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The Journal of Immunology, 2003, 170: 41-47.
Copyright © 2003 by The American Association of Immunologists

Guanine Exchange-Dependent and -Independent Effects of Vav1 on Integrin-Induced T Cell Spreading1 ,2

Miguel Angel del Pozo{dagger},{ddagger}, Martin A. Schwartz, Junru Hu*, William B. Kiosses{dagger}, Amnon Altman3,* and Martin Villalba3,*,§

* Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; {dagger} Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037; {ddagger} Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain; and § Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Institut Fédératif de Recherche 24, Montpellier, France. Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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. {beta}1 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 {beta}1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and {beta}1 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Vav family has three known members in mammalian cells (Vav1, Vav2, and Vav3). Although all vav family genes are expressed in hemopoietic cells, vav 1 transcripts are present at significantly higher levels than those of vav 2 and vav 3 (1). Vav1, a 95-kDa member of the Dbl superfamily of guanine exchange factors (GEFs)4 and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, is essential for lymphocyte development and activation (2). In addition to the characteristic tandem Dbl homology/pleckstrin homology (PH) domains, it displays a cysteine-rich domain, one SH2 and two SH3 domains, and a calponin-like domain (Ref. 2 ; see also Fig. 5GoA). Vav1-deficient mice display a severe reduction in the number of T and B lymphocytes (3, 4). Vav1 is essential for: the actin polymerization that leads to TCR cap formation after TCR/CD3 ligation (3, 4, 5), lipid raft clustering at the immune synapse (6), TCR-induced T cell adhesion and integrin clustering (7), and protein kinase C (PKC) {theta} membrane translocation and its subsequent activation (8). Vav1 associates with an array of cytoskeletal, cytoplasmic, and nuclear proteins. It is believed that Vav1 controls cytoskeleton rearrangements because it displays GEF activity toward Rac in vitro that can be stimulated by tyrosine kinases and phosphoinositides (9, 10, 11). Vav1 also causes Rac, Rho, and Cdc42 activation in vivo (12, 13). In Jurkat T cells, Pak1 activation occurs downstream of Vav1 and is required for activation of extracellular signal-regulated kinase 2 (ERK2) and NF-AT (14). Vav1 interacts with a ternary complex composed by signal linker protein 76 (SLP-76)/Nck/Pak1, facilitating the activation of Rac in the vicinity of its target Pak1 to induce maximal activation of this kinase (15, 16). In contrast, SLP-76 is not required for Rac and Pak1 activation after TCR stimulation (17). In addition, Vav couples TCR to serum response factor-dependent transcription via a Rac/Cdc42-Pak1-mitogen-activated protein 1-dependent pathway (18).



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  		FIGURE 5. Cooperation of Vav1 with Rac does not require GEF activity. A, Scheme of Vav1 mutants used in this report. B, Expression of these mutants in Jurkat cells. Jurkat-TAg cells were transfected with 5 µg of Vav1 wt or 10 µg of the different mutants, and 2 days later whole cell extracts from 1 x 106 cells of the different transfections were subjected to Western blotting, and the expression of the different mutants was analyzed with an anti-c-Myc. C, Jurkat-TAg were transfected as in B, some cells cotransfected with 5 µg of V12Rac, and 2.5 µg of GFP were added to all cells. After 2 days, cells were plated for 10 min on FN-coated slides. Cells were fixed, stained with phalloidin-tetramethylrhodamine isothiocyanate, and analyzed by confocal microscopy. D, Surface area was quantified for GFP+ cells. Values are means ± SD from two experiments in which >100 cells were scored per condition.

 
Integrins are required for leukocyte extravasation into sites of inflammation and mediate the formation of large complexes of cytoskeletal and signaling proteins (19, 20), and their role in the formation of the immune synapse has recently been revised (21). Syk and ZAP-70 are activated through interaction with {beta} integrin cytoplasmic domains (22, 23). Integrins induce tyrosine phosphorylation of Vav family proteins in cells that express Syk. Additionally, Syk and Vav1 cooperatively induced activation of c-Jun N-terminal kinase (JNK), ERK2, and Akt in nonhemopoietic cells attached to fibrinogen (24, 25, 26), and Vav is essential for integrin-induced activation of Pyk2 (7), a focal adhesion kinase that is inducible phosphorylated downstream of the TCR and integrins (27) and that is thought to be important in integrin-mediated pathways. In NK cells, Vav1 and Rac control the activation of p38 mitogen-activated protein kinase triggered by integrins (28). Rac, but not other small Rho GTPases like Rho or Cdc42, regulates integrin-mediated spreading and adhesion of T cells (29). However, it remains unknown whether Vav cooperates with integrins to stimulate mitogen-activated protein kinase activation, gene transcriptional activation and cytoskeleton changes in cell lineages where Vav1 is normally expressed, e.g., T lymphocytes. Here, we investigated the role of Vav1 in T cell spreading after integrin occupancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

The anti-CD3 mAb OKT3 and the anti-Myc mAb 9E10 were purified from culture supernatants of the corresponding hybridomas by protein A-Sepharose chromatography. The anti-hemagglutinin (HA; 12CA5) mAb was from Boehringer Mannheim (Indianapolis, IN). The anti-Vav1, anti-Rac, and the anti-phosphotyrosine mAb were obtained from Upstate Biotechnology (Lake Placid, NY). The anti-JNK mAb was from BD PharMingen (San Diego, CA). Polyclonal anti-Pak1 Ab (R626) was described previously (30). Fibronectin (FN) was prepared from human plasma as described (31), poly-L-lysine was from Sigma-Aldrich (St. Louis, MO), and rhodamine-phalloidin was from Molecular Probes (Eugene, OR).

