Article |
Address correspondence to Christopher Carpenter, Division of Signal Transduction, Harvard Institutes of Medicine, Room 1026, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. Tel.: (617) 667-0948. Fax: (617) 667-0957. E-mail: ccarpent{at}caregroup.harvard.edu
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Abstract |
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Key Words: Vav; Rac; spreading; integrins; growth factors
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Introduction |
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There are >30 RhoGEFs, all of which contain a DH domain, which is necessary to catalyze nucleotide exchange, and an adjacent PH domain (Whitehead et al., 1997). The PH domains in some proteins, including RhoGEFs, bind phosphoinositides and mediate membrane localization (Bottomley et al., 1998). The PH domain of some RhoGEFs is necessary for nucleotide exchange (Freshney et al., 1997). The large number of RhoGEFs and their overlapping specificities for GTP binding proteins raise the questions of how Rho familydependent functions are coordinated in vivo and whether RhoGEFs are redundant or have specific functions. Although RhoGEFs have been extensively studied, these questions remain largely unanswered.
The RhoGEF Vav1 has been well characterized and two other mammalian family members have been identified recently, Vav2 and Vav3 (Katzav et al., 1989; Henske et al., 1995; Schuebel et al., 1996; Trenkle et al., 1998; Movilla and Bustelo, 1999). The expression of Vav proteins is highly conserved: both Caenorhabditis elegans and Drosophila melanogaster have Vav homologues (Dekel et al., 2000). All mammalian Vav proteins share the same domain structure. Within the NH2 terminus of Vav family members is an acidic domain and a region related to calponin that is present in some actin binding proteins. These domains are followed by the DH and PH domains and a COOH terminus with a cysteine-rich region and a single SH2 domain flanked by SH3 domains.
A truncated form of Vav1 was originally identified as an oncogene and Vav1 is expressed primarily in the hematopoietic system, as well as in the pancreas and lung (Bustelo et al., 1993). Vav1 activates Rac1 and perhaps RhoA and Cdc42 (Crespo et al., 1997; Han et al., 1997). Vav2 is widely expressed in tissues and cell lines. The literature is inconsistent with regards to the GTPases activated by Vav2. Schuebel et al. (1998) reported that Vav2 catalyzed exchange for RhoA, RhoB, and RhoG in vitro, whereas Abe et al. (2000) reported that Vav2 catalyzed exchange for Rac1, RhoA, and Cdc42 in vitro. The development of assays based on the known binding interaction between Rac/Cdc42 and p21-activated kinase (PAK) binding domain (PBD) (Sander et al., 1998), and based on the known binding interaction between RhoA and the Rho binding domain (RBD) of rhotekin (Ren et al., 1999), has permitted the determination of Vav2 exchange activity in vivo. Liu and Burridge (2000) showed that Vav2 is an exchange factor for Rac1, Cdc42, and RhoA in CHO cells in vivo using these assays. Vav3 is expressed predominantly in brain and hematopoietic cells and to a lesser extent in other tissues, and activates RhoG, RhoA, and Rac1 in vitro (Movilla and Bustelo, 1999; Trenkle et al., 2000) and RhoA and Rac1 in vivo (Zeng et al. 2000).
Tyrosine phosphorylation is necessary for the exchange activity of the Vav proteins in vitro and has been used as a surrogate for activation in vivo (Crespo et al., 1997). Vav1 is tyrosine phosphorylated in response to many signals, including B or T cell receptor activation and integrin cross-linking in myeloid cells and platelets (Bustelo, 2000). The kinases that phosphorylate Vav1 include Syk in B cells, Zap70 and Fyn in T cells, and Janus kinases in response to cytokine receptor stimulation (Bustelo, 2000; Huang et al., 2000). Vav proteins are also tyrosine phosphorylated in response to growth factor stimulation, including EGF, PDGF, and insulin (Bustelo et al., 1992; Moores et al., 2000; Pandey et al., 2000). Src family kinases phosphorylate and activate Vav proteins in vitro, but their role in Vav function in vivo is not established. Phosphorylation of Y174 stimulates exchange activity in vitro (Han et al., 1997) by disrupting the inhibitory interaction of Y174 with the DH domain (Aghazadeh et al., 2000). Mutation of Y174 to F activates Vav1, which likely reflects the inability of F174 to bind to and inhibit the DH domain (Lopez-Lago et al., 2000). The Y174F mutant is activated to the same extent as wild-type Vav1 by tyrosine phosphorylation in in vitro exchange assays, indicating that an additional site(s) is phosphorylated to stimulate exchange activity.
