Mechanism of Epidermal Growth Factor Regulation of Vav2, a Guanine Nucleotide Exchange Factor for Rac*

Péter Tamás, Zita Solti, Petra BauerDagger §, András Illés, Szabolcs Sipeki, András Bauer, Anna Faragó, Julian DownwardDagger , and László Buday

From the Department of Medical Chemistry, Semmelweis University Medical School, 9 Puskin Street, 1088 Budapest, Hungary and Dagger  Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Received for publication, July 26, 2002, and in revised form, November 15, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vav2 is a member of the Vav family that serves as a guanine nucleotide exchange factor for the Rho family of Ras-related GTPases. Unlike Vav1, whose expression is restricted to cells of hematopoietic origin, Vav2 is broadly expressed. Recently, Vav2 has been identified as a substrate for the epidermal growth factor (EGF) receptor; however, the mechanism by which Vav2 is activated in EGF-treated cells is unclear. By the means of an in vitro protein kinase assay, we show here that purified and activated EGF receptor phosphorylates Vav2 exclusively on its N-terminal domain. Furthermore, EGF receptor phosphorylates Vav2 on all three possible phosphorylation sites, Tyr-142, Tyr-159, and Tyr-172. In intact cells we also show that Vav2 associates with the activated EGF receptor in an Src homology 2 domain-dependent manner, with Vav2 Src homology 2 domain binding preferentially to autophosphorylation sites Tyr-992 and Tyr-1148 of the EGF receptor. Treatment of cells with EGF results in stimulation of exchange activity of Vav2 as measured on Rac; however, the intensity of the exchange activity does not show any correlation with the level of Vav2 tyrosine phosphorylation. Introducing a point mutation into the Vav2 pleckstrin homology domain or treatment of cells with the phosphatidylinositol 3-kinase inhibitor LY294002 prior to EGF stimulation inhibits Vav2 exchange activity. Although phosphorylation mutants of Vav2 can readily induce actin rearrangement in COS7 cells, pleckstrin homology domain mutant does not stimulate membrane ruffling. These results suggest that EGF regulates Vav2 activity basically through phosphatidylinositol 3-kinase activation, whereas tyrosine phosphorylation of Vav2 may rather be necessary for mediating protein-protein interactions.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In recent years, evidence has accumulated indicating that Rho-like GTPases are key regulators in signaling pathways that link extracellular growth signals to the assembly and organization of the actin cytoskeleton (1). To date, the Rho family consists of at least 14 members, including the widely expressed RhoA, Rac1, and Cdc42 proteins. RhoA regulates formation of stress fibers and focal adhesions in many types of cells, Rac1 stimulates formation of lamellipodia and membrane ruffling, and Cdc42 controls assembly of filopodia (2). Like other members of the Ras superfamily, Rho proteins act as molecular switches to control cellular processes by cycling between active, GTP-bound and inactive, GDP-bound states. Activation of the GTPases, through GDP-GTP exchange, is promoted by guanine nucleotide exchange factors, whereas inactivation is stimulated by GTPase-activating proteins (1, 3). Rho guanine nucleotide dissociation inhibitors appear to stabilize the inactive, GDP-bound form of the protein. Activated Rho GTPases interact with cellular target proteins, such as rhotekin, rhophilin, citron, and several serine/threonine kinases, to trigger a wide variety of cellular responses, including the reorganization of actin cytoskeleton (1, 3).

Vav was originally identified as a transforming gene when an N-terminally truncated form was expressed in fibroblasts as a result of a recombination event occurring during transfection (4). Vav is normally expressed predominantly if not exclusively in hematopoietic cells where it plays a key role in signaling pathways downstream of the T cell receptor and the B cell receptor (for review see Ref. 5). Vav is rapidly phosphorylated on tyrosine following activation of these and other receptors. In addition to two SH31 domains, an SH2 domain, a cysteine-rich domain, a calponin homology domain, and an acidic domain, Vav also contains a Dbl homology domain followed by a pleckstrin homology domain characteristic of guanine nucleotide exchange factors for proteins of the Rho subfamily of the Ras superfamily of GTPases. Recently Vav has been shown to act enzymatically as a guanine nucleotide exchange factor for Rac1, Cdc42, and RhoA (6, 7). The guanine nucleotide exchange activity of Vav toward Rac1 has been shown to be stimulated both in vitro and in vivo following tyrosine phosphorylation of Vav by the T cell-specific tyrosine kinase Lck (6, 7).

Recently, novel Vav family members, Vav2 and Vav3, have been identified that are widely expressed in human tissues (8-10). Both Vav2 and Vav3 have been shown to be phosphorylated on tyrosine residues in response to growth factors, such as EGF and PDGF (9, 11-15). In addition, Vav2 is capable of associating with autophosphorylated growth factor receptors through its SH2 domain (13, 14). It has been also reported that Vav2 SH2 domain can bind directly to multiple autophosphorylation sites on the PDGF receptor (13). Although the interaction of Vav family members with plasma membrane receptors has been well characterized, little is known about the mechanism by which receptor tyrosine kinases regulate the activity of Vav2 or Vav3.

PI3K is known to play a role in the activation of Rac, both from the study of the actin cytoskeleton (16, 17) and direct measurement of GTP binding to Rac in metabolically labeled cells (18). Activation of PI3K in the absence of other stimuli is sufficient to cause membrane ruffling (19, 20), a downstream effect of Rac activation. The mechanism by which PI3K activation leads to Rac activation is unknown. However, it has been reported recently that 3' phosphorylated phosphoinositides such as phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate can bind to the pleckstrin homology (PH) domain of Vav and promote its exchange activity toward Rac in vitro (21). Although this suggests that the guanine nucleotide exchange activity of Vav toward Rac could be responsive to PI3K activation, the role of Vav PH domain is still controversial.

In this study we have investigated the role of Vav2 as a regulator of Rac activity in EGF signaling pathway. We identified the EGF receptor-dependent phosphorylation sites of Vav2 in vitro and in vivo and investigated the role of Vav2 tyrosine phosphorylation in regulation of Vav2 activity. Our results, based on several tyrosine mutants of Vav2, demonstrate that the role of tyrosine phosphorylation of Vav2 is likely less important than its PI3K-dependent activation and probably more complicated than just one tyrosine phosphorylation site regulates Vav2 activity. In addition, we provide evidence that PI3K stimulated by EGF treatment of cells contributes to Vav2-dependent Rac activation. These results suggest that EGF regulates Vav2 activity through PI3K activation, whereas tyrosine phosphorylation of Vav2 may be necessary for mediating protein-protein interactions.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids and Constructs-- Full-length Vav2 were amplified by PCR and subcloned into SalI/EcoRI site of EGFP-C2 plasmid (Clontech). cDNA corresponding to the N-terminal part of Vav2 (amino acids 1-194; NT domain), Dbl homology domain (amino acids 195-386; DH), PH domain (amino acids 387-590), and the C-terminal SH3-SH2-SH3 domains of Vav2 (amino acids 591-590) were amplified by PCR and subcloned into the SalI/EcoRI site of pGEX-4T vector (Amersham Biosciences). Mutations were created with the QuikChange site-directed mutagenesis kit (Stratagene). In all cases the constructs were verified by DNA sequencing. GST fusion proteins were purified by binding to glutathione-agarose (Sigma) and gave essentially single bands on Coomassie Blue-stained SDS polyacrylamide gels. GFP were transiently expressed in COS7 cells and analyzed by SDS-PAGE followed by Western blotting using monoclonal antibody against GFP.