Plasmids

The cDNAs encoding a c-Myc epitope-tagged Vav1 in the pEF mammalian expression vector have been described (8, 32). The Vav mutants VavGEF* (L213A), Vav{Delta}PH (PH deletion from 405 to 507 aa), and VavSH2* (R695L) were cloned in the same vector. HA-JNK1 was cloned in pcDNA3. A constitutively active Rac1 mutant (Rac1V12) was cloned in pEXV. The green-fluorescent protein (GFP) fusions of the Rac and Cdc42 active mutants have been described (33).

Cell culture and transfection

SV40 T Ag-transfected human leukemic Jurkat T (Jurkat-TAg) cells and the mouse T cell hybridoma A1.1 were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 1x MEM nonessential amino acid solution (Life Technologies), and 100 U/ml each penicillin G and streptomycin. Cells were starved for 24 h in 0.2% serum before stimulation and assays of cell spreading, Rac, Pak, or JNK. Jurkat cells in a logarithmic growth phase were transfected with the indicated amounts of plasmid DNAs by electroporation as described previously (8). In each experiment, cells in different groups were transfected with the same total amount of plasmid DNA by supplementing expression vector DNA with the proper amounts of the corresponding empty vector. Cell lysis and analysis were performed 48 h after transfection as described for each experiment.

Human peripheral blood lymphocytes cells were prepared from healthy volunteers by standard Ficoll-Hypaque centrifugation and cultured in the presence of an activating anti-CD3 mAb (OKT3; 1 µg/ml) plus recombinant human IL-2 (20 U/ml) for 6 days. Before restimulation, cells were deprived of anti-CD3 and IL-2 for 36 h.

FN coating and cell spreading

Bacterial plastic dishes (100 mm) or glass coverslips in 24-well plastic dishes were coated with 20 µg/ml human FN or 1 mg/ml poly-L-lysine at 4°C overnight. The dishes were washed three times with PBS, and the FN-coated ones were blocked with 10 mg/ml heat-denatured BSA. Cells were plated and allowed to spread on the substrates for different times and then fixed with 3% paraformaldehyde-PBS for 30 min, permeabilized in 0.2% Triton X-100 in PBS for 5 min, and blocked with 10% normal goat serum. F-actin was stained with rhodamine-phalloidin. Images were acquired using a Bio-Rad 1024 MRC laser scanning confocal imaging system. Spreading of GFP+ cells was quantified by scoring 0 points for round cells, 1 point for partially spread cells, and 2 points for fully spread cells. The index of cell spreading was calculated as (y + 2z)/(x + y + z) where x, y, and z are the number of cells that were scored as 0, 1, and 2, respectively.

Quantification of cell surface area

The area of the cell surface in contact with the FN-coated dishes was outlined and measured using the Inovision ISEE software (Inovision, Raleigh, NC) running on a UNIX workstation (Silicon Graphics, Mountain View, CA). At least 100 cells of each experimental condition were analyzed.

Immunoprecipitation and immunoblotting

Cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 5 mM Na4P2O7, 1 mM Na3VO4 plus 1% Nonidet P-40, 20 mM NaH2PO4, 3 mM {beta}-glycerophosphate, 10 mM NaF, and 10 µg/ml quantities each of aprotinin and leupeptin) for 10 min on ice. After centrifugation (16,000 x g for 10 min at 4°C), the supernatants were incubated with optimal concentrations of Abs for 30 min at 4°C, followed by 30 µl of protein A/G plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4°C. Samples were washed four times in lysis buffer, and precipitates were dissolved in Laemmli buffer and resolved by SDS-PAGE. Electrophoresed samples were processed for Western blot analysis as previously described (34).

Rac and Cdc42 GTPase assays

Cells were serum starved overnight 24 h after transfection. Jurkat cells were kept in suspension in medium with 0.2% BSA or plated on dishes coated with 20 µg/ml FN. Cells were then chilled on ice, washed with ice-cold PBS, and lysed in buffer containing 0.5% Nonidet P-40, 50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 20 µg of recombinant GST fused to the Rac/Cdc42-binding domain of Pak. Lysates were then incubated with glutathione-agarose beads (Pharmacia, Peapack, NJ) at 4°C for 30 min, washed with lysis buffer, and eluted with SDS sample buffer. Bound GTPase was analyzed by Western blotting using a monoclonal anti-Rac Ab or a polyclonal Ab to Cdc42. Whole cell lysates were also analyzed for the presence of each GTPase for normalization.

Pak kinase assays

Pak was precipitated from cell lysates with anti-Pak1 Abs and kinase activity determined using an in-gel kinase assay with myelin basic protein as substrate as previously described (30). Pak protein in the precipitates was determined by Western blotting with the same Ab. Autoradiographs were quantitated by scanning densitometry, and the specific activity (kinase activity normalized to Pak protein) was calculated.

