From the Departments of Medicine and Pathology, Albert
Einstein College of Medicine, Bronx, New York 10461
Received for publication, October 24, 2000, and in revised form, February 14, 2001
Vascular endothelial growth factor (VEGF)-induced
endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex
assembly and disassembly. We investigated the signaling pathway by
which VEGF regulates focal adhesion complex assembly by examining the
signaling proteins involved. VEGF stimulated the tyrosine
phosphorylation of the SH2 domain-containing signaling proteins NCK and
CRK in human umbilical vein endothelial cells. The signaling pathways
that couple the kinase insert domain-containing receptor to NCK and CRK
is most likely mediated by another cellular protein, as NCK and CRK
were tyrosine-phosphorylated in response to VEGF in cells expressing
receptors mutated at each of several candidate SH2
domain-interacting cytosolic tyrosines. In the absence of VEGF
treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the
recruitment of PAK to focal adhesions, and FAK
immunoprecipitated with both NCK and PAK in VEGF-treated (but not
untreated) human umbilical vein endothelial cells. Inhibition of NCK
protein expression using antisense oligonucleotides led to the
inhibition of both VEGF-induced focal adhesion assembly and
VEGF-induced cell migration, demonstrating a necessary role of NCK in
these cellular responses.
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INTRODUCTION |
Angiogenesis, the formation of new blood capillaries, is an
important component of normal physiological processes such as wound
healing and development (reviewed in Ref. 1). Angiogenesis also
contributes to several pathological situations, including tumor growth
(2, 3), rheumatoid arthritis (4), and degenerative eye
disorders (5, 6). Angiogenesis is a complex process involving
endothelial cell movement and proliferation and endothelial cell-mediated degradation of the extracellular matrix. Multiple angiogenic stimulators and inhibitors regulate these processes (7-9).
Vascular endothelial growth factor
(VEGF)1 has received
attention as a key regulator of endothelial cell functions (10-12). VEGF expression correlates both temporally and spatially with the onset
of angiogenesis in several physiological situations (13-15), and VEGF
elicits a strong angiogenic response in a variety of in vivo
models (16, 17). An essential role of VEGF in tumor angiogenesis and
ischemia-related retinal disorders has been demonstrated by the
findings that neutralizing anti-VEGF antibodies or
dominant-negative VEGF receptors inhibit both angiogenesis and the
progression of these pathological disorders (18-21).
A major component of the angiogenic response are the changes that occur
in endothelial cell interactions with the extracellular matrix as well
as changes in cell-cell interactions. Endothelial cells are linked to
each other by tight- and adherens-type junctions and are linked to the
extracellular matrix by a variety of integrin and other adhesion
molecules (22-25). VEGF activates endothelial cells, in part, through
stimulating signal transduction pathways that regulate the enzymatic
components of adhesion complexes. VEGF-induced tyrosine
phosphorylation of VE-cadherins (26), a component of adherens-type
cell-cell junctions, has been implicated as a key step in endothelial
cell migration. VEGF-induced phosphorylation of the tight junction
proteins occludin and zonula occluden-1 (27, 28) is a potential
mechanism by which the growth factor enhances vascular permeability.
Experimental evidence indicating a role for VEGF in regulating
cell-matrix interactions includes the findings that VEGF enhances the
expression of
1
1 and
2
2 integrins (29) and that neutralizing
antibodies to
v
5 integrins block growth
factor-induced neovascularization (30, 31).
VEGF exhibits high affinity binding to two distinct receptor tyrosine
kinases, the fms-like tyrosine kinase Flt-1 (32, 33) and the kinase insert domain-containing receptor (KDR) (34, 35). Both
receptors possess insert sequences within their catalytic domains and
seven immunoglobulin-like domains in the extracellular regions and are
related to the platelet-derived growth factor family of receptor
tyrosine kinases. Although expression of both VEGF receptor types
occurs in adult endothelial cells, including human umbilical vein
endothelial (HUVE) cells, recent findings suggest that KDR,
and not Flt-1, is able to mediate the mitogenic and chemotactic effects
of VEGF in endothelial cells (36). The key targets that mediate the
diverse biological functions of VEGF in endothelial cells remain
incompletely understood for either VEGF receptor. Somewhat varying
results as to the downstream effects for the VEGF receptors have been
obtained. VEGF stimulates the tyrosine phosphorylation of phospholipase
C
, MAPK, phosphatidylinositol 3-kinase, FAK, and paxillin in HUVE
cells and of phospholipase C
, p120GAP, and NCK in bovine
aortic endothelial cells (37, 38). In porcine aortic endothelial cells
transfected with KDR and Flt-1, VEGF has no effect on
phosphatidylinositol 3-kinase activity and only a weak effect on
p120GAP tyrosine phosphorylation (39).