Antibodies and Peptides-- Monoclonal antibody raised against the GFP was supplied by the Cancer Research UK Hybridoma Development Unit. Anti-phosphotyrosine antibody 4G10 was obtained from Upstate Biotechnology, Inc. Anti-Rac antibody and anti-EGF receptor antibody were purchased from BD Biosciences Transduction Laboratories. Anti-GST, anti-PI3K p110alpha , and anti-phospho-Akt (Ser-473) rabbit polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. Monoclonal anti-Vav2 antibody was purchased from Babraham Bioscience Technologies. Five phosphopeptides, pY992, pY1068, pY1086, pY1148, and pY1087, derived from the autophosphorylation sites of EGF receptor, were synthesized with the following sequences: DADE(pTyr)LIPQ, VQNP(pTyr)HNQP, PVPE(pTyr)INQS, DNPD(pTyr)QQDF, and ENAE(pTyr)LRVA, respectively.

Cell Lines and Stimulation-- COS7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (50 µg/ml). For stimulation, cells, 80% confluent, were serum-starved overnight and stimulated with EGF (Sigma-Aldrich) at 50 ng/ml for 5 min. Alternatively, the cells were pre-treated with the PI3K inhibitor LY294002 at 20 µM for 60 min and then stimulated with EGF as above.

Transient Transfection-- LipofectAMINE was obtained from Invitrogen and used for transfection of COS7 cells according to the manufacturer's instructions. Briefly, 4 × 105 cells were plated on 6-well dishes 24 h prior to transfection. 1 µg of the various Vav2 constructs and 7 µl of LipofectAMINE were added to each well in 1 ml of Dulbecco's modified Eagle's medium. After 5 h, cells were washed once with Dulbecco's modified Eagle's medium and cultured in their regular medium. Prior to stimulation with EGF, cells were serum-starved for 18 h.

Immunoprecipitation and Western Blotting-- Immunoprecipitation and Western blotting were performed as described previously (22). Briefly, after stimulation, COS7 cells were washed with ice-cold phosphate-buffered saline and lysed in 0.5 ml of ice-cold 50 mM Hepes puffer, pH 7.4, containing 100 mM NaCl, 1% Triton X-100, 20 mM NaF, 1 mM EGTA, 1 mM Na3VO4, 1 mM p-nitrophenyl phosphate, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml each of leupeptin, soybean trypsin inhibitor, and aprotinin. Lysates were clarified by centrifugation at 15,000 × g for 10 min at 4 °C. Lysates were then precleared with protein G-Sepharose, and proteins were immunoprecipitated with 8 µg of monoclonal anti-GFP antibody. Immunoprecipitates were collected by incubating with protein G-Sepharose (Amersham Biosciences) for 1 h at 4 °C. Immunoprecipitates were washed three times with ice-cold 50 mM Hepes buffer, pH 7.4, containing 250 mM NaCl, 0.2% Triton X-100, and 0.1 mM Na3VO4. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with the indicated antibodies. Blots were developed by the enhanced chemiluminescence (ECL; Amersham Biosciences) system. For precipitation with GST fusion proteins, 5 µg of GST-Vav2-SH2 domain was used as non-covalently-bound adducts to glutathione-agarose beads. Separation of bound proteins and immunoblotting were performed as described above.

Kinase Assay-- For Vav2 phosphorylation, 5-5 µg of GST-tagged fragments of Vav2 immobilized on glutathione-agarose beads were washed with phosphorylation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MnCl2, 0.1% Triton X-100, 0.1 mM Na3VO4, 0.1 mM ATP) and resuspended in 50 µl of phosphorylation buffer. Kinase reaction was initiated by addition of 1 µg of EGF receptor purified by affinity chromatography from lysates of EGF-treated A431 cells, as described previously (22). After 20 min at room temperature, immobilized GST proteins were placed on ice and washed three times with ice-cold solution containing 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 0.2% Triton X-100, and 0.1 mM Na3VO4. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-phosphotyrosine antibody 4G10.

PI3K Assay-- PI3K activity was measured as described previously (23, 24). After appropriate treatments, cell lysates were incubated with anti-PI3K p110alpha antibody as indicated for 1 h at 4 °C. Immunoprecipitates were collected by incubating with protein G-Sepharose (Amersham Biosciences) for 30 min at 4 °C. Immunoprecipitates were washed three times with ice-cold 50 mM Hepes buffer, pH 7.4, containing 250 mM NaCl, 0.2% Triton X-100, and 0.1 mM Na3VO4 and then 2 times with kinase buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, and 10 µM Na3VO4. The kinase activity was measured by resuspending the immunoprecipitates in 30 µl of kinase buffer and incubating with 10 µl of 2 mg/ml phosphatidylinositol, 1 µl of 1 M MgCl2, 1 µl of 100 mM ATP, and 10 µCi of [gamma -32P]ATP for 10 min at 22 °C. The reaction was terminated by addition of 20 µl of 5 N HCl and 200 µl of chloroform:methanol (1:1) mix. The aqueous and organic phases were separated by centrifugation at 2000 rpm for 10 min. The organic phase containing the phosphoinositol phosphates were spotted onto a silica gel TLC plate coated with 1% potassium oxalate and separated in a solvent system consisting of chloroform:methanol:water:ammonium hydroxide (90:70:14.6:5.4). The TLC plate was exposed to x-ray film for 1-5 h at -80 °C and developed.

Activity Assay for Rac1-- GST-N-PAKalpha fusion proteins were expressed in and purified from Escherichia coli on glutathione-Sepharose beads. For the pull outs, transiently transfected COS7 cells were grown in 6-cm dishes, starved overnight, and harvested 48 h after transfection in lysis buffer containing 10 mM MgCl2. The precleared lysates were added to the GST-N-PAKalpha beads (~20 µg of N-PAK proteins per point) and tumbled on a wheel for 60 min at 4 °C. After a single wash in ice-cold phosphate-buffered saline containing 0.1% (v/v) Triton X-100 and 5 mM MgCl2 the beads were drained and subjected to SDS-PAGE. Bound and activated Rac was identified following Western blotting.

