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INTRODUCTION |
Vascular endothelial growth factor
(VEGF)1 exerts its cellular
responses by binding to one of its receptors, VEGFR-2/fetal liver
kinase-1, and stimulating its autophosphorylation. VEGFR-2 belongs to a
subfamily of receptor tyrosine kinases (RTKs) whose activation plays an
essential role in a large number of biological processes such as
embryonic development, wound healing, and pathological angiogenesis (1,
2). Although many cellular events involved in angiogenesis, including
endothelial cell proliferation, migration, and differentiation, have
been extensively characterized, the signal transduction pathways
downstream of VEGFR-2, which might mediate these events, are largely
limited. Elucidating the precise molecular mechanisms of signal
transduction relays involved in angiogenesis is required for design of
better anti-angiogenic strategies.
Recent studies on VEGFR-2 signal transduction relay have shown that
many well characterized signaling proteins such as phospholipase C
1
(PLC
1) and phosphatidylinositol 3-kinase (PI3K) are activated following stimulation of endothelial cells with VEGF (3, 4). However,
the role of individual tyrosine residues of VEGFR-2 that might
contribute to association and activation of PLC
1 is not clear and is
subject to a great deal of inconsistency within the literature. For
example, Cunningham et al. (5) suggested that tyrosines 801 and 1175 of human VEGFR-2 (corresponding to tyrosines 799 and 1173 of
mouse VEGFR-2, respectively) are binding sites for PLC
1. A study by
Takahashi et al. (3) showed that tyrosine 1175 is a
PLC
1-binding site in VEGFR-2 and that phosphorylation of tyrosine
1175 is required for association and activation of PLC
1 by VEGFR-2.
In contrast, Wu et al. (6) suggested that tyrosine 952 (but
not tyrosines 801 and 1175) is the PLC
1-binding site in VEGFR-2. How
can these discrepancies within the literature be explained? One
possibility is that VEGF-mediated VEGFR-2 autophosphorylation and the
ability of VEGFR-2 to recruit signaling proteins are influenced by
other endothelial cell-surface receptors such as VEGFR-1, neuropilin-1, and neuropilin-2, which are normally expressed by endothelial cells.
Because VEGF binds to all of these receptors, it is highly possible that activation of these receptors and their signal
transduction relays are influenced by the presence of these receptors
likely due to establishment of receptor homo- and heterodimerization in
endothelial cells (7-10). A second possibility is that activation of
VEGFR-2 and stimulation of its associated signaling proteins are
affected by endothelial cadherins and integrins, adding an additional level of complexity to VEGFR-2-induced signal transduction relays in endothelial cells (11, 12). Finally, it is also possible that
individual autophosphorylation sites in VEGFR-2 are not stringently
required for the recruitment and association of PLC
1. If this is
true, VEGFR-2 autophosphorylation sites may be compensatory in their
ability to associate with PLC
1.
In this study, we have addressed the enigma concerning the
recruitment and activation of PLC
1 by VEGFR-2 by using a unique system of a VEGFR-2 chimera and constructing a panel of VEGFR-2 tyrosine mutants, including tyrosines 799, 820, 949, 994, 1006, 1080, and 1173. In this system, VEGFR-2 is selectively activated by CSF-1
without any contributions from other VEGFRs such as VEGFR-1 and
neuropilins. Here, we report the following results. 1) Tyrosine 1006 (but not tyrosines 799, 949, 994, 820, 1080, 1173, and 1221) of VEGFR-2
is responsible for association with and activation of PLC
1. 2)
Association of PLC
1 with VEGFR-2 is established primarily by the
C-terminal SH2 domain of PLC
1. 3) PLC
1 activation is required for
endothelial cell tubulogenesis and differentiation, but not for
VEGFR-2-induced endothelial cell proliferation.