The goal of this study was to clarify the signaling mechanism by which
VEGF stimulates endothelial cell migration. As there is compelling
evidence that the assembly and disassembly of focal adhesions play a
key role in the mechanism by which several extracellular stimuli
regulate both cell morphology and movement (40-42), and VEGF
stimulates the tyrosine phosphorylation of FAK (37), we took the
strategy of exploring the cell signaling proteins that couple VEGF
binding to its receptors with focal adhesion assembly. Our results
demonstrate that the SH2 domain-containing signaling protein NCK and
the p21 GTPase-activated serine/threonine kinase PAK play necessary
roles in this signaling pathway.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HUVE cells were isolated as previously
reported (43). Passage 2 cells were cultured on 0.2% gelatin-coated
tissue culture plates in medium 199 containing 20% newborn calf serum,
5% human serum, and 7.5 µg/ml endothelial growth factor (Sigma).
Antibodies--
The rabbit anti-KDR antibody was isolated in our
laboratory and targets a polypeptide domain within the KDR cytosolic
domain (44). Anti-NCK, anti-CRK, anti-FAK, and anti-phosphotyrosine (PY20) monoclonal antibodies were from Transduction Laboratories. Anti-FAK and anti-PAK polyclonal antibodies were from Santa Cruz Biotechnology, Inc. Peroxidase-conjugated donkey anti-rabbit and sheep
anti-mouse immunoglobulins were from Amersham Pharmacia Biotech.
Western Blotting--
HUVE cells were grown on 10-cm dishes
until subconfluent. The cells were incubated in serum-free DMEM with 1 mM Na3VO4 for 1 h at either 4 or 37 °C; VEGF (50 ng/ml) was then added for either 4 h at
4 °C or 0-30 min at 37 °C. Cells were lysed in lysis buffer (10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.2 mM Na3VO4, 30 mM
Na4P2O7, 30 mM NaF, 1 mM EDTA, 1 mg/ml leupeptin, 0.7 mg/ml pepstatin A, and 1%
Triton X-100), and primary antibodies were added to the lysates. After
a 1-h incubation at 4 °C, the antibodies were immobilized on protein
A-Sepharose beads (Sigma), and the beads were washed three times with
lysis buffer and boiled in 40 µl of SDS-polyacrylamide gel
electrophoresis sample loading buffer. Proteins were separated on
SDS-polyacrylamide gels, transferred to nitrocellulose (Bio-Rad), and
blotted with appropriate antibodies. The filters were then blocked in
Tris-buffered saline containing 0.2% Tween plus 5% dry milk at
4 °C overnight and probed with specific antibodies for 2 h at
room temperature. After washing, the filter was incubated with
horseradish peroxidase-linked anti-rabbit or anti-mouse IgG, and
reactions were visualized through enhanced chemiluminescence (ECL,
Amersham Pharmacia Biotech).
Site-directed Mutagenesis of the KDR cDNA--
For
substitution of tyrosine residues with phenylalanine,
site-directed mutagenesis was carried out on KDR cDNA using the U.S.E. mutagenesis kit (Amersham Pharmacia Biotech). The
following oligonucleotides were used for mutagenesis:
5'-TTGCTCCAACGAAGTCTTTCCCT-3' (Y951F),
5'-GGAAGTCCTTAAACAGATCTTCA-3' (Y996F), 5'-TTACCCCGAGGAATGGAGAAGCA-3' (Y1106F), 5'-CTGGTGTAGTTTTATCAGGGGCC-3' (Y1130F), and
5'-GAAGAACAATGAAGTCTTTGCCA-3' (Y1175F). All mutations were
confirmed by DNA sequencing. The resulting vectors were transfected
into HEK293 cells, and stable transfectants were selected and expanded
as previously described (44).
In Vitro Kinase Assay--
HUVE cells were grown on 10-cm dishes
until subconfluent. Cells were treated with VEGF (50 ng/ml) for 15 min
at 37 °C. After one wash with ice-cold phosphate-buffered saline,
cells were scraped from the dish and lysed in lysis buffer.
Immunoprecipitation with anti-PAK antibodies was performed as described
above. The immunoprecipitates were washed three times with lysis buffer
and three times with kinase buffer (50 mM HEPES (pH 7.3),
50 mM KCl, 10 mM
-glycerophosphate, 10 mM MgCl2, 5 mM NaF, 2 mM MnCl2, and 0.05% Triton X-100). Kinase reactions were carried out for 20 min at 30 °C with 0.25 mg/ml myelin basic protein as a substrate, 10 µM ATP, and 1 µCi of [
-32P]ATP in 30 µl of kinase buffer. The
reaction was stopped by addition of 0.5 volume of gel loading buffer
and heating at 100 °C for 5 min. The proteins were separated by
SDS-polyacrylamide gel electrophoresis, and radioactively labeled
proteins were identified by autoradiography of dried gels.