Immunofluorescence-- COS7 cells on glass coverslips were fixed in 4% paraformaldehyde/phosphate-buffered saline for 15 min and permeabilized in 0.2% Triton X-100 for 5 min. The cells were then incubated with TRITC-phalloidin (Sigma-Aldrich) at a final concentration of 0.1 µg/ml for 20 min. Coverslips were mounted onto slides in a 100 mM Tris-HCl buffer, pH 8.5, containing 10% Mowiol 4-88 (Calbiochem), 25% glycerol, and 2.5% 1,4-diazabicyclo-[2.2.2]octane (DABCO; Sigma-Aldrich). Expression of various Vav2-GFP constructs was determined by GFP fluorescence. Microscopy was performed on a Nikon Eclipse E400 fluorescence microscope.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epidermal Growth Factor Stimulates Phosphorylation of Vav2 on Three Tyrosine Residues-- It has been well documented that EGF receptor can phosphorylate Vav2 in intact cells (11-14, 25). López-Lago et al. (12) have shown recently that tyrosine kinase Lck can phosphorylate all potential tyrosine residues (Tyr-142, Tyr-160, Tyr-174) in the N-terminal domain of Vav1. In addition, a phosphospecific antibody generated against the phosphorylated Tyr-174 was capable of recognizing the phosphorylated Vav2 and Vav3 proteins in EGF-treated cells, suggesting that this phosphorylation site is conserved in all known member of the Vav family (12). However, the EGF receptor-dependent phosphorylation sites on Vav2 (apart from Tyr-174) have not been identified so far.

To map the phosphorylation site(s) of EGF receptor on Vav2, we cut the Vav2 molecule into four parts and expressed the protein domains as GST fusion proteins. These protein domains represent the NT part of Vav2, the DH domain, the PH domain, and finally the C-terminal part of the molecule that contains two SH3 and one SH2 domains (SH3-SH2-SH3). Activated EGFR was then purified from EGF-treated A431 cells and used for an in vitro phosphorylation assay, as described under "Experimental Procedures." GST fusion proteins were mixed with activated EGF receptor for 20 min in the presence of ATP, washed samples were separated by SDS-PAGE, and immunoblotted proteins were probed with anti-phosphotyrosine antibody. As illustrated in Fig. 1A, EGF receptor can phosphorylate only the N-terminal domain of Vav2 but not the GST fusion protein control or other domains of Vav2.


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Fig. 1.   EGF receptor phosphorylates all three tyrosine residues in the Vav2 N-terminal domain. A, GST-tagged fragments of Vav2 immobilized on glutathione-agarose were incubated with EGF receptor purified from lysates of EGF-treated A431 cells. The kinase reaction was performed as described under "Experimental Procedures." Immobilized GST-Vav2 proteins were then washed and subjected to SDS-PAGE. Phosphorylation of Vav2 fragments was visualized by Western blotting with anti-phosphotyrosine antibody. The positions of different GST fusion proteins are indicated. B, GST-Vav2 and its mutant forms immobilized on glutathione-agarose were incubated with activated and purified EGF receptor. The kinase reaction was performed as described under "Experimental Procedures." Immobilized GST-Vav2 proteins were than washed, subjected to SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody. To assure equal loading of different GST fusion proteins, nitrocellulose membrane was re-probed with anti-GST antibody.

Previous studies have revealed that the Vav can be phosphorylated by a number of tyrosine kinases in activated T and B lymphocytes, including Fyn, Syk, ZAP-70, and Lck (26), and the major phosphorylation site was Tyr-174 (7, 12, 21). This phosphorylation site is in the N-terminal domain of Vav proteins; therefore, we tested whether EGF receptor is also capable of phosphorylating Tyr-172 of Vav2, a tyrosine residue corresponding to Tyr-174 of Vav. A point mutation was introduced into the Vav2 NT domain, changing tyrosine 172 to phenylalanine (Y172F). In the phosphorylation assay, GST-NT and GST-NT-Y172F fusion proteins were incubated with the EGF receptor. After intensive washing, samples were subjected to SDS-PAGE and transferred to nitrocellulose. As the anti-phosphotyrosine immunoblot demonstrates, we could detect a small but significant decrease in the level of mutant NT domain phosphorylation (Fig. 1B). In addition to the Tyr-172, the N-terminal domain of Vav2 contains two more conserved tyrosine residues that can be found in all member of the Vav family (12). Therefore, we introduced additional point mutations into NT-Y172F domain, changing tyrosines 142 and 159 to phenylalanines, respectively. The performed phosphorylation assay and then the anti-phosphotyrosine immunoblot shows that the EGF receptor-dependent phosphorylation of the double mutant NT-Y172/142F significantly decreased compared with the level of NT-Y172F phosphorylation. In addition, the phosphorylation of the other double mutant, NT-Y172/159F, was further decreased (Fig. 1B). These results suggest that activated EGF receptor is likely capable of phosphorylating all three tyrosine residues in the N-terminal domain of Vav2. It is noteworthy that the isoform of Vav2 we used in our experiment possesses another tyrosine residue, Tyr-187, in its N-terminal domain. However, this tyrosine residue is neither conserved among Vav family members nor represents consensus phosphorylation site for EGF receptor. Nevertheless, point mutation was introduced into Vav2 NT domain, changing Tyr-187 to phenylalanine, but this mutation did not affect the phosphorylation level of Vav2 NT domain (data not shown). Immunoblotting of the nitrocellulose membrane with anti-GST antibody revealed that equal amounts of Vav2-NT constructs were immunoprecipitated (Fig. 1B).

To confirm that EGF receptor phosphorylates Vav2 on all three tyrosine residues in vivo, GFP-tagged fusion constructs of Vav2 were generated encoding wild type Vav2 and different Vav2 mutants including Vav2-Y172F, Vav2-Y172/142F, Vav2-Y172/159F, and Vav2-Y159F. These mutants were transiently expressed in COS7 cells and then Vav2 proteins were immunoprecipitated with anti-GFP monoclonal antibodies using lysates prepared from unstimulated or EGF-treated cells. As shown in Fig. 2A, EGF treatment of cells leads to a strong tyrosine phosphorylation of wild type Vav2. However, when single point mutants were used, both Vav2-Y172F and Vav2-Y159F showed decreased tyrosine phosphorylation. In addition, the overall level of tyrosine phosphorylation of double mutant Vav2-Y172/142F was further reduced compared with Vav2-Y159F or Vav2-Y172F. Finally, tyrosine phosphorylation of the double mutant Vav2-Y172/159F was hardly detectable. This experiment demonstrates that in response to EGF all three candidate phosphorylation sites in the N-terminal domain of Vav2 are phosphorylated. Immunoblotting of the same nitrocellulose membrane with anti-GFP antibody revealed that equal amounts of different Vav2 constructs were immunoprecipitated (Fig. 2C).