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MATERIALS AND METHODS |
Reagents and Antibodies--
Human recombinant CSF-1 was
purchased from R&D Systems. Mouse anti-phosphotyrosine (PY-20) and
anti-PLC
antibodies and anti-mouse and anti-rabbit secondary
antibodies were purchased from Transduction Laboratories (Lexington,
KY). Rabbit anti-MAPK and anti-phospho-MAPK antibodies were purchased
from New England Biolabs Inc. (Beverly, MA). Rabbit anti-phospho-PLC
antibody was purchased from BIOSOURCE (Camarillo,
CA). Rabbit anti-VEGFR-2 antibody was made against amino acids
corresponding to the kinase insert or carboxyl terminus of VEGFR-2 (9).
U73122 was purchased from Calbiochem. Mouse anti-phosphotyrosine
antibody 4G10 was purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY).
Cell Lines--
Porcine aortic endothelial (PAE) cells
expressing CKR and tyrosine mutant receptors were established by a
retroviral system as described previously (4, 9). Briefly, cDNAs
encoding CKR and tyrosine mutant receptors were cloned into retroviral vector pLNCX2 and transfected into 293-GPG
cells. Viral supernatants were collected for 7 days, concentrated by
centrifugation, and used as previously described (9).
Site-directed Mutagenesis--
The VEGFR-2 chimera CKR was
used as a template to construct the mutations. CKR was subcloned into
the pGEMT cloning vector, and site-directed mutagenesis was carried out
using a PCR-based site-directed mutagenesis method (4, 9, 30). The
resultant mutations were verified by sequencing and were subsequently
cloned into the pLNCX2 vector at NotI and
SalI sites.
Immunoprecipitation and Western Blotting--
PAE cells
expressing CKR and tyrosine mutant CKRs were grown under semiconfluent
culture conditions in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum and supplemented with glutamate,
penicillin, and streptomycin and serum-starved overnight in DMEM. Cells
were either left resting or stimulated with 40 ng/ml CSF-1 for 10 min
at 37 °C. Cells were washed twice with buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, and 2 mM Na3VO4 and lysed in lysis buffer
(10 mM Tris-HCl, 10% glycerol (pH 7.4), 5 mM
EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100,
1 mM phenylmethylsulfonyl fluoride, 2 mM
Na3VO4, and 20 µg/ml aprotinin). Proteins
were immunoprecipitated using appropriate antibodies. Immunocomplexes
were bound to protein A-Sepharose and washed three times with 1.0 ml of
lysis buffer. Immunoprecipitates were resolved on an
SDS-polyacrylamide gel, and the proteins were transferred to Immobilon
membranes. For Western blot analysis, the membranes were incubated for
60 min in blocking solution containing 10 mM Tris-HCl (pH
7.5), 150 mM NaCl, 10 mg/ml bovine serum albumin, and
0.05% Tween 20. The membranes were then incubated with primary antibodies diluted in blocking solution for another 60 min, washed three times with Western rinse, incubated with horseradish
peroxidase-conjugated secondary antibodies, washed, and developed with
ECL (Amersham Biosciences). Finally, the membranes were stripped by
incubation in stripping buffer containing 6.25 mM Tris-HCl
(pH 6.8), 2% SDS, and 100 mM
-mercaptoethanol at
50 °C for 30 min; washed with Western rinse; and reprobed with the
antibody of interest.
Cell Proliferation--
The proliferation assay was performed as
described (4, 9). Briefly, cells were plated at 2 × 104/ml in 24-well plates containing DMEM supplemented with
10% fetal bovine serum and incubated at 37 °C for 12 h. Cells
were then washed once with phosphate-buffered saline and serum-starved
overnight in DMEM, and various concentrations of CSF-1 were added.
During the last 4 h of incubation, cells were pulsed with
[3H]thymidine (0.2 µCi/ml) and harvested. Results for
each group were collected from four samples. Each experiment was
repeated three times, and essentially the same results were obtained.
The data are presented as -fold increase over the control.
Calcium Flux Assay--
PAE cells expressing either CKR or
tyrosine mutant CKRs were grown on 25-mm round glass coverslips and
serum-starved for 12-18 h. Cells were incubated in HEPES-buffered
saline solution (137 mM NaCl, 5 mM KCl, 4 mM MgCl2, 3 mM
CaCl2·2H2O, 25 mM glucose, and 10 mM HEPES) with 4 µM Fluo-3/AM supplemented
with 0.02% pluronic acid in Me2SO for 30 min at 37 °C.