Antisense Oligoribonucleotides--
Oligonucleotides were 19-mer
2'-O-methyl oligoribonucleoside phosphorothioates
(Oligos Etc., Wilsonville, OR). The oligonucleotides were targeted
against the NCK translation initiation site (codons 2-7). The
sequences of the oligonucleotides were as follows: antisense NCK, 5'-CTACCACCACTTCTTCTGC; and sense NCK (control),
5'-GATGGTGGTGAAGAAGACG.
HUVE cells were grown until 50% confluent. Oligonucleotides and
LipofectAMINE reagent (Life Technologies, Inc.)
(oligonucleotide/LipofectAMINE ratio of 1:5) were incubated for 20 min
at room temperature. Cells were washed with serum-free medium 199, and
oligonucleotide-LipofectAMINE complexes were added directly to the
cells, with a final oligonucleotide concentration of 0.4 nM/ml. After a 4-h incubation at 37 °C, an equal volume
of complete medium containing a 2-fold excess of oligonucleotides was
added. Experiments were typically performed 3 days later.
Immunostaining Assay--
HUVE cells were plated on 4- or 8-well
Permanox plastic chamber slides coated with 0.2% gelatin and grown
until 50% confluent. Transfection by antisense oligonucleotides was
performed as described above, and cells were incubated in the presence
of oligonucleotides for 3 days. Cells were fixed in 3.5% formaldehyde
in phosphate-buffered saline for 10 min at room temperature, and
nonspecific binding was blocked with Hanks' solution containing 1%
bovine serum albumin and 10% goat serum for 1 h at room
temperature. Anti-FAK (1 µg/ml) or anti-PAK (10 µg/ml) antibodies
were added and incubated for 1 h at room temperature. The slides
were washed three times with Hanks' solution containing 1% bovine
serum albumin, followed by a 1-h incubation with Cy3-conjugated goat
anti-mouse or fluorescein isothiocyanate-conjugated goat anti-rabbit
secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.). After
washing three times with Hanks' solution containing 1% bovine serum
albumin, cells were viewed under a Nikon fluorescent microscope.
Cell Migration Assay--
The migration assay utilized a
modified Boyden chamber and was performed essentially as previously
described (45). HUVE cells were grown to 50% confluency and then
mock-transfected or transfected with sense or antisense
oligonucleotides as described above. Three days later, the cells were
lifted from the dish using nonenzymatic cell dissociation solution
(Sigma), centrifuged for 2 min at 1000 rpm, and resuspended in medium
199 containing 0.5% bovine serum albumin. The cells were then plated
on fibronectin-precoated Transwells (Costar Corp.) at a density of
105 cells/well. Medium 199 with VEGF (10 ng/ml) was used as
a chemoattractant in the lower wells. Cell migration assays were
performed for 8 h at 37 °C in a CO2 incubator.
Cells that had migrated trough the Transwells were stained with
CMFDA dye (Sigma) and counted under a fluorescent microscope.
Four different fields were counted for each experiment, and all
samples were performed in triplicate.
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RESULTS |
VEGF Stimulates the Recruitment of NCK and CRK to KDR--
It was
previously shown that NCK tyrosine phosphorylation and the amount of
NCK present in anti-KDR immunoprecipitates are increased in
VEGF-treated bovine aortic endothelial cells and KDR-transfected
porcine aortic endothelial cells (38, 39). The experimental data shown
in Fig. 1 (A and B)
demonstrate that similar results were obtained using HUVE cells.

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Fig. 1.
VEGF stimulates NCK and CRK tyrosine
phosphorylation and association of NCK and CRK with KDR in HUVE
cells. Subconfluent HUVE cells were incubated for 1 h in
serum-free DMEM containing 1 mM
Na3VO4 and then with or without 50 ng/ml VEGF
for the times indicated (A) or for 20 min (B and
C) at 4 °C. The cells were lysed, and anti-Tyr(P)
(pTyr) (A and C) or anti-KDR
(B) immunoprecipitates (IP) were prepared and
immunoblotted (IB) with anti-NCK (A and
B) or anti-CRK (C) antibodies. Arrows
in A and B indicate the expected positions of
NCK. The molecular masses given in C were derived from
standards run on the gel shown. The numbers in
parentheses in B and C indicate the
-fold stimulation caused by VEGF of the intensity of the NCK and CRK
proteins as determined using a PhosphorImager. The LYSATE
lane in C is derived from HUVE cells boiled in
gel sample buffer and loaded directly on the gel.
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NCK is a member of a family of cell proteins that lack enzymatic
activity and that participate in cell signaling by acting as adaptor
proteins (45). Fig. 1C demonstrates that VEGF treatment of
HUVE cells enhanced the tyrosine phosphorylation of another adaptor
protein, CRK. The 42-kDa isoform (CRKII), but not 28-kDa CRKI, was
detected in the anti-KDR immunoprecipitates after VEGF treatment, even
though both isoforms are expressed in these cells.