JNK assays

Transfected Jurkat cells were washed twice in PBS, lysed for 10 min at 4°C in 1 ml of lysis buffer B (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 5 mM Na4P2O7, 1 mM Na3VO4, 10 µg/ml quantities each of aprotinin and leupeptin), and spun at 15,000 x g for 10 min at 4°C; the supernatants were incubated overnight with 2 µg of anti-HA mAb plus 25 µl of protein G-Sepharose 4B beads (Pharmacia Biotech, Uppsala, Sweden) to immunoprecipitate JNK. The kinase activities of washed immunoprecipitates were assayed using 2 µg GST-c-Jun fusion proteins as substrate, in 20 µl of JNK kinase buffer (25 mM HEPES (pH 7.5), 25 mM MgCl2, 50 µM cold ATP, 25 mM {beta}-glycerophosphate, 1 mM DTT, and 0.1 mM Na3VO4). Kinase reactions were incubated for 20 min at 30°C with gentle shaking and were stopped by addition of 20 µl of 2x Laemmli buffer. Proteins were resolved by SDS, 13% PAGE, transferred to nitrocellulose, and subjected to Western blotting. The nitrocellulose membranes were routinely reprobed with anti-JNK Ab and quantified by NIH image software. The level of phosphorylation was normalized to JNK protein levels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Integrin engagement induces Vav1 phosphorylation in T lymphocytes

T cells plated on integrin ligands undergo cell polarization, which involves profound changes in the structure and shape of their cytoskeleton (33, 35). Vav1 is a major protein involved in cytoskeleton reorganization in T cells (1). To analyze the interaction between Vav1 and integrins in T lymphocytes, primary T cells and two T cell lines were plated on FN-coated dishes. FN binds to {alpha}5{beta}1 and {alpha}4{beta}1 integrins in T cells and induces downstream signals. We observed that Vav1 was phosphorylated on tyrosine by integrin occupancy in all T cell types tested: activated human PBLs, Jurkat T cells, and the mouse T cell hybridoma A1.1 (Fig. 1Go). Jurkat T cells showed a transient Vav1 phosphorylation with a peak ~10 min after stimulation and return to near baseline in 1 h. Human PBLs showed a peak after 30 min, and a plateau that remains for at least 1 h. A1.1 cells showed a slower but progressive increase in Vav1 phosphorylation. We conclude that integrin occupancy leads to Vav1 phosphorylation in T cells.



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  		FIGURE 1. FN induces Vav1 phosphorylation. Jurkat T cells, anti-CD3-stimulated human PBLs, and A1.1 mouse T cells were plated on FN-coated plates for different times. Cells were lysed, Vav1 was immunoprecipitated, and its phosphorylation was analyzed with an anti-phosphotyrosine ({alpha}-PTyr) mAb. Similar results were obtained in two experiments. {alpha}-Vav, Anti-Vav1.

 
FN cooperates with Vav1 to induce T cell spreading

Next, we examined effects of FN and/or Vav1 on T cell spreading. Transfected cells were recognized by cotransfection with GFP. Jurkat T cells plated on FN showed very little spreading or filopodia at 10 min after plating (Fig. 2GoA). At 30–60 min, cells started to show mainly filopodia and short lamellipodia (see Fig. 2GoA, insets) Longer incubation did not increase spreading (Fig. 2Go, A and B). By contrast, Vav1-transfected cells showed clear lamellipodia formation after only 10 min on FN (Fig. 2GoA). Very large lamellipodia were found after 30 min that corresponded to maximal spreading (Fig. 2GoB). We failed to observe cell spreading when Jurkat T cells were plated on poly-L-lysine, even after 4 h (data no shown), suggesting that integrin engagement is essential for T cell spreading even when overexpressing Vav. Conversely, two dominant negative Vav constructs, Vav{Delta}PH and VavGEF* (see scheme in Fig. 5GoA and Refs. 6, 8) blocked integrin-induced T cell spreading (Fig. 2GoA). Both mutants showed a 50% reduction in spreading after 30 min of plating on FN (Fig. 2GoC). These mutants were expressed at slightly lower levels than the wild type (Fig. 2GoC, bottom), but they were still capable of blocking FN-induced T cell spreading. These results suggest that Vav activity is essential for integrin-induced spreading in hemopoietic T cells. We therefore decided to study in more detail the mechanism of Vav1-induced T cell spreading. Because Vav1-transfected cells replated on FN for 10 min showed maximal spreading differences relative to mock-transfected cells, this time point was used in the rest of the study.



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  		FIGURE 2. FN cooperates with Vav1 to induce T cell spreading. (A), Jurkat-TAg cells (1 x 107) were transfected with 2.5 µg of GFP plus 10 µg of empty vector or c-Myc-Vav1. After 2 days, the cells were plated on poly-L-lysine (time 0) or for different times on FN-coated slides. Cells were fixed, stained with phalloidin-tetramethylrhodamine isothiocyanate, and analyzed by confocal microscopy. Selected areas are magnified to allow comparison. Bars, 10 µm. White arrowheads, lamellipodia. B and C, Spreading of GFP+ cells was calculated as described in Materials and Methods. Values are means ± SD from two experiments in which >100 cells were scored per condition. C (bottom), Expression of the different Vav constructs.

 
Vav1 activated Rac, Cdc42, Pak, and JNK in T cells plated on FN

Cytoskeletal changes induced by Vav1 have been linked to its ability to activate the small GTPase Rac (9), including studies in Jurkat cells (13, 36). Vav1 overexpression led to increased activity of Rac and Cdc42 (Fig. 3Go, A and B), two known targets of Vav1 in vivo (12). We then studied the effects of Vav-1 on two downstream targets of these GTPases, Pak and JNK. Pak has been linked to cytoskeletal rearrangements and is downstream of Vav1 (14) and was also activated by Vav1 in Jurkat cells (Fig. 3GoC). JNK, which is activated by onco-Vav1 in nonhemopoietic cells and by Rac (9, 37), was also activated by Vav1 in Jurkat cells (Fig. 3GoD). These data suggested that Vav1 overexpression leads to activation of the JNK pathway located downstream of its main targets Rac and Cdc42. In contrast, {beta}1 integrin engagement by FN failed to activate Rac, Cdc42, or JNK and induced a partial activation of Pak. In addition, {beta}1 integrin engagement on cells transfected with Vav1 induced a significant increase on Pak activity, but not of Rac, Cdc42, or JNK activities. These data suggested that Pak, but not other enzymes of this pathway, integrated signals derived from Vav1 and integrins.