After rinsing two times in HEPES-buffered saline solution, the live
cells were placed in an open chamber (Molecular Probes, Inc., Eugene,
OR) with 500 µl of HEPES solution and positioned on the stage
of a Zeiss LSM 510 Axiovert confocal laser scanning microscope equipped
with an argon laser. For each experiment, cells were scanned for at least 5-10 s before the addition of CSF-1 to establish a base-line fluorescence reading. All readings were made while continuously scanning the cells every 789 ms (33).
Measurement of Inositol Phosphate Production--
The
production of inositol phosphates was measured using 60-mm dishes of
PAE cells expressing CKR and tyrosine mutant CKRs labeled for
48 h in DMEM supplemented with 0.1% bovine serum albumin and 1 µCi/ml myo-[H]inositol. The cultures were washed and
incubated for 15 min with DMEM containing 0.1% bovine serum albumin,
15 mM HEPES (pH 7.5), and 20 mM LiCl. The
medium was then aspirated, and fresh medium with or without 40 ng/ml
CSF-1 was added and incubated at 37 °C for 20 min. The culture
plates were placed on ice, and the medium was removed before the
addition of 1.5 ml of ice-cold methanol/HCl (100:1). The quenched
samples were collected, and the plates were rinsed with an additional
1.5 ml of methanol/HCl (100:1). To each sample were added 1.5 ml of HO and 3 ml of CHCl3, and the tubes were mixed by vortexing
and left on ice for 30 min. The water-soluble phase was collected,
diluted with 2 volumes of water and 1 ml of AG 1-X8 formate resin
(Bio-Rad), and incubated for 2 h. The samples were extensively
washed with water and then with a solution containing 5 mM
disodium tetraborate and 60 mM sodium formate. Finally, the
total inositol phosphates were eluted from the resin with a solution of
0.1 M formic acid and 1.0 M ammonium formate
and subjected to scintillation counting. The CHCl3-phase
samples containing the phospholipids were dried, redissolved in
methanol, and subjected to scintillation counting.
In Vitro Angiogenesis/Tubulogenesis
Assay--
Endothelial cell spheroids were generated as previously
described (31). A defined number of cells were suspended in DMEM containing 1% fetal bovine serum and 0.24% (w/v)
carboxymethylcellulose (4000 centipoise) in non-adherent round-bottom
96-well plates under standard cell culture conditions. After 24 h,
all cells formed one single spheroid per well (750 cells/spheroid).
Spheroids were cultured for 2 days before using them in the in
vitro angiogenesis assay in the following manner. Spheroids
containing 750 cells were embedded in collagen gels. Eight volumes of
collagen were mixed with 1 volume of 10× HEPES-buffered saline
solution containing 10% 10× DMEM with phenol red. The pH was adjusted
to 7.4 with 0.1 N NaOH. Spheroids were centrifuged and
suspended in 9 ml of DMEM containing 0.96% carboxymethylcellulose.
Collagen and spheroids were mixed and transferred to prewarmed 24-well
plates, and the gels were allowed to polymerize in the incubator. After
30 min, 100 µl of DMEM containing various concentrations of CSF-1
were added on top of the gel. Sprouting and tubulogenesis were observed after 2 days under an inverted phase-contrast microscope (Nikon), and
pictures were taken using the SPOT camera system.
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RESULTS |
Role of Individual Tyrosine Residues of VEGFR-2 in the Activation
of PLC
1--
VEGF binds to multiple endothelial cell-surface
receptors, including VEGFR-1, VEGFR-2, neuropilin-1, and neuropilin-2
(7-10, 13), by generating potentially homologous and heterologous
signaling networks. Because these receptors are very often expressed in endothelial cells, it is difficult to address the activation of specific signaling molecules by the individual receptors. We have recently constructed a VEGFR-2 chimera containing the extracellular domain of human CSF-1 receptor/c-fms fused with the
transmembrane and cytoplasmic domains of murine VEGFR-2 (9). This model
allowed us to dissect the function of VEGFR-2 in endothelial cells by selectively stimulating the receptor with CSF-1 (4, 29). Throughout
this work, the VEGFR-2 chimera is called CKR. In this study, we used
PAE cells expressing CKR and aimed to address the role of individual
tyrosine residues in the recruitment and activation of PLC
1.