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Fig. 2.   Activation of Rac1 by phosphorylation mutants of Vav2 in vivo. A, GFP-tagged fusion constructs of Vav2 were transiently expressed in COS7 cells and then Vav2 was immunoprecipitated with monoclonal anti-GFP antibody using lysates prepared from unstimulated or EGF-treated cells. Immunoprecipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-phosphotyrosine antibody. B, COS7 cells were transiently transfected with various GFP fusion constructs of Vav2 as indicated. The nucleotide exchange activity of Vav2 in vivo was determined indirectly by the ability of Rac1 to bind to GST-N-PAK fusion protein. Bound Rac1 protein was then immunoblotted with anti-Rac1 monoclonal antibody. C, the amount of Vav2 proteins immunoprecipitated is detected by anti-GFP monoclonal antibody. D, endogenous Vav2 protein was immunoprecipitated with monoclonal anti-Vav2 antibody from lysates of quiescent and EGF-treated cells. Prior to stimulation the cells were treated with specific EGF receptor inhibitors PD168393 and PD153095. Immunoprecipitated proteins were than washed, subjected to SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody. To assure equal immunoprecipitation of Vav2 proteins, nitrocellulose membrane was re-probed with anti-Vav2 antibody. These results are typical of five experiments.

To confirm that endogenous Vav2 can be tyrosine-phosphorylated in COS7 cells in response to EGF stimuli, and EGF acts specifically through its receptor, Vav2 was immunoprecipitated with monoclonal anti-Vav2 antibody from lysates of unstimulated and EGF-treated cells. Fig. 2D demonstrates that EGF stimulates phosphorylation of endogenous Vav2 in COS7 cells, and autophosphorylated EGF receptor is co-immunoprecipitated with Vav2. In addition, the effect of EGF could be inhibited by addition of specific EGF receptor inhibitors PD168393 and PD153035 (Calbiochem).

The SH2 Domain of Vav2 Binds to Autophosphorylation Sites Tyr-992 and Tyr-1148 of the EGF Receptor-- Although it has been reported recently that interaction of Vav2 with activated EGF receptor is mediated in an SH2 domain-dependent manner, Pandey et al. (14) used only GST fusion proteins to investigate the nature of this interaction. To examine whether Vav2-EGFR association is an SH2 domain-dependent interaction in vivo, we introduced point mutations into the SH2 domain of GFP-Vav2 protein, as described previously for Vav (27). Tryptophan at position 673 of Vav2 was mutated to arginine (W673R), and glycine at position 693 was changed to arginine (G693R). GFP-Vav2 proteins were transiently expressed in COS7 cells and then GFP proteins were immunoprecipitated from lysates of untreated and EGF-stimulated cells. The immunoprecipitates were blotted with anti-phosphotyrosine antibody 4G10. As illustrated in Fig. 3A, wild type Vav2 becomes tyrosine-phosphorylated upon EGF treatment of cells and co-immunoprecipitates with the autophosphorylated EGF receptor. However, when SH2 mutant proteins were used, interactions of both W673R and G693R mutants with the EGF receptor were strongly decreased. These data clearly suggest that Vav2 associates with the autophosphorylated EGF receptor in an SH2 domain-dependent manner in vivo. Interestingly, tyrosine phosphorylation levels of those Vav2 mutants that are not capable of associating with the activated EGF receptor are markedly decreased.


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Fig. 3.   The SH2 domain of Vav2 binds to autophosphorylation sites Tyr-992 and Tyr-1148 of the EGF receptor. A, GFP fusion proteins of wild type and mutant Vav2 were transiently expressed in COS7 cells and then serum-starved cells were stimulated with EGF or left untreated. Lysates were immunoprecipitated with anti-GFP antibody. After SDS-PAGE and transfer to nitrocellulose, samples were analyzed by anti-phosphotyrosine antibody. B, COS7 cells were stimulated with EGF at 50 ng/ml for 5 min. Proteins were then precipitated with GST-Vav2-SH2 fusion protein immobilized on glutathione-agarose in the presence of different concentrations of the indicated phosphopeptides. Precipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-phosphotyrosine antibody. Nitrocellulose membrane was later re-probed with anti-EGF receptor antibody. The control lane represents the amount of autophosphorylated EGF receptor bound to the Vav2 SH2 domain without addition of any phosphopeptide.

To investigate further the Vav2/EGF receptor interactions, specific phosphopeptides were synthesized based on the major autophosphorylation sites of the EGF receptor. Untransfected COS7 cells were stimulated with EGF for 2 min, and cell lysates were then mixed with GST-Vav2-SH2 fusion protein immobilized on glutathione-agarose beads in the presence of different phosphopeptides. Fig. 3B shows that DADE(pTyr)LIPQ and DNPD(pTyr)QQDF phosphopeptides corresponding to autophosphorylation sites pY992 and pY1148 of EGF receptor, respectively, could compete binding of activated EGF receptor to the Vav2 SH2 domain. Other phosphopeptides, however, based on the Tyr-1068, Tyr-1086, and Tyr-1173 autophosphorylation sites of the EGF receptor were unable to inhibit interaction of EGF receptor with the Vav2 SH2 domain. Furthermore, unphosphorylated forms of the Tyr-992 and Tyr-1148 peptides were not capable of competing with tyrosine-phosphorylated EGF receptor (Fig. 3B). These results suggest that Vav2 SH2 domain may bind to either the autophosphorylation residues Tyr-992 or Tyr-1148 on the EGF receptor.

Tyrosine Phosphorylation Mutants of Vav2 Are Capable of Activating Rac1 in Vivo-- To determine whether the exchange activity of the Vav2 protein toward Rac might be regulated by post-translational modifications such as phosphorylation, an assay was used that would measure the exchange activity of Vav2 in intact cells. The N-terminal domain of PAKalpha binds to Rac in a GTP-dependent manner (28); this was expressed as GST fusion protein in bacteria, and the purified protein used as an affinity resin to "pull out" GTP-bound Rac from cell lysates. Endogenous Rac1 was then detected by immunoblotting with anti-Rac1 monoclonal antibody.

First, we tested whether overexpression of GFP-Vav2 in COS7 cells results in Rac activation in vivo in response to EGF stimulation. GFP or GFP-Vav2 were transiently expressed in COS7 cells, serum-starved cells were stimulated with EGF for 5 min or left untreated and then activated, GTP-bound Rac1 was precipitated by GST-PAK or GST alone immobilized on beads. As Fig. 4 demonstrates, when GFP alone was used for transfection, no GTP-bound Rac1 could be detected. Even following EGF treatment of the cells we could not see any activated Rac1. However, when full-length Vav2 is overexpressed a small amount of Rac1 can be recovered in GST-PAK pull outs. When the Vav2-expressing cells are treated with EGF prior to lysis the amount of Rac1 in a GTP-bound state increases dramatically, suggesting that EGF treatment can activate the guanine nucleotide exchange activity of Vav2 toward Rac1 in intact cells (Fig. 4).


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Fig. 4.   Overexpression of Vav2 activates Rac1 in an EGF-dependent manner in COS7 cells. COS7 cells were transiently transfected with GFP or GFP-Vav2 and stimulated with EGF or left untreated. The nucleotide exchange activity of Vav2 in vivo was determined indirectly by the ability of Rac1 to bind to GST-N-PAK fusion protein. Bound Rac1 protein was then immunoblotted with anti-Rac1 monoclonal antibody.