Experiments were performed to determine if the interaction of NCK and
CRK with VEGF-activated KDR is direct or mediated through another
protein. The strategy was to express receptor protein in which specific
tyrosines had been mutated to phenylalanine in HEK293 cells (which do
not express endogenous KDR) and test for VEGF-induced NCK and CRK
tyrosine phosphorylation. As there is currently only a limited amount
of information on the identity of the KDR autophosphorylation sites
(46-48), the tyrosines that were targeted for mutagenesis were chosen
based upon whether their adjacent amino acid sequences conformed to a
consensus-binding site (pYDE(P/D/V)) for the NCK SH2 domain (49, 50).
Seven separate cell lines were established. These are denoted KDR,
KDR(Y938F), KDR(Y951F), KDR(Y1106F), KDR(Y1130F), KDR(Y1175F), and
KDR(Y1305F).
Fig. 2A shows the KDR
expression level for some of the generated cell lines; similar
expression levels were observed for KDR(Y1130F) (data not shown). VEGF
treatment led to both KDR autophosphorylation (Fig. 2B) and
an increased amount of NCK and CRK in anti-Tyr(P) immunoprecipitates (Fig. 2, C and D) in cells
expressing native KDR as well as KDR(Y938F), KDR(Y951F), KDR(Y1130F),
and KDR(Y1175F). A similar effect was seen for KDR(Y1106F) and
KDR(Y1130F) (data not shown). VEGF also increased the amount of NCK in
anti-KDR immunoprecipitates for KDR(Y951F), KDR(Y1130F), and
KDR(Y1175F) (Fig. 3A) and each
of the other cell lines expressing a mutant KDR (data not shown).

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Fig. 2.
NCK and CRK tyrosine phosphorylation in
HEK293 cells expressing native and mutant KDRs. A,
cells expressing native or mutant KDR were grown to confluence on
24-well dishes. The cell medium was aspirated, and 200 µl of gel
sample buffer was added to the wells. The samples were transferred to
Eppendorf tubes and boiled for 5 min, and 25 µl was subjected
to 7% SDS-polyacrylamide gel electrophoresis. Immunoblotting was done
using anti-KDR antibodies. B-C, cells expressing native or
mutant KDR were grown to confluence on 10-cm dishes and then incubated
overnight in DMEM containing 1% fetal calf serum. After a 1-h
incubation in serum-free DMEM containing 1 mM
Na3VO4, the cells were incubated at 4 °C for
4 h with or without 50 ng/ml VEGF. The cells were lysed, and
anti-Tyr(P) (PTyr) immunoprecipitates (IP) were
prepared and immunoblotted (IB) with anti-KDR
(B), anti-NCK (C), or anti-CRK (D) antibodies. Aliquots of
cells not immunoprecipitated with antibody were loaded on the
SDS-polyacrylamide gels and are indicated as LYSATE. The
experiment done for A was different that those done for
B-D. Results identical to those shown in B-D
were observed in three separate experiments.
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Fig. 3.
VEGF-induced NCK and SHC activation in HEK293
cells expressing native and mutant KDRs. The experiments were done
exactly as described for Fig. 2 (B-D), except that
immunoprecipitation (IP) was with anti-KDR (A and
B) or anti-Tyr(P) (PTyr) (C)
antibodies, and immunoblotting (IB) was done with anti-NCK
(A) or anti-SHC (C) antibodies. The blot shown in
A was reprobed with the anti-KDR antibody (B) to
ensure that equal amounts of the immunoprecipitates were loaded for the
control and VEGF-treated samples. Results identical to those shown in
each panel were observed in three separate experiments.
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To validate the experimental strategy, we also examined VEGF-induced
SHC tyrosine phosphorylation in the various cell lines because there is
evidence that this signaling protein interacts directly with KDR at
tyrosine 1175 (48). SHC was immunoprecipitated by the anti-Tyr(P)
antibody from VEGF-treated cell extracts expressing native KDR and each
of the mutant KDR-expressing cell lines except KDR(Y1175F) (Fig.
3B shows the results for KDR, KDR(Y1130F), and KDR(Y1175F)).
The experimental result examining VEGF-induced SHC tyrosine
phosphorylation in the various cell lines strengthens confidence in the
overall approach, and it was concluded that neither NCK nor CRK binds
to KDR at the tyrosines targeted by mutagenesis. Since none of the KDR
tyrosines that were not targeted by mutagenesis contain an NCK- or
CRK-interacting consensus sequence, we conclude that NCK and CRK
interact with the receptor through a protein intermediate or bind
directly to KDR at a previously not recognized sequence.
VEGF Binding to Endothelial Cells Leads to PAK Activation and
Recruitment to Focal Adhesions--
We then examined whether PAK
participates in VEGF-induced signal transduction by acting downstream
from NCK. Western blot analysis demonstrated the presence of PAK in
anti-NCK immunoprecipitates (Fig.
4A), but not anti-CRK
immunoprecipitates (data not shown), isolated from HUVE cell extracts.