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  		FIGURE 3. Effects of Vav1 on Rac, Cdc42, Pak, and JNK activities in T cells. A, Jurkat-TAg cells (2 x 107) were transfected with empty vector or c-Myc-Vav1. After 2 days, Jurkat cells were plated on FN for 10 min (Fn) or analyzed on suspension (Sus). Cells were then lysed, and endogenous Rac-GTP loading was determined using a pull-down assay. Proteins were electrophoresed and blotted onto a nitrocellulose membrane. The membrane was then immunoblotted with an anti-Rac Ab, and the amount of Rac was quantified by densitometry. B, Same as A for Cdc42. C, Cells were treated as in A, but Pak was immunoprecipitated with an anti-Pak Ab and the IPs subjected to an in-gel kinase assay. D, Cells were treated as in C, and JNK in vitro kinase assay was performed. Densitometry was analyzed as described in Materials and Methods. Data represent average ± SD of three separate experiments.

 
GEF-independent Vav1 effects on T cell spreading

T cell spreading is barely detectable in T cells plated for 10 min on FN but is substantially increased by Vav overexpression (Figs. 2Go, 4GoA, and 5C). To determine whether these effects of Vav1 on the cytoskeleton were dependent on Rac and/or Cdc42 activity, we used GFP chimeras of constitutively active mutants of both GTPases. Expression of active Rac or Cdc42 induced T cell spreading when plated for 10 min on FN (Fig. 4GoA). Coexpression of Vav1 and constitutively active Rac induced a further increase in T cell spreading (Fig. 4GoA), whereas Vav1 and V12Cdc42 showed almost no cooperation. Western blot analysis showed that expression of active Rac or Cdc42 did not alter Vav1 expression and vice versa (Fig. 4GoA, bottom). Direct measurement of cell area showed that Vav1, V12Rac, and V12Cdc42 individually induced a ~3-fold increase in T cell area (Fig. 4GoB), whereas expression of Vav1 and V12Rac together resulted in a nearly 7-fold increase. Similar results were obtained when the cell spreading index was calculated as in Fig. 2Go (data not shown). Because V12Rac is maximally GTP loaded, these results strongly suggest that Vav1 activates additional pathways leading to T cell spreading apart from Rac activation.



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  		FIGURE 4. Vav1 cooperates with V12Rac, but not with V12Cdc42, to induce T cell spreading. A, Jurkat-TAg cells (1 x 107) were transfected with 2.5 µg of GFP plus 10 µg of empty vector or c-Myc-Vav1 and the indicated active GTPase/GFP fusions. After 2 days, the cells were plated on FN-coated slides for 10 min. Cells were fixed, stained with phalloidin-tetramethylrhodamine isothiocyanate, and analyzed by confocal microscopy. A (bottom), Expression of the different constructs. B, Surface area was quantified for GFP+ cells. Values are means ± SD from two experiments in which >100 cells were scored per condition.

 
To further investigate this possibility and identify the domain(s) of Vav involved in this process, we examined different Vav mutants (Fig. 5GoA). The L213A mutation (VavGEF*) does not display GEF activity toward Rac and/or Cdc42. Deletion of residues 405–507 (Vav{Delta}PH) eliminates the region responsible for the correct localization of Vav1. A point mutation (R695L) in the SH2 domain, which is responsible for the interaction with adaptor proteins such as SLP-76, makes the VavSH2* mutant defective in some of the Vav1 adaptor functions. All these mutants were expressed in Jurkat T cells at levels similar to those of wild-type Vav1 (Fig. 5GoB).

To study the effect of these mutants on spreading, we plated Jurkat T cells on FN-coated plates for 10 min, at which time nontransfected cells showed minimal spreading (Fig. 5GoC). VavSH2* induced an increase in spreading that was only slightly smaller than that of wild-type Vav1 (Fig. 5Go, C and D), whereas {Delta}PH or GEF* Vav1 mutants did not show any effect. Mock-transfected cells plated on FN for only 10 min exhibit a very low level of spreading, so that no inhibition (as described at later time points; see Fig. 2Go) could be observed. In summary, GEF activity toward Rac and/or Cdc42 is required for Vav-mediated T cell spreading.

To identify the domain(s) of Vav1 involved in cooperation with GTP-loaded Rac, we coexpressed these mutants with V12Rac and then plated cells on FN for 10 min (Fig. 5Go, C and D). Coexpression of VavGEF* with V12Rac still cooperated to induce T cell spreading, further demonstrating that Vav1 can regulate the actin cytoskeleton independently of its GEF activity (as suggested in Fig. 4Go). Strikingly, VavSH2* failed to cooperate with V12Rac, strongly suggesting that the SH2 domain of Vav1 mediates an additional, GEF-independent pathway. Coexpression of Vav{Delta}PH had no effect. This mutant entirely localizes to the insoluble compartment (38), suggesting that its function is impaired by mislocalization. In fact, Vav{Delta}PH behaves as the most efficient dominant negative Vav1 mutant (Fig. 2Go; Refs. 6, 8, 32 and 38).