Initially, we evaluated the kinetics of tyrosine phosphorylation of
PLC
1 by CKR following CSF-1 stimulation. For this purpose, cells
were stimulated with CSF-1, and the kinetics of phosphorylation of
PLC
1 was evaluated by subjecting total cell lysates to Western blot
analysis using anti-phospho-PLC
1 antibody that specifically recognizes the active form of PLC
1. CKR-mediated phosphorylation of
PLC
1 peaked after 10 min of stimulation, was significantly reduced
after 30 min, and was not detectable after 45 min of stimulation (Fig.
1A). To test the role of
individual tyrosine sites in VEGFR-2-mediated activation of PLC
1 in
PAE cells, we individually replaced tyrosines 799, 820, 949, 994, 1006, 1173, and 1212 with phenylalanine. Fig. 1C shows the
schematic location of these tyrosine sites in VEGFR-2. We initially
tested the ability of F799/CKR and F1173/CKR to stimulate PLC
1 activation. These tyrosine sites are conserved in both human and mouse VEGFR-2, and the corresponding tyrosines in human VEGFR-2 are
at positions 801 and 1175, respectively. These tyrosine sites have
previously been suggested to bind PLC
1 (3). As demonstrated in Fig.
1D, individual mutation of tyrosines 799 and 1173 in mouse VEGFR-2 had no significant effect on their ability to activate PLC
1.
The data suggest that tyrosines 799 and 1173 are not responsible for
PLC
1 activation and furthermore argue that other tyrosine sites in
VEGFR-2 might mediate its activation. To test the contribution of other
tyrosine sites in VEGFR-2 to the activation of PLC
1, additional
sites were mutated and expressed in PAE cells. To this end, PAE cells
expressing tyrosine mutant CKRs, including tyrosines 820, 949, 994, 1006, and 1221, were stimulated with CSF-1 and analyzed for activation
of PLC
1. As demonstrated in Fig. 1F, all of the tyrosine
mutant receptors, viz. F820/CKR, F949/CKR, F994/CKR, and
F1221/CKR, were able to stimulate PLC
1 activation. In contrast,
F1006/CKR failed to stimulate robust activation of PLC
1 compared
with the wild-type receptor and other tyrosine mutant CKRs (Fig.
1F). The data also suggest that, in addition to tyrosines
799 and 1173, the presence of tyrosines 820, 949, 994, and 1221 is not
required for PLC
1 activation by VEGFR-2.

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Fig. 1.
Role of individual tyrosine residues of
VEGFR-2 in PLC 1 activation.
Serum-starved semiconfluent PAE cells expressing CKR were either
non-stimulated or stimulated with 40 ng/ml CSF-1 for the indicated
times, washed, and lysed, and cell extracts were normalized for
protein. Total cell lysates were subjected to Western blot analysis
using anti-phospho-PLC 1 antibody (A). The same membrane
was reprobed with anti-PLC 1 antibody (B). A schematic
representation of tyrosine residues located in the cytoplasmic region
of VEGFR-2 is shown in C. Serum-starved PAE cells expressing
wild-type CKR, F799/CKR, or F1173/CKR were treated with CSF-1
for 10 min, washed, lysed, and subjected to Western blot
analysis using anti-phospho-PLC 1 (D). The same membrane
was reprobed with anti-PLC 1 antibody (E). Serum-starved
PAE cells expressing wild-type CKR, F820/CKR, F949/CKR,
F994/CKR, F1006/CKR, or F1221/CKR were treated with CSF-1, and total
cell lysates were resolved by SDS-PAGE and blotted with
anti-phospho-PLC 1 antibody (F). The same membrane was
reprobed with anti-PLC 1 antibody for protein levels
(G).