The anti-PAK antibody recognizes at least three PAK isoforms (
-PAK
(PAK1),
-PAK (PAK3), and
-PAK (PAK2)), and multiple isoforms were
detected after immunoprecipitation with NCK antiserum. VEGF had no
effect on the amount of PAK associated with NCK (Fig. 4A);
this conclusion was confirmed by a complementary experiment in which
cell extracts were immunoprecipitated with the anti-PAK antibody, and
Western blotting was performed with the anti-NCK antibody (Fig.
4B).

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Fig. 4.
Interaction of NCK and PAK in HUVE
cells. Subconfluent HUVE cells were incubated for 1 h in
serum-free DMEM containing 1 mM
Na3VO4, and half the plates were stimulated
with 50 ng/ml VEGF for 20 min at 37 °C. Cell extracts were prepared,
and anti-NCK (A) or anti-PAK (B)
immunoprecipitates (IP) were immunoblotted (IB)
with anti-PAK (A) or anti-NCK (B)
antibodies.
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These results indicate that PAK is bound to NCK, even in the absence of
growth factor. Experiments were then done to determine the consequence
of VEGF treatment on the enzymatic activity of PAK. HUVE cells were
treated with or without VEGF, and anti-PAK immunoprecipitates were
prepared from cell lysates. PAK activity was measured by monitoring the
phosphorylation of myelin basic protein using
[
-32P]ATP, SDS-polyacrylamide gel electrophoresis, and
autoradiography. As shown in Fig.
5A, the phosphorylation of
myelin basic protein was significantly increased in samples from
VEGF-treated compared with untreated cells.

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Fig. 5.
PAK activity is increased upon VEGF
stimulation of HUVE cells. Subconfluent HUVE cells were incubated
for 1 h in serum-free DMEM containing 1 mM
Na3VO4 and then stimulated with 50 ng/ml VEGF
for 0, 5, or 20 min at 37 °C. Cell extracts were immunoprecipitated
(IP) with the anti-PAK antibody. One-half of the
immunoprecipitates were subjected to kinase assays in the presence of
myelin basic protein (MBP) and [32P]ATP
(A). The other half of the immunoprecipitates were
immunoblotted (IB) with anti-Tyr(P) (p-Tyr)
antibody (B). The numbers in
parentheses in A are the VEGF-induced -fold
stimulation of the amount of 32P-labeled myelin basic
protein as determined using a PhosphorImager.
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32P labeling of one other protein (60 kDa) was enhanced in
anti-PAK immunoprecipitates prepared from the VEGF-treated samples (Fig. 5A). Proteins of 60 and 90 kDa were observed upon
blotting anti-PAK immunoprecipitates with the anti-Tyr(P) antibody
(Fig. 5B). The identities of the 90- and 60-kDa proteins are
not known, and neither is the tyrosine kinase responsible for their phosphorylation.
It was previously shown that the expression of a constitutively active
PAK leads to recruitment of the recombinant protein to focal adhesions
(51, 52). We therefore tested whether this is also the case for
VEGF-activated PAK. In the absence of VEGF, immunofluorescent staining
with the anti-PAK antibody (Fig.
6C) revealed no focal
adhesion-like structures. Focal adhesions, as observed using anti-FAK
(Fig. 6A) or anti-Tyr(P) (data not shown) antibodies, were
present in these cells, but they were ~25% the size of those seen in
the presence of VEGF (Fig. 6B). Within 15 min of VEGF
treatment, PAK staining (Fig. 6D) was identical to the
staining with the anti-FAK or anti-Tyr(P) antibody, demonstrating the
recruitment of PAK to focal adhesions.

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Fig. 6.
VEGF activation of HUVE cells leads to
recruitment of PAK to focal complexes. HUVE cells were
plated on 4- or 8-well Permanox plastic chamber slides coated with
0.2% gelatin and grown until they were confluent. Cells were either
untreated (A and C) or treated with 50 ng/ml VEGF
(B and D) for 20 min. Cells were fixed,
permeabilized, and incubated with antibody to FAK (A and
B) or PAK (C and D). Immunofluorescent
staining was performed as described under "Experimental
Procedures." The photographic fields shown in B and
D are the same. The results shown are representative of at
least 10 different fields observed in each experiment and of four
similar independent experiments.
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Both the KDR and Flt-1 VEGF receptor subtypes are express by HUVE
cells. Although it was previously demonstrated that VEGF-induced endothelial cell migration is mediated by KDR and not Flt-1 (36), it
cannot be ruled out that Flt-1 does not participate in the effects of
VEGF on PAK activation and focal adhesion assembly. To address this
issue, we asked whether PlGF treatment of HUVE cells leads to focal
adhesion assembly. The rationale for this experiment was that PlGF,
which shares 50% amino acid homology with VEGF, binds to Flt-1, but
not KDR. The results shown in Fig. 7
demonstrate that immunofluorescent staining of PlGF-treated cells (Fig.