Altogether, these results place Rac both downstream and parallel to Vav1 in the integrin-mediated T cell spreading pathway. Vav1 could activate two independent pathways leading to T cell spreading after integrin occupancy: 1) Vav1 activates Rac through its GEF domain; 2) Vav1 facilitates Rac function through its SH2 adaptor domain, likely by bringing Rac targets to its vicinity (15, 16).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that Vav1 activity is essential for integrin-induced T cell spreading. Vav1-induced cytoskeletal reorganization has been linked to its ability to activate Rho family small GTPases (9, 10, 11, 12). However, our data establish the novel concept that Vav1 can regulate the actin cytoskeleton independently of its GEF activity. Vav1 activates at least two pathways leading to cytoskeletal rearrangements. Whereas the PH/Dbl homology domains are required for Rac activation, the C-terminal adapter modules mediate a separate pathway.

Our data place Vav1 downstream of integrins in the pathway leading to T cell spreading, as has been shown in other cellular systems (15, 16, 24). Thus, inhibition of Vav1 activity by two dominant negative constructs, VavGEF* and Vav{Delta}PH, blocks T cell spreading induced by FN. Conversely, Vav1 greatly increases T cell spreading after integrin occupancy. However, our attempts to show defective spreading on FN of vav1-/- mouse T lymphocytes were unsuccessful, suggesting that other members of the Vav family could compensate in vivo. In fact, Martinez-Gakidis et al. (39) have recently shown that Vav1/Vav3 double knockout macrophages were unable to migrate on FN, whereas macrophages deficient in either one of the genes exhibited no significant alterations. This contrasts with the fact that VavPH and VavGEF* block T cell spreading (Fig. 2Go). The two more likely explanations are that these mutants also block the activity of other members of the Vav family or that in vav1-/- T cells there is a mechanism of compensation for Vav function that is not present in normal T cells.

Vav1 overexpression combined with {beta}1 integrin ligation leads to substantial changes in the cytoskeleton (Figs. 1Go and 2Go), whereas changes in Cdc42 and Rac activity were much more moderate (Fig. 3Go). These modest changes could suggest that these GTPases are constitutively active in these cells. However, the induction of cell spreading by Vav-1, V12 Rac, and V12 Cdc42 (Figs. 2Go, 4Go, and 5Go) and the absence of constitutive JNK activation in these cells (see, e.g., Refs. 8, 40 and 41) rule out this simplistic explanation. A more likely explanation for this result is that T cells can have more than one pool of Rac/Cdc42. Integrin engagement can induce tyrosine phosphorylation of Vav1 that would activate a specific Rac/Cdc42 pool, leading to a modest increase in total Rac/Cdc42 activity. However, this fraction could be critical for cytoskeletal reorganization. Consistent with this idea, different Rac-GEFs are able to selectively induce certain downstream targets but not others (42). It is also possible that certain Rac/Cdc42 effectors (i.e., Pak) might be sensitive to slight increases in the level of GTPase activation. In fact, T cell spreading after combined Vav1 and integrin stimulation correlated better with activity levels of Pak than with those of Rac or Cdc42 (Fig. 3Go). This suggests that Pak could be integrating signals coming from several domains of Vav1. These domains would include those that directly activate Rac/Cdc42, plus an adapter function that may facilitate Pak activation by the active GTPases.

Both V12Rac and V12Cdc42 were able to induce T cell spreading, although the latter was slightly less efficient (Fig. 4Go). The effect of V12Cdc42 was not detected in a previous study where T cells were incubated on FN for periods of time up to 16 h after overexpression (29). Our assay was probably more sensitive, because we observed cells at longer time points after transfection. Strikingly, Vav1 cooperates with V12Rac but not with V12Cdc42 to induce T cell spreading (Fig. 4Go). Because these mutants are constitutively active, Vav1 cannot further increase their GTP loading. Therefore, synergy between Vav1 and maximally GTP-loaded V12Rac suggests that Vav1 activates pathways distinct from GTPase activation. Some Vav1 functions have already been reported to be independent of its GEF activity, i.e., NF-AT activation (43). Furthermore, our results show, for the first time, that Vav1 can cooperate with Rac to induce cytoskeletal rearrangements in a GEF activity-independent manner. Moreover, VavSH2*, which induces cytoskeletal changes in resting or FN-activated cells, fails to cooperate with V12Rac, strongly suggesting that the SH2 domain of Vav1 is required for its cooperation with V12Rac. Thus, the SH2 domain is likely mediating activation of a pathway different from Vav1 enzymatic activity. The SH2 domain of Vav1 is necessary for JNK activation (44), and for its translocation to the membrane and subsequent filopodia formation (45). This domain binds SLP-76, which facilitates Pak activation downstream of integrins (15, 16), although not downstream of the TCR (17). An attractive possibility is that the SH2 domain of Vav1 would bring the complex SLP-76/Nck/Pak to the vicinity of Rac (15), facilitating Pak activation by Rac. Consistent with this idea, our results show a synergistic effect of Vav1 and FN on Pak activation (Fig. 3GoC). Additionally, T cell spreading correlates better with Pak than with Rac or Cdc42 activity levels (Fig. 3Go). Consequently, Pak could be integrating signals coming from the Vav1 SH2 and GEF domains. However, our results show that GEF activity is absolutely essential for Vav-mediated T cell spreading, whereas the SH2 domain is not absolutely required (Figs. 2Go and 5Go). By using V12Rac, we have unmasked this second function that is probably related to the adaptor function of Vav1. In summary, we favor a model in which 1) the GEF activity of Vav is first required for T cell spreading and 2) the SH2 domain recruits the final components.