To determine the effect of Vav2 phosphorylation on its exchange activity toward Rac1, various phosphorylation mutants of Vav2 were expressed in cells, and their ability to activate Rac1 in response to EGF stimulation was tested. In the same experiment where tyrosine phosphorylation of Vav2 mutants was evaluated, cell lysates were also used for the Rac activity assay, as described above. Fig. 2B shows that following EGF treatment of cells overexpressing wild type Vav2 significant increase of GTP-bound Rac1 can be detected. However, in those cells that overexpress different phosphorylation mutant forms of Vav2 no correlation was found between their phosphorylation level and their ability to activate Rac1 in vivo. Moreover, the Vav2-Y159/172F double mutant whose phosphorylation is markedly reduced in response to EGF is capable of activating Rac1.

EGF-dependent Vav2 Activity Requires an Intact PH Domain-- It has been reported earlier that the activity of Vav protein family is stimulated by products of PI3K binding to the PH domain; however, the role of the PH domain in the regulation of Vav proteins is still controversial. For example, mutations in the PH domain of Vav1 do not completely inhibit exchange activity, and PH mutant Vav3 was as active as wild type Vav3 assessed by its ability to induce morphological changes in transfected cells (9, 21). On the other hand, it has been shown recently that mutations of the PH domain of Vav2 impaired Vav2 signaling, transforming activity, and membrane association; however, these mutations did not influence exchange activity measured on Rac (29). To address the possible role of PI3K in Vav2 regulation in intact cells, wild type Vav2 was transiently expressed in COS7 cells stimulated with EGF or left untreated. As shown in Figs. 5 and 6, in EGF-stimulated cells Vav2-dependent Rac1 activation could be inhibited by addition of a specific PI3K inhibitor LY294002.


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Fig. 5.   Effect of PI3K inhibitor on Rac activation by Vav2 in intact cells. A, COS7 cells were transiently transfected with GFP-Vav2 construct. They were serum-starved overnight and stimulated with EGF or left untreated. Prior to stimulation the cells were treated with the PI3K inhibitor LY294002. The status of GTP-loaded Rac1 was examined by binding to GST-N-PAK fusion protein and anti-Rac1 immunoblotting. B, immunoblot of the lysates used in A showing the expression levels of GFP-Vav2. C, PI3K was immunoprecipitated with anti-PI3K p110alpha antibody using cell lysates of unstimulated and EGF-treated cells. Prior to stimulation the cells were treated with the PI3K inhibitor LY294002. PI3K assay was then performed with the immunoprecipitates in the presence of phosphatidylinositol and [gamma -32P]ATP as described under "Experimental Procedures." D, COS7 cells were pretreated with LY294002 and then stimulated with EGF or left untreated. Cell lysates were then immunoblotted with phosphospecific anti-phospho-Akt (anti-P-Akt) antibody. These results are representative of three experiments.


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Fig. 6.   PH domain mutant Vav2 fails to activate Rac1 in response to EGF. A, COS7 cells were transiently transfected with GFP-Vav2 or GFP-Vav2-R425C constructs. Following serum starvation, cells were stimulated for 5 min with EGF as indicated. Prior to stimulation the cells were treated with the PI3K inhibitor LY294002. The cleared lysates were subjected to a binding reaction with the GST-N-PAK fusion protein, and specifically bound GTP-loaded Rac1 was detected in an immunoblot with anti-Rac1 antibody. B, lysates from COS7 cells expressing the GFP-Vav2 constructs were immunoblotted with anti-GFP antibody. These results are representative of three experiments.

To confirm that LY294002 indeed inhibits PI3K activity in our system, endogenous PI3K was immunoprecipitated from lysates of quiescent and EGF-treated cells, and PI3K activity was measured as described under "Experimental Procedures." As shown in Fig. 5C, EGF is capable of inducing activity of PI3K, and this effect was inhibited by addition of the specific inhibitor LY294002. It has been well documented that Akt activation occurs through phosphorylation of threonine 308 in the activation loop, followed by autophosphorylation of serine 473 in the C-terminal region (30). To monitor the activation of PI3K within the cell, we evaluated phosphorylation of serine 473 as a marker of PI3K activity. Fig. 5D demonstrates that EGF can stimulate phosphorylation of threonine 473 on Akt. In addition, pretreatment of cells with LY294002 could completely inhibited endogenous activity of PI3K.

The role of PI3K in the activation of Rac by Vav2 in intact cells was further investigated by introducing point mutation into the PH domain of Vav2, changing the conserved arginine 425 to cysteine (R425C). PH domains of several enzymes carrying this mutation are not capable of binding phosphatidylinositol 3,4,5-trisphosphate (31, 32). COS7 cells were transiently transfected with Vav2-R425C, and Rac1 activation was measured in response to EGF stimulation. Interestingly, in quiescent cells expressing mutant Vav2 slight increase of GTP-bound Rac1 was detected. (Fig. 6). In EGF-stimulated cells Vav2-R425C did not activate further Rac1, and LY2940042 did not show any effect on Rac1 activation, indicating that in EGF signaling pathway PH domain is required for Vav2 activity.

Recently, tyrosine phosphorylation of Vav in NIH-3T3 cells in response to serum stimulation has been shown to be Wortmannin-sensitive, suggesting that PI3K contributes to Vav phosphorylation (33). In addition, products of PI3K have been reported to enhance Lck-dependent phosphorylation of Vav in vitro (21). To address whether tyrosine phosphorylation of Vav2 upon EGF treatment depends on the activity of PI3K, point mutation was introduced into the PH domain of GFP-Vav2, as described above. GFP-Vav2 and GFP-Vav2-R425C constructs were transiently expressed in COS7 cells and then Vav2 proteins were immunoprecipitated with anti-GFP antibody using lysates from unstimulated or EGF-treated cells. Fig. 7 demonstrates that pretreatment of cells with the PI3K inhibitor LY294002 failed to decrease the phosphorylation levels of either GFP-Vav2 or GFP-Vav2-R420C. Based on these data it appears that products of PI3K are not involved in the EGF-dependent regulation of Vav2 tyrosine phosphorylation.


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Fig. 7.   PI3K is not involved in the EGF-dependent phosphorylation of Vav2. COS7 cells were transiently transfected with GFP fusion constructs of Vav2 as indicated. Prior to EGF stimulation the cells were treated with specific PI3K inhibitor LY294002 for 60 min. Lysates were then subjected to immunoprecipitation with anti-GFP antibody. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibody.