7B) using the anti-FAK antibody was identical to that observed for control cells not treated with growth factor (Fig. 7A). As shown previously, VEGF treatment stimulated focal
adhesion assembly (Fig. 7C).

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Fig. 7.
VEGF (but not PlGF) activates the assembly of
focal adhesions in HUVE cells. HUVE cells were plated on 8-well
Permanox plastic chamber slides coated with 0.2% gelatin and grown
until they were confluent. Cells were either untreated (A)
or treated with 50 ng/ml PlGF (B) or 50 ng/ml VEGF
(C) for 20 min. Cells were fixed, permeabilized, and
incubated with antibody to FAK. Immunofluorescent (IF)
staining was performed as described under "Experimental
Procedures." The results shown are representative of at least 10 different fields observed in each experiment and of four similar
independent experiments.
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Further evidence indicating that VEGF stimulates the recruitment of PAK
to focal adhesions is shown in Fig. 8.
The results demonstrate that FAK was detected in both anti-PAK (Fig.
8A) and anti-NCK (Fig. 8B) immunoprecipitates
obtained from VEGF-treated (but not untreated) HUVE cell extracts.

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Fig. 8.
PAK and NCK associate with FAK after VEGF
activation of HUVE cells. Subconfluent HUVE cells were incubated
for 1 h in serum-free DMEM containing 1 mM
Na3VO4 and then stimulated with 50 ng/ml VEGF
at 37 °C for 5 min. Cell extracts were immunoprecipitated
(IP) with anti-NCK (A) or anti-PAK (B)
antibodies. Immunoblotting (IB) was done using the anti-FAK
antibody.
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NCK Antisense Oligonucleotides Block VEGF-induced Focal Adhesion
Formation and Cell Migration--
The experimental results shown in
Figs. 6 and 7 demonstrate that VEGF treatment leads to NCK and PAK
localization to focal adhesions, but do not clarify the functional
requirement of these signaling proteins in either the formation of
focal adhesions or biological activities associated with these cellular
complexes. To address these issues, we took the strategy of inhibiting
NCK expression using antisense oligonucleotides and testing the
consequence on the formation of focal adhesions. A 19-mer
2'-O-methyl oligoribonucleoside phosphorothioate targeting
the NCK translation initiation site (codons 2-7) was used. The
corresponding sense oligonucleotide was used as a control. The
transfection protocol utilized LipofectAMINE and resulted in >95% of
cells being transfected as determined using a fluorescently labeled
oligonucleotide (data not shown). The transfection protocol using
either sense or antisense oligonucleotide (0.4 nmol/ml) had only a
small effect on the viability of the cells compared with cells
subjected to transfection with no oligonucleotide, with a <10% loss
in cell number after 3 days. There was a significant (25%) increase in
the number of rounded cells 3 days post-transfection with either
antisense or sense oligonucleotide compared with
mock-transfected cells, indicating that the oligonucleotides
do have some nonspecific effects.
The amount of cellular NCK protein was determined at several time
points following transfection. From analyzing several experiments (n = 6; 3 days post-transfection), we found that the
level of NCK protein was reduced between 60 and 80% by antisense
oligonucleotides compared with cells subjected to the protocol without
oligonucleotides (Fig. 9A).
There was also a 20-25% decrease in the level of NCK protein after
transfection with sense oligonucleotides (Fig. 9A). As a
control, we compared the level of CRK protein in NCK antisense versus NCK sense oligonucleotide-transfected cells (Fig.
9B) and found no differences in any of the experiments.

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Fig. 9.
Inhibition of NCK expression using antisense
oligonucleotides. HUVE cells were grown to 50% confluency on
10-cm dishes. Cells were transfected with antisense or sense
oligonucleotides or mock-transfected using LipofectAMINE
(NO-oligo) as described under "Experimental Procedures."
Three days later, the cells were detached from the dishes using trypsin
and counted, and an equivalent number of cells were microcentrifuged
and suspended in gel sample buffer. Immunoblotting was done using
either anti-NCK (A) or anti-CRK (B) antibodies.
The experiment is representative of six separate experiments.
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Prior transfection of HUVE cells with sense oligonucleotide had no
effect on VEGF activation of focal adhesion formation as determined by
immunofluorescent staining with anti-FAK antibodies (Fig.
10, A and B). As
is the case with cells not subjected to the transfection protocols,
cells transfected with sense oligonucleotides and not treated with VEGF
contained small focal adhesions, and VEGF caused an increase in both
their number and size. VEGF-induced redistribution of PAK to focal
adhesions was also not affected in the sense
oligonucleotide-transfected cells (Fig. 10, C and D). In the absence of VEGF treatment, cells transfected with
the antisense oligonucleotides contained a similar number of the small focal adhesions seen in cells not transfected or transfected with sense
oligonucleotide. However, in the presence of VEGF, transfection with
the NCK antisense oligonucleotides significantly reduced the number of
large focal adhesions seen in control cells (Fig. 10, E and
F).