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Role of Tyrosines 1006 and 1080 of VEGFR-2 in PLC
1 and MAPK
Activation--
Tyrosines 1006 and 1080 are located in the kinase
domain of VEGFR-2, adjacent to the N and C termini of the activation
loop, respectively (Fig. 2A).
To test the putative contribution of tyrosines 1006 and 1080 to PLC
1
activation, we generated a double tyrosine mutant receptor in which
both tyrosines 1006 and 1080 were replaced with phenylalanine. The
resultant receptor was similarly expressed in PAE cells and tested for
its ability to activate PLC
1. As Fig. 2B shows,
ligand-stimulated cells expressing the double mutant receptor
(F1006/F1080/CKR) maintained their ability to induce residual
activation of PLC
1. The ability of the double mutant receptor to
activate PLC
1 was not completely diminished and indeed was similar
to that of F1006/CKR (Fig. 2B), suggesting that tyrosine 1080 is not involved in the activation of PLC
1 by VEGFR-2. In addition, the single tyrosine 1080 mutant was also fully capable of
activating PLC
1, similar to the wild-type receptor (data not shown).

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Fig. 2.
Mutation of tyrosine 1006 impairs the ability
of VEGFR-2 to stimulate PLC 1 activation, but
not MAPK activation. Shown is a schematic representation of
tyrosines 1006 and 1080 of VEGFR-2 (A). Serum-starved
semiconfluent PAE cells expressing CKR, F1006/CKR, F1006/F1080/CKR were
either non-stimulated or stimulated with 40 ng/ml CSF-1 for 10 min,
washed, and lysed, and cell extracts were normalized for protein. Total
cell lysates were subjected to Western blot analysis using
anti-phospho-PLC 1 antibody (B). The same membrane was
reprobed with anti-PLC 1 antibody (C). Serum-starved cells
were stimulated with CSF-1 as described for B,
immunoprecipitated with anti-VEGFR-2 antibody, and subjected to Western
blot analysis using anti-phosphotyrosine antibody (D). The
same membrane was reprobed with anti-VEGFR-2 antibody (E).
Serum-starved cells were stimulated with CSF-1 as described for
B, and total cell lysates were subjected to Western blot
analysis using anti-phospho-MAPK (p44/42) antibody (F). The
same membrane was reprobed with anti-MAPK antibody
(G).
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To test the role of tyrosines 1006 and 1080 in the
ligand-dependent autophosphorylation VEGFR-2, we also
analyzed their ligand-dependent tyrosine phosphorylation.
As Fig. 2D demonstrates, mutation of tyrosines 1006 and 1080 had no significant effect on the tyrosine phosphorylation of the
receptor, suggesting that these tyrosine residues are not involved in
the regulation of ligand-dependent receptor
autophosphorylation. To test whether tyrosine 1006 plays a role in
VEGFR-2-depdendent activation of MAPK and also to test whether
activation of PLC
1 modulates MAPK activation in this system, we
analyzed the ability of this receptor to stimulate MAPK activation. As
Fig. 2F shows, both single mutant (F1006/CKR) and double
mutant (F1006/F1080/CKR) receptors were fully able to activate MAPK.
The data strongly suggest that activation PLC
1 is not required for
VEGFR-2-mediated phosphorylation of MAPK.
The SH2 Domains of PLC
1 Cooperatively Associate with
Ligand-stimulated VEGFR-2--
Association of PLC
1 with RTKs is
established by its N- and C-SH2 domains (14). However, the N- and C-SH2
domain requirement of PLC
1 for its activation appears to be
distinctive among RTKs whose stimulation leads to PLC
1 activation
(14, 15, 31). To test which SH2 domain of PLC
1 is involved in
association with VEGFR-2, we made recombinant GST fusion proteins
consisting of the N-SH2, C-SH2, or C- and N-SH2 domains of PLC
1 and
tested their ability to associate with ligand-stimulated CKR in
vitro. As shown in Fig.