Phosphorylation Mutants but Not PH Domain Mutants of Vav2 Induce Morphological Changes in COS7 Cells-- The morphological changes induced by overexpression of Vav2 have been studied intensively. Several laboratories (13, 34) demonstrated that Vav2 overexpression induced lamellipodia and membrane ruffling. However, other laboratories (11, 35) found that overexpression of Vav2 resulted in stress fiber formation. To study the morphological changes caused by our Vav2 mutants, first, GFP-Vav2 was transiently expressed in COS7 cells. Twenty-four h after transfection cells were starved-starved for overnight and then stimulated with EGF for 10 min or left untreated. Cells were then fixed, permeabilized, and incubated with TRITC-labeled phalloidin to visualize the F-actin filaments. Microscopic analysis revealed that Vav2 overexpression resulted in membrane ruffle formation even in the absence of serum (30% of Vav2-expressing cells showed membrane ruffling) (Fig. 8, C and D). Following EGF stimulation, more the 90% of cells expressing Vav2 induced strong membrane ruffling (Fig. 8, E and F). It has to be noticed that upon EGF treatment ~30% of untransfected COS7 cells displayed membrane ruffle formation. In contrast to other reports, in cells expressing GFP-Vav2 we could not see any sign of stress fiber formation (11, 35).


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Fig. 8.   The effects of Vav2 expression on the actin cytoskeleton. COS7 cells were transiently transfected with GFP alone (A and B) and with wild type Vav2 fused to GFP (C-F). The cells were maintained for 24 h in the presence of serum and then they were starved for a further 24 h before fixation. Prior to fixation, cells were stimulated for 10 min with EGF (E and F) or left untreated (A-D). Fixed cells were visualized for GFP (A, C, and E). The distribution of actin was also visualized by staining with TRITC-phalloidin (B, D, and F). These results are representative of three independent experiments.

To further examine the effect of different GFP-Vav2 constructs, including phosphorylation and PH mutants, on cell morphology and cytoskeletal organization, these constructs were transiently expressed in COS7 cells. Expression of the double phosphorylation mutant Vav2-Y172/159F that shows basal phosphorylation level even in EGF-stimulated cells resulted in reorganization of actin. This mutant induced extensive membrane ruffling in serum-starved cells (Fig. 9, A and B). Other Vav2 constructs such as Vav2-Y172F, Vav2-Y159F, and Vav2-Y142,172F were also capable of inducing membrane ruffling in COS7 cells in the absence of serum (data not shown). In response to EGF stimulation, both untransfected and Vav2-expressing cells showed intensive membrane ruffle formation (Fig. 9, C and D). When PH domain mutant Vav2 was expressed in serum-starved COS7 cells, no specific morphological changes were detected in the actin cytoskeleton (Fig. 9, E and F). In addition, following EGF stimulation, only a small percentage of cells (between 5-10%) expressing Vav2-R425C showed membrane ruffling (Fig. 9, G and H). Microscopic analysis of Vav2 constructs reflects our previous findings in which we have shown that tyrosine phosphorylation mutants of Vav2 were capable of activating Rac1. Furthermore, a mutation introduced into the PH domain of Vav2 leads to the impaired function of Vav2 tested in either a Rac activity assay or microscopic analysis of F-actin regulation.


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Fig. 9.   The effects of transiently expressed Vav2 mutants on cell morphology. COS7 cells were transiently transfected with plasmids encoding phosphorylation mutant GFP-Vav2-Y159,172F (A-D) or with PH domain mutant GFP-Vav2-R425C (E-H). The cells were maintained for 24 h in the presence of serum and then they were starved for a further 24 h before fixation. Prior to fixation, cells were stimulated for 10 min with EGF (C, D, G, and H) or left untreated (A, B, E, and F). Fixed cells were visualized for GFP (A, C, E, and G). The distribution of actin was also visualized by staining with TRITC-phalloidin (B, D, F, and H). Arrows indicate membrane ruffling. These results are representative of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To understand the mechanism of EGF-dependent Vav2 activation, we examined first the EGF-dependent phosphorylation sites on Vav2 and the nature of interaction of Vav2 with the activated EGF receptor. Based on our in vitro and in vivo data all three tyrosine residues (142, 159, and 172) in the N-terminal domain of Vav2 can be phosphorylated by the EGF receptor. López-Lago et al. (12) have shown recently that tyrosine kinase Lck can phosphorylate all potential tyrosine residues (Tyr-142, Tyr-162, Tyr-174) in the N-terminal domain of Vav1 (12). This means that receptor and non-receptor kinases may phosphorylate not only the Tyr-174, which was the first phosphorylation site on Vav family proteins to be identified, but also that other phosphorylation sites may exist. Unfortunately, from our experiments based on mutational analysis we could not determine the contribution of individual phosphorylation site to the overall phosphorylation level of full-length Vav2. In addition, using a phosphopeptide competition assay we show here that Vav2 SH2 domain may bind directly to either the autophosphorylation residues Tyr-992 or Tyr-1148 on the EGF receptor or can couple with proteins that bind to these phosphorylation sites on the receptor. This is very similar to previous results seen with the PDGF receptor. Investigating the interaction of Vav2 with the autophosphorylated PDGF receptor, multiple phosphorylation sites were identified on the PDGF receptor that can recruit Vav2 to the plasma membrane (13).

The current model of Vav2 regulation suggests that Tyr-174 of Vav binds to DH domain and inhibits Vav exchange activity. Phosphorylation or mutation of that site to any other residue that lacks the hydroxyl group activates Vav by preventing the inhibited conformation (36). This model is further supported by many laboratories. For example, mutation of 174 of Vav2 to phenylalanine led to a higher level of Vav1 phosphorylation in fibroblasts, to oncogenic activation, and to enhancement of other Vav-mediated signals such as JNK activation and stimulation of the nuclear factor of T lymphocytes (12). Another study also demonstrated that mutation of Tyr-174 augmented the ability of Vav to up-regulate nuclear factor of activated T cells activation, as well as the Vav exchange activity leading to Rac activation (37). In agreement with the above model, we report here that in COS7 cells overexpressing Vav2, EGF stimulates Rac1 activity measured in vivo in a Vav2-dependent manner. This is the first time that Vav2-dependent endogenous Rac1 activation is shown in an inducible system. It is likely that tyrosine phosphorylation of Vav2 in response to EGF relieves autoinhibition by exposing the GTPase interaction surface of the Vav2 DH domain leading to Rac1 activation (36). In addition, we measured the ability of different Vav2 phosphorylation mutants to activate Rac1 in vivo. All mutants tested were capable of activating Rac to the same level as wild type Vav2 did in response to EGF. In the current model of Vav1 regulation Tyr-174 (or Tyr-172 in Vav2) is the key regulatory site (36). However, our findings that all phosphorylation mutants were able to activate Rac1 to the same level independently of the nature of the mutations suggest that all three tyrosine phosphorylation sites on Vav2 may relieve autoinhibition in the structure of Vav2, or tyrosine phosphorylation represents a more complicated mechanism in the regulation of Vav2 activity.