Another possibility is that the SH2 domain could be targeting a Rac-specific effector, such as WAVE/Scar (46). This would account for the lack of synergy between Vav1 and V12Cdc42 in T cell spreading. In contrast, more than one mechanism could explain the lack of cooperation between V12Rac and VavSH2*. For example, phosphorylation of VavSH2* is deficient after integrin and anti-CD3 stimulation (M. A. del Pozo and M. Villalba, unpublished results). Therefore, it is possible that other adaptor proteins with SH2 domains, which bind tyrosine-phosphorylated residues of wild-type Vav1, do not recognize this mutant, thus impairing Vav1 cooperation with V12Rac.

Intriguingly, Vav1 also activates two intracellular pathways involved in cytoskeletal reorganization after TCR engagement (7). One of them regulates TCR capping via Wiscott-Aldrich syndrome protein (WASP), and the other regulates integrin clustering via a yet unidentified signaling component. We also describe herein a dual effect of Vav1 (GEF dependent and independent) in integrin-mediated T cell spreading. It is feasible that the WASP-dependent pathway could account for the GEF-dependent effects of Vav1, whereas the WASP-independent pathway could be similar to the GEF-independent pathway unmasked in the present work. Clearly, further analysis is required to test this possibility.

We propose that Vav1 influences Rac downstream signaling specificity. There are several examples in which other GEFs were found to influence which effector proteins become activated when a Rho GTPase is GTP loaded. Thus, Tiam1 and Dbl stimulate Pak but not JNK activity mediated by Rac and Cdc42, whereas FGD1 and GFKG1 show the opposite effect (42). Their large size, subcellular targeting motifs, and interaction with other proteins might explain these effects. GEFs may form complexes with GTPase downstream targets influencing the biological outcome, as we propose here for Vav1 and Rac effectors. Sos1 acts both upstream and downstream of Ras (47, 48). PIX transduces signals from Cdc42 to Rac, forming part of a complex that also involves Pak (49, 50), and Dbl is activated by the Cdc42 effector ACK1 (51).


   Acknowledgments
 
We thank Drs. M. Karin for plasmids, S. Shattil for critical reading, and Natasha Weaver and Bette Cessna for manuscript preparation.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants R01 GM47214 (to M.A.S.) and R01 GM50819 (to A.A.) and SAF 2002-02425 (to M.A.d.P.) was originally supported by HFSPO (LT0019/1998-M), Lady Tata Memorial Trust International Award for Research in Leukemia, and is now a Leukemia and Lymphoma Society Special Fellow (Grant 3347-02). M.V. was a special fellow of the Leukemia and Lymphoma Society (Grant 3143-00) and is now supported by the Fondation de France. Back

2 This is publication number 475 from the La Jolla Institute for Allergy and Immunology and publication number 15180-VB from The Scripps Research Institute. Back

3 Address correspondence and reprint requests to Dr. Miguel A. del Pozo, Departments of Immunology and Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, E-mail address: mdelpozo{at}scripps.edu; Dr. Amnon Altman, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, E-mail address: amnon{at}liai.org; or Dr. Martin Villalba, Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5535, Institut Fédératif de Recherche 24, 1919 Route de Mende, 34293 Montpellier, France, E-mail address: villalba{at}igm.cnrs-mop.fr Back

4 Abbreviations used in this paper: GEF, guanine exchange factor; PH, pleckstrin homology domain; PKC, protein kinase C; FN, fibronectin; GFP, green-fluorescent protein; WASP, Wiscott-Aldrich syndrome protein; JNK, c-Jun N-terminal kinase; ERK2, extracellular signal-regulated kinase 2; SLP-76, signal linker protein 76; HA, hemagglutinin; Jurkat-TAg, SV40 T Ag-transfected human leukemic Jurkat T cells. Back

