CrossRef][Medline]
Benard, V., and G.M. Bokoch. 2002. Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345:349359.[CrossRef][Medline]
Bunting, M., E.S. Harris, T.M. McIntyre, S.M. Prescott, and G.A. Zimmerman. 2002. Leukocyte adhesion deficiency syndromes: adhesion and tethering defects involving beta 2 integrins and selectin ligands. Curr. Opin. Hematol. 9:3035.[CrossRef][Medline]
Bustelo, X.R. 2000. Regulatory and signaling properties of the Vav family. Mol. Cell. Biol. 20:14611477.
Caron, E., and A. Hall. 1998. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science. 282:17171721.
Cichowski, K., J.S. Brugge, and L.F. Brass. 1996. Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of the proto-oncogene product, p95vav, in platelets. J. Biol. Chem. 271:75447550.
Clark, E.A., W.G. King, J.S. Brugge, M. Symons, and R.O. Hynes. 1998. Integrin-mediated signals regulated by members of the rho family of GTPases. J. Cell Biol. 142:573586.
Coxon, A., P. Rieu, F.J. Barkalow, S. Askari, A.H. Sharpe, U.H. von Andrian, M.A. Arnaout, and T.N. Mayadas. 1996. A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity. 5:653666.[Medline]
del Pozo, M.A., M.A. Schwartz, J. Hu, W.B. Kiosses, A. Altman, and M. Villalba. 2003. Guanine exchange-dependent and -independent effects of Vav1 on integrin-induced T cell spreading. J. Immunol. 170:4147.
Doody, G.M., S.E. Bell, E. Vigorito, E. Clayton, S. McAdam, R. Tooze, C. Fernandez, I.J. Lee, and M. Turner. 2001. Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation. Nat. Immunol. 2:542547.[CrossRef][Medline]
D'Souza-Schorey, C., B. Boettner, and L. Van Aelst. 1998. Rac regulates integrin-mediated spreading and increased adhesion of T lymphocytes. Mol. Cell. Biol. 18:39363946.
Fischer, K.D., Y.Y. Kong, H. Nishina, K. Tedford, L.E. Marengere, I. Kozieradzki, T. Sasaki, M. Starr, G. Chan, S. Gardener, et al. 1998. Vav is a regulator of cytoskeletal reorganization mediated by the T- cell receptor. Curr. Biol. 8:554562.[Medline]
Foy, D.S., and K. Ley. 2000. Intercellular adhesion molecule-1 is required for chemoattractant-induced leukocyte adhesion in resting, but not inflamed, venules in vivo. Microvasc. Res. 60:249260.[CrossRef][Medline]
Fujikawa, K., A.V. Miletic, F.W. Alt, R. Faccio, T. Brown, J. Hoog, J. Fredericks, S. Nishi, S. Mildiner, S.L. Moores, et al. 2003. Vav1/2/3-null mice define an essential role for Vav family proteins in lymphocyte development and activation but a differential requirement in MAPK signaling in T and B cells. J. Exp. Med. 198:15951608.
Glogauer, M., C.C. Marchal, F. Zhu, A. Worku, B.E. Clausen, I. Foerster, P. Marks, G.P. Downey, M. Dinauer, and D.J. Kwiatkowski. 2003. Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions. J. Immunol. 170:56525657.
Gotoh, A., H. Takahira, R.L. Geahlen, and H.E. Broxmeyer. 1997. Cross-linking of integrins induces tyrosine phosphorylation of the proto-oncogene product Vav and the protein tyrosine kinase Syk in human factor-dependent myeloid cells. Cell Growth Differ. 8:721729.[Abstract]
Holsinger, L.J., I.A. Graef, W. Swat, T. Chi, D.M. Bautista, L. Davidson, R.S. Lewis, F.W. Alt, and G.R. Crabtree. 1998. Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction. Curr. Biol. 8:563572.[Medline]
King, W.G., M.D. Mattaliano, T.O. Chan, P.N. Tsichlis, and J.S. Brugge. 1997. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. Mol. Cell. Biol. 17:44064418.[Abstract]
Krawczyk, C., A. Oliveira-dos-Santos, T. Sasaki, E. Griffiths, P.S. Ohashi, S. Snapper, F. Alt, and J.M. Penninger. 2002. Vav1 controls integrin clustering and MHC/peptide-specific cell adhesion to antigen-presenting cells. Immunity. 16:331343.[Medline]
Laudanna, C., J.J. Campbell, and E.C. Butcher. 1996. Role of Rho in chemoattractant-activated leukocyte adhesion through integrins. Science. 271:981983.[Abstract]
Ley, K. 2003. Arrest chemokines. Microcirculation. 10:289295.[CrossRef][Medline]
Ley, K., D.C. Bullard, M.L. Arbones, R. Bosse, D. Vestweber, T.F. Tedder, and A.L. Beaudet. 1995. Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J. Exp. Med. 181:669675.[Abstract]
Li, S., A. Yamauchi, C.C. Marchal, J.K. Molitoris, L.A. Quilliam, and M.C. Dinauer. 2002. Chemoattractant-stimulated Rac activation in wild-type and Rac2-deficient murine neutrophils: preferential activation of Rac2 and Rac2 gene dosage effect on neutrophil functions. J. Immunol. 169:50435051.
Lu, H., C.W. Smith, J. Perrard, D. Bullard, L. Tang, S.B. Shappell, M.L. Entman, A.L. Beaudet, and C.M. Ballantyne. 1997. LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice. J. Clin. Invest. 99:13401350.