3B, no significant association
between CKR and GST-N-SH2 was observed. Only a modest association of
CKR with the C-SH2 domain was detected. In contrast, when both the N-
and C-SH2 domains were fused to GST, a strong association between CKR
and the GST fusion proteins containing the N- and C-SH2 domains was
detected. These results suggest that the presence of the N-SH2 domain
of PLC
1 alone is not sufficient to mediate the association of
PLC
1 with VEGFR-2. In contrast, the C-SH2 domain is, in part, able
to associate with VEGFR-2 without the N-SH2 domain. However, the
presence of the N-SH2 domain greatly facilitated the ability of the
C-SH2 domain to interact with VEGFR-2 (Fig. 3B), suggesting that the C- and N-SH2 domains of PLC
1 cooperatively associate with
VEGFR-2.

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Fig. 3.
The C-SH2 domain of
PLC 1 associates with tyrosine 1006 of
VEGFR-2. Shown is a schematic presentation of PLC 1 and GST
fusion proteins containing the SH2 domains of PLC (A).
Serum-starved semiconfluent PAE cells expressing CKR were stimulated
with 40 ng/ml CSF-1, washed, and lysed, and cell extracts were
normalized for protein. Total cell lysates were incubated with
Sepharose-bound GST-N-SH2, GST-C-SH2, or GST-N+C-SH2. After extensive
washing, the precipitated proteins were resolved by SDS-PAGE and
immunoblotted with anti-VEGFR-2 antibody (B). Serum-starved
semiconfluent PAE cells expressing CKR, F1006/CKR, and F1173/CKR were
stimulated with 40 ng/ml CSF-1, washed, and lysed, and cell extracts
were normalized for protein. Total cell lysates were incubated with
either GST-C-SH2 or GST-C+N-SH2 as described for B and
subjected to Western blot analysis using anti-VEGFR-2 antibody
(C).
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To test the contribution of tyrosines 1006 and 1173 to complex
formation between CKR and the SH2 domains of PLC
1, PAE cells expressing either F1006/CKR or F1173/CKR were stimulated with CSF-1,
and cell lysates were incubated with GST-C-SH2 and GST-C+N-SH2 recombinant proteins. As shown in Fig. 3C, the ability of
CKR to associate with the SH2 domains of PLC
1 was significantly
reduced when tyrosine 1006 was mutated. In contrast, mutation of
tyrosine 1173 did not alter the association of CKR and the SH2 domains of PLC
1. Altogether, the data suggest the following. (i) The N-SH2
domain of PLC
1 alone is not sufficient to bind VEGFR-2. (ii) The
C-SH2 domain of PLC
1 binds to VEGFR-2 weakly, but its association
with VEGFR-2 is greatly enhanced by the presence of the N-SH2 domain.
This suggests that the C- and N-SH2 domains of PLC
1 are engaged in
association with VEGFR-2 in a cooperative manner. (iii) The presence of
tyrosine 1006 (but not tyrosine 1173) is required for association of
PLC
1 with VEGFR-2.
The Presence of Tyrosine 1006 Is Required for
VEGFR-2-mediated Inositol 1,4,5-Triphosphate (IP3)
Generation and Intracellular Calcium Release--
The immediate
consequence of PLC
1 activation by RTKs is accumulation of
IP3 and diacylglycerol. IP3 accumulation
stimulates the release of calcium from intracellular stores (14, 15). To test the requirement for tyrosines 1173 and 1006 of VEGFR-2 in
IP3 production and calcium release, we tested the ability
of F1173/CKR and F1006/CKR to stimulate IP3 production and
intracellular calcium release in PAE cells. As depicted in Fig.
4A, stimulation of both CKR
and F1173/CKR resulted in robust production of IP3. In
contrast, F1006/CKR failed to stimulate a significant amount of
IP3 production (Fig. 4A). In addition, we also
evaluated the ability of CKR and tyrosine mutant CKRs to stimulate the
intracellular calcium release in PAE cells after stimulation with
CSF-1. Fig. 4B shows that stimulation of PAE cells
expressing either CKR or F1173/CKR with CSF-1 caused rapid
intracellular calcium release as measured using Fluo-3/AM as a probe.