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Fig. 10.
Inhibition of NCK expression blocks
VEGF-induced assembly of focal complexes. HUVE cells were plated
on 8-well Permanox plastic chamber slides coated with 0.2% gelatin and
grown until 50% confluent. Mock transfection (A and
B) or transfection by sense (C and D)
or antisense (E and F) oligonucleotides was
performed as described under "Experimental Procedures," and the
cells were incubated for 3 days. Cells were either untreated
(A, C, and E) or treated with 50 ng/ml
VEGF for 20 min (B, D, and F). Cells
were fixed, permeabilized, and incubated with the anti-FAK antibody.
Immunofluorescent staining was performed as described under
"Experimental Procedures." The results shown are representative of
at least 10 different fields observed in each experiment and of four
similar independent experiments.
|
|
VEGF stimulates the directed migration of HUVE cells, and FAK has been
implicated as playing a necessary role in this response (40). In view
of our data (Fig. 10) demonstrating a necessary role of NCK in focal
adhesion formation, we examined whether inhibition of NCK using
antisense oligonucleotides would block VEGF-induced migration. The
results shown in Fig. 11 confirm that
this is in fact the case. HUVE cells transfected with NCK antisense
oligonucleotides were significantly less responsive to VEGF than either
mock-transfected or NCK sense oligonucleotide-transfected cells. These
results demonstrate that VEGF signaling through NCK is required for
endothelial cell migration.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 11.
Inhibition of NCK expression blocks
VEGF-induced HUVE cell migration. HUVE cells were grown to 50%
confluency and then mock-transfected or transfected with sense or
antisense oligonucleotides as described under "Experimental
Procedures." Three days later, the cells were replated on
fibronectin-precoated Transwells at a density of 105
cells/well. Medium 199 with VEGF (10 ng/ml) was used as a
chemoattractant in the lower wells. Cell migration assays were
performed for 8 h at 37 °C in a CO2 incubator.
Cells that had migrated through the Transwells were stained with CMFDA
dye and counted under a fluorescent microscope. Four different fields
were counted for each experiment, and all samples were performed in
triplicate.
|
|
 |
DISCUSSION |
In this study, we describe a signal transduction pathway that
couples VEGF binding to its receptor with focal adhesion formation and
cell migration. The model predicts that VEGF binding to KDR leads to
the recruitment of an NCK-PAK complex to the receptor, with subsequent
tyrosine phosphorylation of NCK. PAK catalytic activity is then
augmented, accompanied by increased phosphorylation of 60- and 90-kDa
proteins. The NCK-PAK complex is recruited to focal adhesions, which is
followed by enhancement of cell migration. Each of these steps is now
discussed in greater detail.
HUVE cells express both the KDR and Flt-1 VEGF receptor subtypes, and
NCK associates with both receptors after VEGF treatment (55). Although
Fig. 7 shows that PlGF treatment of these cells does not stimulate
focal adhesion assembly, it is not clear whether PlGF-induced Flt-1
signaling is identical to that for VEGF. Further evidence consistent
with the conclusion that VEGF binding to KDR mediates the effects
described in this study is the fact that Flt-1 does not participate in
VEGF-induced cell migration (36) and results demonstrating that VEGF
treatment of KDR-transfected porcine aortic endothelial cells (which do
not express Flt-1) leads to both NCK tyrosine phosphorylation and an
increase in focal adhesion assembly (53).
It was concluded from the data in Figs. 3 and 4 that NCK and CRK do not
interact directly with KDR after VEGF treatment or interact with the
receptor at a previously not recognized peptide sequence. The tyrosines
that were targeted for mutagenesis in these experiments were chosen
based upon whether their adjacent amino acid sequences conformed to a
consensus-binding site (pYDE(P/D/V)) for the NCK SH2 domain (49, 50).
Neither of the known NCK-binding sites on the platelet-derived growth
factor receptor tyrosine kinase (SVDYpYVPMLD) or Flt-1
(SVVLpYSTPPI) contains this consensus recognition sequence, which
indicates that there is binding promiscuity. None of the 18 tyrosines
within the cytosolic domain of KDR are contained within peptides
conforming to this sequence. The KDR peptide that most closely
matches the consensus amino acid sequence is RAPDY1130TTPE,
and other peptides (QGKDY951VGAIP,
GASPY1106PGVK, and DGKDY1175IVLP) contain
prolines within 1-5 amino acids carboxy-terminal to tyrosines. These
KDR tyrosines were targeted for mutagenesis, and no differences in NCK
or CRK activation were observed for any of the resulting cell lines
compared with cells expressing the native receptor.