Received for publication June 11, 2002. Accepted for publication October 21, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bustelo, X. R.. 2000. Regulatory and signaling properties of the Vav family. Mol. Cell. Biol. 20:1461.[Free Full Text]
  2. Collins, T., M. Deckert, A. Altman. 1997. Views on Vav. Immunol. Today 18:221.[Medline]
  3. Fischer, K. D., Y. Y. Kong, H. Nishina, K. Tedford, L. E. Marengère, I. Kozieradzki, T. Sasaki, M. Starr, G. Chan, S. Gardener, M. P. Nghiem, D. Bouchard, M. Barbacid, A. Bernstein, J. M. Penninger. 1998. Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor. Curr. Biol. 8:554.[Medline]
  4. Holsinger, L. J., I. A. Graef, W. Swat, T. Chi, D. M. Bautista, L. Davidson, R. S. Lewis, F. W. Alt, G. R. Crabtree. 1998. Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction. Curr. Biol. 8:563.[Medline]
  5. Wülfing, C., A. Bauch, G. R. Crabtree, M. M. Davis. 2000. The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte-antigen-presenting cell interface. Proc. Natl. Acad. Sci. USA 97:10150.[Abstract/Free Full Text]
  6. Villalba, M., K. Bi, F. Rodriguez, Y. Tanaka, S. Schoenberger, A. Altman. 2001. Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells. J. Cell Biol. 155:331.[Abstract/Free Full Text]
  7. Krawczyk, C., A. Oliveira-dos-Santos, T. Sasaki, E. Griffiths, P. S. Ohashi, S. Snapper, F. Alt, J. M. Penninger. 2002. Vav1 controls integrin clustering and MHC/peptide-specific cell adhesion to antigen-presenting cells. Immunity 16:331.[Medline]
  8. Villalba, M., N. Coudronniere, M. Deckert, E. Teixeiro, P. Mas, A. Altman. 2000. Functional interactions between Vav and PKC{theta} are required for TCR-induced T cell activation. Immunity 12:151.[Medline]
  9. Crespo, P., K. E. Schuebel, A. A. Ostrom, J. S. Gutkind, X. R. Bustelo. 1997. Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product. Nature 385:169.[Medline]
  10. Han, J., B. Das, W. Wei, L. Van Aelst, R. D. Mosteller, R. Khosravi-Far, J. K. Westwick, C. J. Der, D. Broek. 1997. Lck regulates Vav activation of members of the Rho family of GTPases. Mol. Cell. Biol. 17:1346.[Abstract]
  11. Han, J., K. Luby Phelps, B. Das, X. Shu, Y. Xia, R. D. Mosteller, U. M. Krishna, J. R. Falck, M. A. White, D. Broek. 1998. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 279:558.[Abstract/Free Full Text]
  12. Olson, M. F., N. G. Pasteris, J. L. Gorski, A. Hall. 1996. Fasciogenital dysplasia protein (FGD1) and Vav, two related protein required for normal embryonic development, are upstream regulators of Rho GTPases. Curr. Biol. 6:1628.[Medline]
  13. Michel, F., G. Mangino, G. Attal-Bonnefoy, L. Tuosto, A. Alcover, A. Roumier, D. Olive, O. Acuto. 2000. CD28 utilizes Vav-1 to enhance TCR-proximal signaling and NF-AT activation. J. Immunol. 165:3820.[Abstract/Free Full Text]
  14. Yablonski, D., L. P. Kane, D. Qian, A. Weiss. 1998. A Nck-Pak1 signaling module is required for T-cell receptor-mediated activation of NFAT, but not of JNK. EMBO J. 17:5647.[Medline]
  15. Bubeck Wardenburg, J., R. Pappu, J. Y. Bu, B. Mayer, J. Chernoff, D. Straus, A. C. Chan. 1998. Regulation of PAK activation and the T cell cytoskeleton by the linker protein SLP-76. Immunity 9:607.[Medline]
  16. Obergfell, A., B. A. Judd, M. A. del Pozo, M. A. Schwartz, G. A. Koretzky, S. J. Shattil. 2001. The molecular adapter SLP-76 relays signals from platelet integrin {alpha}IIb{beta}3 to the actin cytoskeleton. J. Biol. Chem. 276:5916.[Abstract/Free Full Text]
  17. Ku, G. M., D. Yablonski, E. Manser, L. Lim, A. Weiss. 2001. A PAK1-PIX-PKL complex is activated by the T-cell receptor independent of Nck, Slp-76 and LAT. EMBO J. 20:457.[Medline]
  18. Charvet, C., P. Auberger, S. Tartare-Deckert, A. Bernard, M. Deckert. 2002. Vav1 couples T cell receptor to serum response factor-dependent transcription via a MEK-dependent pathway. J. Biol. Chem. 21:21.
  19. Giancotti, F. G., E. Ruoslahti. 1999. Integrin signaling. Science 285:1028.[Abstract/Free Full Text]
  20. Schwartz, M. A., M. H. Ginsberg. 2002. Networks and crosstalk: integrin signalling spreads. Nat. Cell Biol. 4:65.
  21. Sims, T. N., M. L. Dustin. 2002. The immunological synapse: integrins take the stage. Immunol. Rev. 186:100.[Medline]
  22. Woodside, D. G., A. Obergfell, A. Talapatra, D. A. Calderwood, S. J. Shattil, M. H. Ginsberg. 2002. The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains. J. Biol. Chem. 270:39401.
  23. Woodside, D. G., A. Obergfell, L. Leng, J. L. Wilsbacher, C. K. Miranti, J. S. Brugge, S. J. Shattil, M. H. Ginsberg. 2001. Activation of Syk protein tyrosine kinase through interaction with integrin {beta} cytoplasmic domains. Curr. Biol. 11:1799.[Medline]
  24. Miranti, C. K., L. Leng, P. Maschberger, J. S. Brugge, S. J. Shattil. 1998. Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Curr. Biol. 8:1289.[Medline]
  25. Moores, S. L., L. M. Selfors, J. Fredericks, T. Breit, K. Fujikawa, F. W. Alt, J. S. Brugge, W. Swat. 2000. Vav family proteins couple to diverse cell surface receptors. Mol. Cell. Biol. 20:6364.[Abstract/Free Full Text]