Unlike CKR and F1173/CKR, F1006/CKR completely failed to stimulate
intracellular calcium release (Fig. 4B). Collectively, these
results suggest that the presence of tyrosine 1006 (but not tyrosine
1173) of VEGFR-2 is required for IP3 production and
intracellular calcium release.

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Fig. 4.
The presence of tyrosine 1006 of VEGFR-2 is
required for inositol phosphate production and calcium flux. PAE
cells expressing CKR, F1173/CKR, and F1006/CKR were incubated with
1µCi/ml myo-[H]inositol for 48 h in DMEM
supplemented with 0.1% bovine serum albumin, and phospholipid
production was measured by scintillation counting as described under
"Materials and Methods" (A). Serum-starved PAE cells
expressing CKR, F1173/CKR, and F1006/CKR were grown on glass coverslips
and stimulated with CSF-1, and calcium flux was measured by confocal
microscopy using Fluo-3/AM as a probe as described under "Materials
and Methods" (B). IPs, inositol
phosphates.
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Mutation of Tyrosine 1006 of VEGFR-2 Enhances Its Ability to
Stimulate Proliferation of PAE Cells--
One of the functions of
VEGFR-2 in endothelial cells is the induction of endothelial cell
proliferation (9). To test the potential role of tyrosine 1006 of
VEGFR-2 in endothelial cell proliferation, we subjected cells
expressing CKR, F1173/CKR, F949/CKR, and F1006/CKR to a proliferation
assay. As shown in Fig. 5, stimulation of
PAE cells expressing CKR, F949/CKR, and F1173/CKR with CSF-1 induced
proliferation of PAE cells in a CSF-1-dependent manner. As
previously reported, mutation of tyrosine 1173 partially abolishes VEGFR-2-mediated proliferation of PAE cells (4). In contrast to
wild-type CKR, F949/CKR, and F1173/CKR, stimulation of
F1006/CKR-expressing cells resulted in augmented proliferation of PAE
cells. Interestingly, an increase in the concentration of CSF-1
resulted in even greater cell proliferation. This observation was in
contrast to what we observed with wild-type VEGFR-2 or CKR. At
subsaturated concentrations of ligand (1-5 ng/ml CSF-1 and 10-20
ng/ml VEGF), stimulation of CKR and VEGFR-2 induced cell proliferation.
However, at saturated concentrations of ligand (10-40 ng/ml CSF-1 and
20-100 ng/ml VEGF), VEGFR-2 stimulation caused cell differentiation
(Ref. 4 and this work). Collectively, these results suggest that
phosphorylation of tyrosine 1006 and its involvement in the recruitment
of PLC
1 to VEGFR-2 may negatively regulate the ability of VEGFR-2 to
stimulate endothelial cell proliferation.

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Fig. 5.
Mutation of tyrosine 1006 enhances the
ability of VEGFR-2 to stimulate cell proliferation. Serum-starved
PAE cells expressing wild-type CKR and tyrosine mutant CKRs were
treated with different concentrations of CSF-1, and DNA synthesis was
measured by [3H]thymidine uptake. The results are
expressed as the mean (cpm/well) ± S.D. of quadruplicates. The
data are expressed as a ratio of stimulated versus
non-stimulated samples (A). Serum-starved semiconfluent PAE
cells expressing CKR or F1006/CKR were either non-stimulated or
stimulated with 40 ng/ml CSF-1 for 20 or 30 min, washed, and lysed, and
cell extracts were normalized for protein. Total cell lysates were
subjected to Western blot analysis using anti-phospho-AKT antibody
(B). The same membrane was reprobed with anti-AKT antibody
(C).
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We have recently shown that activation of the PI3K pathway is required
for VEGFR-2-mediated endothelial cell growth (4). To test the ability
of F1006/CKR to stimulate the PI3K pathway, we measured AKT
phosphorylation using anti-phospho-AKT antibody. The result shows that
F1006/CKR retained its ability to activate AKT. Indeed, AKT activation
appeared to be increased by F1006/CKR compared with wild-type CKR (Fig.