The hypothesis that the binding of NCK with KDR is indirect is
consistent with evidence that has been reported for the epidermal growth factor receptor (54, 55), in which the binding of epidermal growth factor leads to the recruitment of the Dok-related (DokR) protein to the epidermal growth factor receptor, with subsequent binding of NCK to DokR. We did not detect DokR in anti-NCK
immunoprecipitates prepared from cell lysates after VEGF treatment
(data not shown). Other receptor tyrosine kinases bind NCK directly
after growth factor activation. For example, VEGF binding to the Flt-1
receptor subtype leads to a direct interaction of both NCK and CRK with Flt-1 Tyr(P)1333 (56). The amino acid sequence containing
Flt-1 Tyr1333 (SVVLYSTPPI) is quite similar to the sequence
containing KDR Tyr1130 (RAPDYTTPEM), but we conclude that
there are sufficient structural differences so that NCK does not bind
to the KDR peptide.
An interaction of NCK with PAK in quiescent cells and the ability of
activated growth factor (e.g. platelet-derived growth factor
and epidermal growth factor) receptors to recruit the complex to the
cells' surface have been noted previously (57, 58). The association
between NCK and PAK is mediated by the second SH3 domain of NCK and a
proline-rich sequence in the amino terminus of PAK (58, 59). Our
finding that PAK activity is enhanced by VEGF is consistent with
previous studies documenting that localization of the second NCK SH3
domain, or of PAK itself, to the cell-surface membrane results in PAK
activation; this is independent of other signals. These previous
studies showed that PAK activation by a membrane-targeted NCK SH3
domain is blocked by negative regulators of the Cdc42 or Rac
GTP-binding proteins (60, 61). The activation of PAK by VEGF treatment
is therefore likely to involve Cdc42 or Rac, although
G-protein-independent mechanisms of PAK activation have also been
reported (62).
The VEGF-stimulated increase in PAK activity is accompanied by the
phosphorylation of 60- and 90-kDa proteins. Both of these proteins
associate with PAK as demonstrated by their immunoprecipitation using
anti-PAK antibodies. The PAK family of serine/threonine kinases ranges
in molecular mass from 62 to 68 kDa (63, 64), and PAK
autophosphorylation is an important mechanism by which PAK cellular
function is regulated (65). The 60-kDa protein seen in Fig.
5A is most likely not a PAK family member because it is
tyrosine-phosphorylated in response to VEGF. We cannot rule out the
possibility that the [32P]ATP phosphorylation of the
60-kDa protein is due to a tyrosine kinase that immunoprecipitates with
the anti-PAK antibody, and not PAK itself. The identity of the
tyrosine-phosphorylated 90-kDa PAK substrate is also not known, but
most likely it is the 90-kDa protein identified in anti-PAK
immunoprecipitates by other investigators (57, 66).
It is not known whether the catalytic activity of PAK participates in
either focal adhesion assembly/disassembly or if it has some other role
in VEGF-induced signaling. Studies from the literature have yielded
seemingly conflicting results on the precise roles that the PAKs play
in regulating reorganization of the actin cytoskeleton. For example,
Cdc42 and Rac mutants defective for PAK binding can still form
filopodia and lamellipodia (67, 68), suggesting that PAK activity is
not required for these effects. On the other hand, an activated PAK1
mutant that is defective in Cdc42 or Rac binding promotes formation of
polarized membrane ruffles and vinculin-containing focal adhesions in
Swiss 3T3 cells (51, 52). This effect of PAK is independent of its
kinase activity. The confusion regarding the precise role of PAK in
Cdc42- and Rac-dependent morphological changes has been
partially resolved by the finding that a family of PAK-binding proteins
(PIX and COOL) exists that acts to enhance the coupling between Cdc42
and PAK and is required for PAK recruitment to focal adhesions
(66).
Our finding that NCK participates in the signal transduction pathway by
which VEGF stimulates endothelial cells to migrate is consistent with
the results obtained by Kiosses et al. (69). These authors
studied the role of PAK in endothelial cell migration after activating
endogenous PAK either by replating serum-starved cells on fibronectin
or by microinjecting cells with recombinant catalytically active or
dominant-negative protein. The conclusion of the study was that PAK
plays a role in coordinating leading edge adhesion formation and
trailing edge detachment to produce polarized cell movement.
Significantly, the role of PAK in cell migration was found to be
dependent upon a short proline-rich sequence that is known to bind the
SH2 domain of NCK, as a recombinant PAK lacking this domain acted as a
dominant-negative inhibitor of cell migration after microinjection into
endothelial cells.
We thank Dr. Anthony Ashton for valuable
assistance with the cell migration assays and Dona Wu, Harry Ma, Tina
Calderon, and Lillie Lopez for assistance with culturing HUVE cells.
The abbreviations used are:
VEGF, vascular
endothelial growth factor;
KDR, kinase insert domain-containing
receptor;
HUVE, human umbilical vein endothelial;
MAPK, mitogen-activated protein kinase;
FAK, focal adhesion kinase;
p120GAP, p120 GTPase-activating protein;
PAK, p21-activated kinase;
DMEM, Dulbecco's modified Eagle's medium;
Dok, downstream of tyrosine kinases;
PlGF, placenta-derived growth
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