Vav proteins activate pathways dependent on Rho family GTP-binding proteins, including protein kinases in the mitogen-activated protein kinase and PAK families, actin rearrangement, and stimulation of transcription by serum response factor and nuclear factor kappa B (Bustelo, 2000). Interestingly, the activation of nuclear factor of activated T cells does not require the exchange activity of Vav1 (Kuhne et al., 2000). In contrast, Vav2 potentiation of nuclear factor of activated T cells requires exchange activity in B cells (Doody et al., 2000). Studies of cells from mice lacking Vav1 indicate that Vav1 is critical for B and T cell signaling (Fischer et al., 1995, 1998; Tarakhovsky et al., 1995; Zhang et al., 1995; Turner et al., 1997). B cells from Vav-/- mice have a defective proliferative response to B cell receptor activation and fail to mount an immune response to nonrepetitive antigens. Cytotoxic T cells from Vav-/- mice show a reduction in CD3- plus CD28-mediated proliferation, a decrease in IL-2 production, and a reduction in cytotoxic T cell responses. Little is known about the biological roles of Vav2 or Vav3.
To better understand the function of Vav2, we determined the GTPases activated by Vav2 in vivo and investigated the role of Src and the Vav2 DH and PH domains in regulating Vav2 exchange activity. We used a mutant form of Vav2 that lacks exchange activity to inhibit the function of endogenous Vav2 and found that this mutant blocked spreading of NIH3T3 cells on fibronectin. The cells form filopodia, but do not form lamellipodia. This mutant did not block Rac-dependent effects of PDGF or EGF, suggesting that Vav2 is necessary for integrin-dependent activation of Rac during cell spreading, but not for growth factordependent Rac activation.
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Results |
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Src is necessary for Vav2 activity
Coexpression of activated Lck with wild-type Vav2 promotes colony formation and induces lamellipodia (Schuebel et al., 1998), and the exchange activity of Vav proteins is activated by Src-like kinases in vitro, but it is not known whether Src kinases are necessary for Vav2 activation in vivo. To determine whether Src family members are necessary for Vav2 activity, HEK293T cells were transfected with Vav2 and kinase-dead (KD) Src, and Rac activation was compared with cells transfected with Vav2 alone. As shown in Fig. 3
A, KD Src blocked Vav2-dependent Rac activation. We did not detect increased Rac activation when Vav2 was cotransfected with activated Src, although Vav2 tyrosine phosphorylation was increased (data not shown), indicating that endogenous Src activity is sufficient to fully activate Vav2. We also determined the effect of KD Src and the Src inhibitor, PP2, on the formation of Vav2-dependent lamellipodia. NIH3T3 were transfected with Vav2 and KD Src or with Vav2 alone and treated with PP2. PP2 treatment or cotransfection of Vav2 and KD Src reduced the number of Vav2 transfected cells with lamellipodia by 80 and 70%, respectively, and in those cells in which lamellipodia were present, they were much less prominent (Fig. 3 B). These data strongly suggest that Src or an Src family member is necessary for Vav2 activation of Rac in vivo.
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Mutation of the Vav2 DH domain
All exchange factors for Rho family of GTPases contain a DH domain that is required to catalyze nucleotide exchange. We wished to make a Vav2 DH mutant that lacked exchange activity and thus might function as a dominant negative to allow us to identify Vav2-dependent functions. Mutations in the DH domain of several RhoGEFs disrupt catalysis of GDP/GTP exchange (Hart et al., 1994; Liu et al., 1998; Ma et al., 1998). Based on these mutations, we mutated R335 to G and the L342/L343 residues to R342/S343 in the DH domain of Vav2. The R335G mutant functioned very much like wild-type Vav2 in assays of Rac activation, stimulation of Jnk and induction of lamellipodia (data not shown), which is somewhat surprising since a similar mutant in the Trio DH domain markedly inhibited exchange activity in vitro (Liu et al., 1998). The L342R/L343S mutant did not activate Rac (Fig. 5 A), or Rho (data not shown) and did not cause lamellipodia in transfected NIH3T3 cells (Fig. 5 B) or Jnk activation in transfected COS7 cells (data not shown, see Fig. 7
C).