  26. Yron, I., M. Deckert, M. E. Reff, A. Munshi, M. A. Schwartz, A. Altman. 1999. Integrin-dependent tyrosine phosphorylation and growth regulation by Vav. Cell Adhes. Commun. 7:1.[Medline]
  27. Avraham, H., S. Y. Park, K. Schinkmann, S. Avraham. 2000. RAFTK/Pyk2-mediated cellular signalling. Cell Signal 12:123.[Medline]
  28. Mainiero, F., A. Soriani, R. Strippoli, J. Jacobelli, A. Gismondi, M. Piccoli, L. Frati, A. Santoni. 2000. RAC1/P38 MAPK signaling pathway controls {beta}1 integrin-induced interleukin-8 production in human natural killer cells. Immunity 12:7.[Medline]
  29. D’Souza-Schorey, C., B. Boettner, L. Van Aelst. 1998. Rac regulates integrin-mediated spreading and increased adhesion of T lymphocytes. Mol. Cell. Biol. 18:3936.[Abstract/Free Full Text]
  30. del Pozo, M. A., L. S. Price, N. B. Alderson, X. D. Ren, M. A. Schwartz. 2000. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19:2008.[Medline]
  31. Renshaw, M. W., X. D. Ren, M. A. Schwartz. 1997. Growth factor activation of MAP kinase requires cell adhesion. EMBO J. 16:5592.[Medline]
  32. Villalba, M., J. Hernandez, M. Deckert, A. Altman. 2000. Vav modulation of the Ras/MEK/ERK signaling pathway plays a role in NFAT activation and CD69 upregulation. Eur. J. Immunol. 30:1587.[Medline]
  33. del Pozo, M. A., M. Vicente-Manzanares, R. Tejedor, J. M. Serrador, F. Sanchez-Madrid. 1999. Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes. Eur. J. Immunol. 29:3609.[Medline]
  34. Villalba, M., S. Kasibhatla, L. Genestier, A. Mahboubi, D. R. Green, A. Altman. 1999. PKC{theta} is a necessary component, and cooperates with calcineurin, to induce FasL expression during activation-induced T cell death. J. Immunol. 163:5813.[Abstract/Free Full Text]
  35. Sanchez-Madrid, F., M. A. del Pozo. 1999. Leukocyte polarization in cell migration and immune interactions. EMBO J. 18:501.[Medline]
  36. Kaminuma, O., M. Deckert, C. Elly, Y. C. Liu, A. Altman. 2001. Vav-Rac1-mediated activation of the c-Jun N-terminal kinase/c-Jun/AP-1 pathway plays a major role in stimulation of the distal NFAT site in the interleukin-2 gene promoter. Mol. Cell. Biol. 21:3126.[Abstract/Free Full Text]
  37. Crespo, P., X. R. Bustelo, D. S. Aaronson, O. A. Coso, B. M. Lopez, M. Barbacid, J. S. Gutkind. 1996. Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav. Oncogene 13:455.[Medline]
  38. Villalba, M., K. Bi, J. Hu, Y. Altman, P. Bushway, E. Reits, J. Neefjes, G. Baier, R. T. Abraham, A. Altman. 2002. Translocation of PKC{theta} in T cells is mediated by a nonconventional, PI3-K- and Vav-dependent pathway, but does not absolutely require phospholipase C. J. Cell Biol. 157:253.[Abstract/Free Full Text]
  39. Martinez-Gakidis, M. A., B. Zhang, T. Mayadas, F.W. Alt, W. Swat, and J. S. Brugge. 2001. Role for Vav family proteins in macrophage functions. Poster 1476. In 41st Annual Meeting of the American Society for Cell Biology, Washington, DC, December 8–12, p. 270a.
  40. Werlen, G., E. Jacinto, Y. Xia, M. Karin. 1998. Calcineurin preferentially synergizes with PKC-{theta} to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
  41. Ghaffari-Tabrizi, N., B. Bauer, A. Altman, G. Utermann, F. Uberall, G. Baier. 1999. Protein kinase C{theta}, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T-cells. Eur. J. Immunol. 29:132.[Medline]
  42. Zhou, K., Y. Wang, J. L. Gorski, N. Nomura, J. Collard, G. M. Bokoch. 1998. Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42. J. Biol. Chem. 273:16782.[Abstract/Free Full Text]
  43. Kuhne, M. R., G. Ku, A. Weiss. 2000. A guanine nucleotide exchange factor-independent function of Vav1 in transcriptional activation. J. Biol. Chem. 275:2185.[Abstract/Free Full Text]
  44. Arudchandran, R., M. J. Brown, M. J. Peirce, J. S. Song, J. Zhang, R. P. Siraganian, U. Blank, J. Rivera. 2000. The Src homology 2 domain of Vav is required for its compartmentation to the plasma membrane and activation of c-Jun NH2-terminal kinase 1. J. Exp. Med. 191:47.[Abstract/Free Full Text]
  45. Kranewitter, W. J., C. Danninger, M. Gimona. 2001. GEF at work: Vav in protruding filopodia. Cell Motil. Cytoskeleton 49:154.[Medline]
  46. Miki, H., H. Yamaguchi, S. Suetsugu, T. Takenawa. 2000. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 408:732.[Medline]
  47. Scita, G., J. Nordstrom, R. Carbone, P. Tenca, G. Giardina, S. Gutkind, M. Bjarnegard, C. Betsholtz, P. P. Di Fiore. 1999. EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401:290.[Medline]
  48. Nimnual, A. S., B. A. Yatsula, D. Bar-Sagi. 1998. coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. Science 279:560.[Abstract/Free Full Text]
  49. Manser, E., T. H. Loo, C. G. Koh, Z. S. Zhao, X. Q. Chen, L. Tan, I. Tan, T. Leung, L. Lim. 1998. PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1:183.[Medline]
  50. Obermeier, A., S. Ahmed, E. Manser, S. C. Yen, C. Hall, L. Lim. 1998. PAK promotes morphological changes by acting upstream of Rac. EMBO J. 17:4328.[Medline]
  51. Kato-Stankiewicz, J., S. Ueda, T. Kataoka, Y. Kaziro, T. Satoh. 2001. Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner. Biochim. Biophys. Acta 284:470.



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