Another intriguing question is why does EGF receptor phosphorylate Vav2 on three tyrosine residues if one of them (Tyr-172) is sufficient as a regulatory site? Previous reports have recognized tyrosine 174 of Vav1 as a potential docking site for the SH2 domain of tyrosine phosphatase SHP-1 (38). Therefore, it is likely that phosphotyrosine residues in the N-terminal domain of Vav2 might be implicated in protein-protein interactions. We have found a number of SH2 domain-containing molecules to be associated with tyrosine-phosphorylated Vav2 in response to EGF stimulation.2

PI3K plays a critical role in the regulation of Rac by several different stimuli. Much of our knowledge of Rac regulation comes from the study of the actin cytoskeleton, where induction of the formation of lamellipodia and membrane ruffles has been equated with the activation of Rac (39). The ability of PDGF, insulin-like growth factor I, and insulin to induce ruffling has been shown to be inhibited by drugs and receptor mutations that inhibit PI3K activation (16, 17). It is suggested, therefore, that many growth factors signal Rac activation via a PI3K-dependent mechanism, and studies of nucleotide binding to Rac in permeabilized porcine aortic endothelial cells confirm that PDGF activates a PI3K inhibitor-sensitive guanine nucleotide exchange factor for Rac (18). The identity of such a factor remains obscure, but recent observations that phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate can bind to the PH domain of Vav and promote its exchange activity toward Rac in vitro have lead to the proposition that Vav may be a PI3K-dependent Rac exchange factor in cells (21). However, mutations in the PH domain of Vav1 do not completely inhibit exchange activity, and PH mutant Vav3 was as active as wild type Vav3 assessed by its ability to induce morphological changes in transfected cells (9, 21). Although it has been shown recently that the PH domain is necessary for Vav2 activity, PI3K itself was not required for Vav2 activation of Rac or lamellipodia formation based on experiment performed in the presence of Wortmannin (34). In an independent study, it has been shown that mutations of the PH domain of Vav2 impaired Vav2 signaling, transforming activity, and membrane association; however, these mutations did not influence exchange activity measured on Rac (29). In addition, a cysteine-rich domain was found to be important for Vav2 function (29). Our studies demonstrate that both intact PH domain and activity of PI3K are required for Vav2-dependent Rac1 activation in EGF signaling pathway. We show here for the first time that, upon EGF treatment, Vav2-dependent Rac1 activity can be inhibited by a PI3K-specific inhibitor LY294002. In addition, a mutation introduced into the PH domain resulted in the inability of Vav2 to activate Rac1.

Previous studies found that stable overexpression of Vav2 in NIH-3T3 cells induced the formation of stress fibers characteristic for Rho activation (35). In contrast, recent findings demonstrate that cells expressing activated Vav2 display lamellipodia and membrane ruffling (40) or membrane ruffling and stress fiber formation (11). Here we report that serum-starved COS7 cells expressing Vav2 seem to be more rounded and display membrane ruffling. The intensity of membrane ruffle formation can be further increased in these cells upon stimulation of cells with EGF, suggesting that EGF can induce morphological changes in a Vav2-dependent manner. In addition, a PH domain mutant form of Vav2 that failed to activate Rac1 in a pull out assay was not capable of inducing membrane ruffling in either serum-starved or EGF-treated COS7 cells. This is in agreement with previous reports in which a Vav2 construct carrying similar point mutation did not activate Rac and cause lamellipodia in human embryonic kidney 293T cells (34). It has been well established that growth factors, such as EGF and PDGF, rapidly induce membrane ruffling (11, 34, 35, 39-41). Therefore, it is likely that Vav2 may contribute to the growth factor-dependent Rac activation and then the concomitant morphological changes in the actin cytoskeleton. Recently, it has been shown that a Vav2 DH domain mutant that was not capable of activating Rac in vivo may function as a dominant negative mutant specific for Vav2 (34). This dominant negative mutant failed to inhibit Vav1-dependent Rac activation and PDGF- and EGF-dependent lamellipodia formation and also did not inhibit EGF-dependent JNK activation (34). It was concluded that Vav2 could not mediate EGF- and PDGF-dependent Rac activation, morphological changes, and JNK activation. A number of possible explanations for differences between our data and the observation of Marignani et al. (34) are possible. First of all, different assay systems have been used; we measured Rac1 activation or membrane ruffling in COS7 cells expressing Vav2 or its mutant forms in response to EGF stimulation, whereas for detecting Rac activation they co-express OncoVav1 and the dominant negative form of Vav2. Second, although they show that the dominant negative Vav2 failed to inhibit changes in actin cytoskeleton and JNK activation in response to growth factor treatment of cells, the effect of dominant negative Vav2 directly on EGF- or PDGF-dependent Rac activation was not measured. A third alternative is that the cell system that we use in which Vav2 is overexpressed is artificial and yields an abnormal Rac activation simply because of the high levels of Vav2 expression. However, the Rac activity and morphological changes of the cells are still sensitive to EGF stimulation and can be blocked by inhibitors of PI3K, so this seems unlikely.

The simplest model of EGF regulation of Rac in epithelial and mesenchymal cells is therefore that stimulation of the EGF receptor leads to autophosphorylation, binding to Vav2, and phosphorylation of Vav2 on tyrosine residues 142, 159, and 172. We show here that Vav2 binds to the autophosphorylation sites Tyr-992 and Tyr-1148 of EGF receptor in an SH2 domain-dependent manner. The nucleotide exchange activity of Vav2 toward Rac is then activated, which is likely mediated by lipid products of PI3K rather than tyrosine phosphorylation of Vav2. Tyrosine phosphorylation of Vav2 might be implicated in protein-protein interactions that represent another layer of complexity of Vav2 regulation. Nevertheless, further experiments will be required to further understand the fine regulation of Vav2-dependent Rac activation in growth factor signaling pathways.

    FOOTNOTES

* This work was supported by the Wellcome Trust, the Howard Hughes Medical Institute, the Hungarian Ministry of Social Welfare (Grant ETT 18/2000), and the Hungarian Science Foundation (Grants OTKA 25427 and OTKA 31705).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Post-doctoral fellow of the Deutsche Forschungsgemeinschaft.

To whom correspondence should be addressed. Tel.: 36-1-266-2755 (ext. 4049); Fax: 36-1-266-7480; E-mail: buday@puskin.sote.hu.

Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M207555200

2 P. Tamás and L. Buday, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: SH, Src homology; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; PH, pleckstrin homology; NT, N-terminal; DH, Dbl homology; GST, glutathione S-transferase; GFP, green fluorescent protein; TRITC, tetramethylrhodamine isothiocyanate; JNK, c-Jun NH2-terminal kinase; IP, immunoprecipitated; N-PAK, N-terminal-PAK.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Van Aelst, L., and D'Souza-Schorey, C. (1997) Genes Dev. 11, 2295-2322[Free Full Text]
2. Hall, A. (1994) Annu. Rev. Cell Biol. 10, 31-54[CrossRef]
3. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
4. Katzav, S., Martin-Zanca, D., and Barbacid, M. (1989) EMBO J. 8, 2283-2290[Abstract]
5. Bustelo, X. R. (1996) Crit. Rev. Oncog. 7, 65-88[Medline] [Order article via Infotrieve]
6. Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S., and Bustelo, X. R. (1997) Nature 385, 169-172[CrossRef][Medline] [Order article via Infotrieve]
7. Han, J., Das, B., Wei, W., Van Aelst, L., Mosteller, R. D., Khosravi-Far, R., Westwick, J. K., Der, C. J., and Broek, D. (1997) Mol. Cell. Biol. 17, 1346-1353[Abstract]
8. Henske, E. P., Short, M. P., Jozwiak, S., Bovey, C. M., Ramlakhan, S., Haines, J. L., and Kwiatkowski, D. J. (1995) Ann. Hum. Genet. 59, 25-37[Medline] [Order article via Infotrieve]
9. Movilla, N., and Bustelo, X. R. (1999) Mol. Cell. Biol. 19, 7870-7885[Abstract/Free Full Text]
10. Schuebel, K. E., Bustelo, X. R., Nielsen, D. A., Song, B. J., Barbacid, M., Goldman, D., and Lee, I. J. (1996) Oncogene 13, 363-371[Medline] [Order article via Infotrieve]
11. Liu, B. P., and Burridge, K. (2000) Mol. Cell. Biol. 20, 7160-7169[Abstract/Free Full Text]
12. López-Lago, M., Lee, H., Cruz, C., Movilla, N., and Bustelo, X. R. (2000) Mol. Cell. Biol. 20, 1678-1691[Abstract/Free Full Text]
13. Moores, S. L., Selfors, L. M., Fredericks, J., Breit, T., Fujikawa, K., Alt, F. W., Brugge, J. S., and Swat, W. (2000) Mol. Cell. Biol. 20, 6364-6373[Abstract/Free Full Text]
14. Pandey, A., Podtelejnikov, A. V., Blagoev, B., Bustelo, X. R., Mann, M., and Lodish, H. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 179-184[Abstract/Free Full Text]
15. Zeng, L., Sachdev, P., Yan, L., Chan, J. L., Trenkle, T., McClelland, M., Welsh, J., and Wang, L. H. (2000) Mol. Cell. Biol. 20, 9212-9224[Abstract/Free Full Text]
16. Nobes, C. D., Hawkins, P., Stephens, L., and Hall, A. (1995) J. Cell Sci. 108, 225-233[Abstract/Free Full Text]
17. Wennstrom, S., Hawkins, P., Cooke, F., Hara, K., Yonezawa, K., Kasuga, M., Jackson, T., Claesson-Welsh, L., and Stephens, L. (1994) Curr. Biol. 4, 385-393[Medline] [Order article via Infotrieve]
18. Hawkins, P. T., Eguinoa, A., Qiu, R. G., Stokoe, D., Cooke, F. T., Walters, R., Wennstrom, S., Claesson-Welsh, L., Evans, T., Symons, M., et al.. (1995) Curr. Biol. 5, 393-403[Medline] [Order article via Infotrieve]
19. Reif, K., Nobes, C. D., Thomas, G., Hall, A., and Cantrell, D. A. (1996) Curr. Biol. 6, 1445-1455[Medline] [Order article via Infotrieve]
20. Rodriguez-Viciana, P., Warne, P. H., Khwaja, A., Marte, B. M., Pappin, D., Das, P., Waterfield, M. D., Ridley, A., and Downward, J. (1997) Cell 89, 457-467[Medline] [Order article via Infotrieve]
21. Han, J., Luby-Phelps, K., Das, B., Shu, X., Xia, Y., Mosteller, R. D., Krishna, U. M., Falck, J. R., White, M. A., and Broek, D. (1998) Science 279, 558-560[Abstract/Free Full Text]
22. Buday, L., and Downward, J. (1993) Cell 73, 611-620[Medline] [Order article via Infotrieve]
23. Myers, M. G., Jr., Grammer, T. C., Wang, L. M., Sun, X. J., Pierce, J. H., Blenis, J., and White, M. F. (1994) J. Biol. Chem. 269, 28783-28789[Abstract/Free Full Text]
24. Zeng, Z. Z., Yellaturu, C. R., Neeli, I., and Rao, G. N. (2002) J. Biol. Chem. 277, 41213-41219[Abstract/Free Full Text]
25. Tamas, P., Solti, Z., and Buday, L. (2001) Cell. Signal. 13, 475-481[CrossRef][Medline] [Order article via Infotrieve]
26. Cantrell, D. (1998) Curr. Biol. 8, R535-538[Medline] [Order article via Infotrieve]
27. Katzav, S. (1993) Oncogene 8, 1757-1763[Medline] [Order article via Infotrieve]
28. Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S., and Lim, L. (1994) Nature 367, 40-46[CrossRef][Medline] [Order article via Infotrieve]
29. Booden, M. A., Campbell, S. L., and Der, C. J. (2002) Mol. Cell. Biol. 22, 2487-2497[Abstract/Free Full Text]
30. Toker, A., and Newton, A. C. (2000) J. Biol. Chem. 275, 8271-8274[Abstract/Free Full Text]
31. Varnai, P., Rother, K. I., and Balla, T. (1999) J. Biol. Chem. 274, 10983-10989[Abstract/Free Full Text]
32. Watton, S. J., and Downward, J. (1999) Curr. Biol. 9, 433-436[CrossRef][Medline] [Order article via Infotrieve]
33. Das, B., Shu, X., Day, G. J., Han, J., Krishna, U. M., Falck, J. R., and Broek, D. (2000) J. Biol. Chem. 275, 15074-15081[Abstract/Free Full Text]
34. Marignani, P. A., and Carpenter, C. L. (2001) J. Cell Biol. 154, 177-186[Abstract/Free Full Text]
35. Schuebel, K. E., Movilla, N., Rosa, J. L., and Bustelo, X. R. (1998) EMBO J. 17, 6608-6621[Abstract/Free Full Text]
36. Aghazadeh, B., Lowry, W. E., Huang, X. Y., and Rosen, M. K. (2000) Cell 102, 625-633[Medline] [Order article via Infotrieve]
37. Kuhne, M. R., Ku, G., and Weiss, A. (2000) J. Biol. Chem. 275, 2185-2190[Abstract/Free Full Text]
38. Wu, J., Motto, D. G., Koretzky, G. A., and Weiss, A. (1996) Immunity 4, 593-602[Medline] [Order article via Infotrieve]
39. Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399[Medline] [Order article via Infotrieve]
40. Abe, K., Rossman, K. L., Liu, B., Ritola, K. D., Chiang, D., Campbell, S. L., Burridge, K., and Der, C. J. (2000) J. Biol. Chem. 275, 10141-10149[Abstract/Free Full Text]
41. Xie, H., Pallero, M. A., Gupta, K., Chang, P., Ware, M. F., Witke, W., Kwiatkowski, D. J., Lauffenburger, D. A., Murphy-Ullrich, J. E., and Wells, A. (1998) J. Cell Sci. 111, 615-624[Abstract/Free Full Text]


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