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L342R/L343SVav2 mutant blocks cell spreading on fibronectin
Spreading of cells on fibronectin involves Cdc42-dependent filopodia formation followed by Rac-dependent lamellipodia and then Rho activation (Clark et al., 1998; Price et al., 1998). To determine whether Vav2 is necessary for cell spreading, NIH3T3 cells were transfected with L342R/L343SVav2 and 24 h later they were trypsinized, allowed to recover, and then plated onto fibronectin. At various time points the cells were fixed and stained for Vav2 expression and for actin. The number of cells that were unspread, partially spread, or fully spread was then determined. L342R/L343SVav2 significantly impaired the spreading of NIH3T3 cells on fibronectin, indicating that Vav2 is necessary for this process (Fig. 8
A).
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Since tyrosine phosphorylation has been correlated with Vav family activity, we also determined whether plating of NIH3T3 cells on fibronectin induced tyrosine phosphorylation of Vav2. NIH3T3 cells were trypsinized, rested, and then plated onto fibronectin-coated plates. At the times after plating, Vav2 was immunoprecipitated and tyrosine phosphorylation of Vav2 and total cell lysate was assessed by Western blotting. We detected a decrease in tyrosine phosphorylation of Vav2 at early time points, followed by a return to basal levels (Fig. 8 D). Vav2 protein levels were similar for each time point (data not shown). Tyrosine phosphorylation of proteins in the total cell lysate increased in response to plating as expected.
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Discussion |
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The tyrosine kinases Syk, Zap70, and Janus kinase likely phosphorylate and activate Vav1 in vivo, but the kinase(s) that stimulate Vav2 in vivo are not known. Inhibition of Vav2-dependent Rac exchange and lamellipodia by both KD Src and the Src inhibitor PP2 indicate that Src is necessary for the activity of overexpressed Vav2. Therefore, it is likely that Src, or a family member, is also necessary for the function of endogenous Vav2.
All RhoGEFs contain a PH domain immediately COOH-terminal to the DH domain. A mutation in the PH domain of Vav2 predicted to disrupt phosphoinositide binding based both on the crystal structure of PH domains and the effects of a similar mutation in Vav1 (Ferguson et al., 1995; Hyvonen and Saraste, 1997; Han et al., 1998) lacks exchange activity, indicating that the PH domain of Vav2 is necessary for its function. This is in contrast to Vav1 and Vav3, where mutation of the PH domain does not affect exchange activity (Han et al., 1998; Movilla and Bustelo, 1999). Some exchange factors appear to require the PH domain to properly bind the GTP binding protein (Soisson et al., 1998), but the PH domain could be required to interact with phosphoinositides. However, we found that the PI-3 kinase is not necessary for Rac activation by overexpressed Vav2, in apparent contrast to Vav1, where wortmannin partially inhibits lamellipodia in cells cotransfected with Syk and Vav1 (Miranti et al., 1998). It is likely that PI-3 kinase products are not required for the activity of endogenous Vav2, but we cannot exclude the possibility that PI-3 kinase may further stimulate activated Vav2 or play a role in localization. Neither have we excluded the possibility that PtdIns-4,5-P2 is necessary for Vav2 exchange activity or that a PH domaindependent proteinprotein interaction is required.
We mutated the DH domain of Vav2 to develop a construct that would allow us to investigate the function of endogenous Vav2 using a dominant negative approach. The R335 to G mutant retained exchange activity, but the double mutation of L342/L343 to R/S, respectively, eliminated Vav2 exchange activity for both Rac1 and RhoA. This mutant did not activate Jnk or induce lamellipodia, strongly indicating that the DH domain is necessary for these functions of Vav2. To determine if this mutant could function as a specific dominant negative, we determined whether it blocked Vav1-dependent nucleotide exchange for Rac or actin organization and found that it did not. These results indicate that the DH mutant of Vav2 does not sequester Rac and thereby block all Rac-dependent events.