Luscinskas, F.W., G.S. Kansas, H. Ding, P. Pizcueta, B.E. Schleiffenbaum, T.F. Tedder, and M.A. Gimbrone, Jr. 1994. Monocyte rolling, arrest and spreading on IL-4-activated vascular endothelium under flow is mediated via sequential action of L-selectin, ß1-integrins, and ß2-integrins. J. Cell Biol. 125:14171427.[Abstract]
Manetz, T.S., C. Gonzalez-Espinosa, R. Arudchandran, S. Xirasagar, V. Tybulewicz, and J. Rivera. 2001. Vav1 regulates phospholipase cgamma activation and calcium responses in mast cells. Mol. Cell. Biol. 21:37633774.
Marignani, P.A., and C.L. Carpenter. 2001. Vav2 is required for cell spreading. J. Cell Biol. 154:177186.
Miranti, C.K., L. Leng, P. Maschberger, J.S. Brugge, and S.J. Shattil. 1998. Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Curr. Biol. 8:12891299.[Medline]
Mizgerd, J.P., H. Kubo, G.J. Kutkoski, S.D. Bhagwan, K. Scharffetter-Kochanek, A.L. Beaudet, and C.M. Doerschuk. 1997. Neutrophil emigration in the skin, lungs, and peritoneum: different requirements for CD11/CD18 revealed by CD18-deficient mice. J. Exp. Med. 186:13571364.
Mocsai, A., M. Zhou, F. Meng, V.L. Tybulewicz, and C.A. Lowell. 2002. Syk is required for integrin signaling in neutrophils. Immunity. 16:547558.[Medline]
Mocsai, A., H. Zhang, Z. Jakus, J. Kitaura, T. Kawakami, and C.A. Lowell. 2003. G-protein-coupled receptor signaling in Syk-deficient neutrophils and mast cells. Blood. 101:41554163.
Moores, S.L., L.M. Selfors, J. Fredericks, T. Breit, K. Fujikawa, F. Alt, J.S. Brugge, and W. Swat. 2000. Vav family proteins couple to diverse cell surface receptors. Mol. Cell. Biol. 20:63646373.
Morii, N., T. Teru-uchi, T. Tominaga, N. Kumagai, S. Kozaki, F. Ushikubi, and S. Narumiya. 1992. A rho gene product in human blood platelets. II. Effects of the ADP-ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. J. Biol. Chem. 267:2092120926.
Nobes, C.D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 81:5362.[Medline]
Obergfell, A., K. Eto, A. Mocsai, C. Buensuceso, S.L. Moores, J.S. Brugge, C.A. Lowell, and S.J. Shattil. 2002. Coordinate interactions of Csk, Src, and Syk kinases with []IIb[ß]3 initiate integrin signaling to the cytoskeleton. J. Cell Biol. 157:265275.
Olson, M.F., N.G. Pasteris, J.L. Gorski, and A. Hall. 1996. Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases. Curr. Biol. 6:16281633.[Medline]
Price, L.S., J. Leng, M.A. Schwartz, and G.M. Bokoch. 1998. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell. 9:18631871.
Ren, X.D., W.B. Kiosses, and M.A. Schwartz. 1999. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18:578585.
Reynolds, L.F., L.A. Smyth, T. Norton, N. Freshney, J. Downward, D. Kioussis, and V.L. Tybulewicz. 2002. Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3-kinase-dependent and -independent pathways. J. Exp. Med. 195:11031114.
Ridley, A.J. 1999. Rho family proteins and regulation of the actin cytoskeleton. Prog. Mol. Subcell. Biol. 22:122.[Medline]
Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 70:389399.[Medline]
Roberts, A.W., C. Kim, L. Zhen, J.B. Lowe, R. Kapur, B. Petryniak, A. Spaetti, J.D. Pollock, J.B. Borneo, G.B. Bradford, et al. 1999. Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity. 10:183196.[Medline]
Tedford, K., L. Nitschke, I. Girkontaite, A. Charlesworth, G. Chan, V. Sakk, M. Barbacid, and K.D. Fischer. 2001. Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling. Nat. Immunol. 2:548555.[CrossRef][Medline]
Turner, M., P.J. Mee, A.E. Walters, M.E. Quinn, A.L. Mellor, R. Zamoyska, and V.L. Tybulewicz. 1997. A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes. Immunity. 7:451460.[Medline]
Walzog, B., K. Scharffetter-Kochanek, and P. Gaehtgens. 1999. Impairment of neutrophil emigration in CD18-null mice. Am. J. Physiol. 276:G1125G1130.[Medline]
Webb, D.J., J.T. Parsons, and A.F. Horwitz. 2002. Adhesion assembly, disassembly and turnover in migrating cellsover and over and over again. Nat. Cell Biol. 4:E97E100.[CrossRef][Medline]
Welch, H.C., W.J. Coadwell, C.D. Ellson, G.J. Ferguson, S.R. Andrews, H. Erdjument-Bromage, P. Tempst, P.T. Hawkins, and L.R. Stephens. 2002. P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac. Cell. 108:809821.[Medline]
Woodside, D.G., D.K. Wooten, and B.W. McIntyre. 1998. Adenosine diphosphate (ADP)-ribosylation of the guanosine triphosphatase (GTPase) rho in resting peripheral blood human T lymphocytes results in pseudopodial extension and the inhibition of T cell activation. J. Exp. Med. 188:12111221.
Yron, I., M. Deckert, M.E. Reff, A. Munshi, M.A. Schwartz, and A. Altman. 1999. Integrin-dependent tyrosine phosphorylation and growth regulation by Vav. Cell Adhes. Commun. 7:111.[Medline]
Zigmond, S.H. 1988. Orientation chamber in chemotaxis. Methods Enzymol. 162:6572.[Medline]