From the Department of Medical Chemistry, Semmelweis University
Medical School, 9 Puskin Street, 1088 Budapest, Hungary and
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
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ABSTRACT |
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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.
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.
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 p110 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 p110 Activity Assay for Rac1--
GST-N-PAK 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.
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.
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).
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.
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 PAK
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).
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.
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.
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).
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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 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.
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 [
-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.
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-PAK
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
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.
View larger version (42K):
[in a new window]
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.
View larger version (28K):
[in a new window]
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.
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.
View larger version (22K):
[in a new window]
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.
View larger version (31K):
[in a new window]
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 p110
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 [
-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.
View larger version (37K):
[in a new window]
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.
View larger version (48K):
[in a new window]
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.
View larger version (78K):
[in a new window]
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.
View larger version (61K):
[in a new window]
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
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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