Since Vav2 is tyrosine phosphorylated in response to EGF and PDGF stimulation, we thought that Vav2 might be necessary for stimulation of Rac and thus growth factorinduced lamellipodia and Jnk activation. However, L342R/L343SVav2 Vav2 did not block lamellipodia induced by either PDGF or EGF, indicating that Vav2 is not necessary for Rac activation by these growth factors. L342R/L343SVav2 did not block Jnk activation by EGF. Tyrosine phosphorylation of Vav2 in response to growth factor treatment may reflect activation of Vav2 for a function other than stimulation of lamellipodia or Jnk activation, or it could also be inhibitory.
Cell spreading requires activation of Cdc42 followed by Rac and then Rho (Price et al., 1998). L342R/L343SVav2 blocked the spreading of fibroblasts on fibronectin. Cells expressing L342R/L343SVav2 did not form lamellipodia, indicating that L342R/L343SVav2 acts as a dominant negative to block Rac activation by integrins and thus that Vav2 is required for integrin activation of Rac during cell spreading. Since these cells form filopodia and we detected minimal activation of Cdc42 by Vav2, it is likely that another RhoGEF activates Cdc42 in response to integrin activation.
Since we find that Vav2 is necessary for cell spreading and Src activity is necessary for Vav2 activation of Rac and lamellipodia formation, our data suggest a model of Rac activation by integrins that depends on Src phosphorylation of Vav2. Src is known to be important in cell spreading, although some evidence indicates the kinase activity of Src may not be required (Kaplan et al., 1995). In studies in which Src kinase activity did not appear to be necessary for spreading, phosphorylation of focal adhesion proteins was increased, whereas in cells derived from the Src-/-/Yes-/-/Fyn-/- triple knock-out tyrosine phosphorylation of focal adhesion proteins is minimal (Klinghoffer et al., 1999). These results suggest that some level of Src family kinase activity is necessary for adherence and spreading, which could have been provided by Yes or Fyn in Src-/- cells. We did not detect an increase in tyrosine phosphorylation of Vav2 in response to plating of NIH3T3 cells on fibronectin, confirming the results of Liu and Burridge (2000) and Moores et al. (2000). This may reflect concomitant dephosphorylation of inhibitory sites and phosphorylation of sites that stimulate exchange activity, with little net change in total tyrosine phosphorylation. It is also possible that integrin-dependent activation of Vav2 is primarily due to cellular localization involving the PH or SH2 or SH3 domains. Based on the induction of lamellipodia in cells overexpressing Vav2, assays of GTPase activation in vivo and the ability of a dominant negative Vav2 to block lamellipodia formation in cells plated on fibronectin, we conclude that Vav2 activates Rac in vivo and is necessary for cell spreading.
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Materials and methods |
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Antibodies
The Vav2 antiserum was raised in rabbits using the GST fusion protein of the Vav2 COOH terminus as the antigen. Sera did not recognize Vav1 or Vav3. HA (12CA5) antibody was purchased from Boehringer. T7 antibody was purchased from Novagen. FLAG (M2) monoclonal antibody was purchased from Sigma-Aldrich. Antiphosphotyrosine monoclonal antibody was a gift from Dr. Thomas Roberts (Dana Farber Cancer Institute, Boston, MA). Rac and Rho monoclonal antibodies were purchased from Transduction Laboratories.
Cell culture and transfections
NIH3T3 and COS7 cells were grown in DME containing 10% (vol/vol) calf serum. HEK293T cells were grown in DME with 10% (vol/vol) heat-inactivated fetal calf serum. Transient transfections were done using Superfect (QIAGEN) or Lipofectamine Plus Reagent (GIBCO BRL) according to the manufacturer's guidelines.
Cell stimulation and immunoprecipitation
PP2 was used at a concentration of 2 µM for 30 min. In experiments in which growth factor effects on tyrosine phosphorylation of Vav2 were determined, cells were serum starved for 16 h followed by treatment with PDGF (40 ng/ml) or EGF (33 nM) for 15 min. PDGF was used at a concentration of 15 ng/ml for immunofluorescence studies. Immunoprecipitations were done by first washing the cells in cold PBS and then lysing them in a buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 10 mM EDTA, 0.2 mM sodium orthovanadate, 10% glycerol, 4 µg/ml leupeptin, 4 µg/ml pepstatin, and 4 µg/ml AEBSF. Lysates were cleared by centrifugation and incubated with antibody and protein ASepharose beads for 3 h at 4°C. The beads were washed three times in lysis buffer and associated proteins were resolved by SDS-PAGE followed by transfer to Immobilon-P membrane (Millipore). Membranes were blocked with 2% (wt/vol) BSA in Tris-buffered saline. The blots were probed with primary antibody and then with horseradish peroxidaseconjugated secondary antibody (Boehringer) and visualized by enhanced chemiluminescence (Dupont) according to the manufacturer's instructions.
Microscopy
NIH3T3 cells on glass coverslips were fixed in 3% paraformaldehyde-PBS for 10 min, permeabilized in 0.2% Triton X-100 for 2 min, and blocked in 2% BSA-PBS for 10 min. The cells were incubated with primary antibodies for 1 h, washed, and then incubated with secondary antibodies (Jackson ImmunoResearch Laboratories) and rhodamine-phalloidin (100 ng/ml; Sigma-Aldrich) for 1 h. Coverslips were mounted onto slides with Fluoromount-G (Southern Biotechnology Associates, Inc.). Images of both live and fixed cells were acquired using a microscope (Diaphot 300; Nikon) mounted with a camera (SenSys CCD; Photometrics) and processed using Vaytek imaging software.
Jnk kinase assays
COS7 cells were transfected and then lysed 24 h later in a buffer containing 40 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 25 mM NaF, 1 mM sodium vanadate, 4 µg/ml aprotinin, and 4 µg/ml leupeptin. FLAG-Jnk was immunoprecipitated from cleared lysates with M2 antibody and protein Gsepharose beads for 1 h at 4°C. Beads were washed and kinase activity was assayed in 30 µl of kinase buffer (25 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM ß-glycerophosphate, 0.1 mM sodium vanadate, 2 mM DTT), 20 µM ATP, 5 µCi [-32P]ATP, and 5 µg GST-c-jun. Reactions were incubated at 30°C for 30 min and terminated by the addition of 10 µl 5x Laemmli buffer (Ma et al., 1998). The proteins were separated by SDS-PAGE and phosphorylation quantified with a Molecular Imager (Bio-Rad Laboratories).
GTPase activity assays
GST fusion proteins of PBD (GST-PBD; Sander et al., 1998) or rhotekin (GST-RBD; Ren et al., 1999) were as described by (O'Connor et al., 2000). For Rac and Cdc42 activity assays, cells were lysed 24 h after transfection in a buffer containing 20 mM Hepes, pH 7.5, 100 mM NaCl, 0.5% NP-40, 10 mM MgCl2, 10 mM ß-glycerophosphate, 10% glycerol, 4 µg/ml leupeptin, and 4 µg/ml pepstatin (Sander et al., 1998). Lysates were cleared and incubated with GST-PBD (to assess Rac or Cdc42 activation) or GST-TRBD (to assess Rho activation) bound to glutathione agarose beads for 30 min at 4°C. For the Rho activity assay, lysates were diluted 1:1 in a buffer containing 50 mM Tris, pH 7.2, 500 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF. The beads were washed three times in lysis buffer and analyzed for bound Rac, Cdc42 and Rho by Western blotting.
Cell spreading on fibronectin
Plates or coverslips were coated with fibronectin (10 µg/ml) for 2 h at 37°C. Cells were prepared for plating by trypsinization and then were washed once in DME with 1 mg/ml soybean trypsin inhibitor. Cells were pelleted, washed twice with DME, and then resuspended in DME and allowed to recover for 40 min at 37°C. Cells were then plated onto fibronectin-coated coverslips or plates. Cells were fixed at 15 and 60 min after plating and stained for Vav2 with T7 antibody followed by AMCA antimouse secondary antibody and rhodamine-phalloidin. Vav2-expressing cells were classified as unspread (adherent with no projections), partially spread (adherent with limited lamellipodia), and fully spread.
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health grant GM 54389 (CLC).
Submitted: 28 March 2001
Revised: 25 May 2001
Accepted: 30 May 2001
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References |
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