5B). This suggests that the F1006/CKR-enhanced proliferation
of PAE cells is, in part, associated with more efficient activation of
the PI3K pathway by this receptor.
Tyrosine 1006 of VEGFR-2 Is Required for Differentiation and in
Vitro Angiogenesis of PAE Cells--
Angiogenesis is a complex
cellular process that involves endothelial cell proliferation,
migration, and differentiation. Activation of VEGFR-2 is known to
provoke these cellular events in endothelial cells. To test the
requirement for tyrosines 1006 and 1173 in VEGFR-2-mediated cell
differentiation, PAE cells expressing wild-type CKR, F1006/CKR, and
F1173/CKR were subjected to differentiation and in vitro
angiogenesis assays. As shown in Fig.
6A, stimulation of both
wild-type CKR and F1173/CKR stimulated morphological changes in PAE
cells. In contrast, stimulation of F1006/CKR resulted in no apparent
morphological change in PAE cells, suggesting that tyrosine 1006 of
VEGFR-2 is involved in mediating endothelial cell differentiation. To
further test the contribution of tyrosine 1006 to VEGFR-2-mediated cell
differentiation, we assessed the ability of F1006/CKR to stimulate
tubulogenesis of PAE cells. As shown in Fig. 6B, stimulation
of both wild-type CKR and F1173/CKR induced tubulogenesis; however
F1006/CKR failed to stimulate tubulogenesis (Fig. 6B). To
test the role of PLC
1 in endothelial cell tubulogenesis, we also
attempted to inhibit PLC
1 by a pharmacological approach using
U73122, a selective PLC
1 inhibitor. As shown in Fig. 7, CKR-induced tubulogenesis was
inhibited by pretreatment of cells with U73122. Collectively, these
results suggest that the presence of tyrosine 1006 of VEGFR-2 is
essential for its ability to stimulate endothelial cell differentiation
and tubulogenesis. Furthermore, activation of PLC
1 by VEGFR-2 is
required for these cellular responses in endothelial cells.

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Fig. 6.
Mutation of tyrosine 1006 abrogates the
ability of VEGFR-2 to stimulate differentiation and tubulogenesis of
PAE cells. PAE cells expressing CKR, F1173/CKR, and F1006/CKR were
plated in six-well plates, serum-starved overnight, and either
non-stimulated or stimulated with CSF-1 (20 ng/ml) for 24-30 h.
Morphological changes associated with CSF-1 stimulation were viewed
under an inverted microscope (magnification ×10) and photographed with
the SPOT camera system (A). PAE cells expressing CKR,
F1173/CKR, and F1006/CKR were prepared as spheroids and subjected to an
in vitro angiogenesis/tubulogenesis assay as described under
"Materials and Methods." Sprouting and tubulogenesis were observed
after 2 days under an inverted phase-contrast microscope, and pictures
were taken using the SPOT camera system (B).
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Fig. 7.
Inhibition of PLC 1
by pharmacological means inhibits the ability of CKR to stimulate
tubulogenesis. PAE cells expressing CKR were prepared in spheroid
forms and subjected to an in vitro
angiogenesis/tubulogenesis assay as described for Fig. 6B.
Spheroids were treated with different concentrations of the PLC 1
inhibitor U73122 as indicated. Sprouting and tubulogenesis were
observed after 2 days under an inverted phase-contrast microscope, and
pictures were taken using the SPOT camera system.
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DISCUSSION |
In recent years, it has been suggested that PLC
1 activation is
associated with VEGFR-2-mediated endothelial cell proliferation and
MAPK activation (3), and there are several studies that have also
investigated the VEGFR-2 tyrosine residues that are important in
PLC
1 activation. However, the mechanism by which PLC
1 is
activated by VEGFR-2 and the biological importance of PLC
1 in
endothelial cells remain elusive and controversial (3-6). We found
that tyrosine 1006 of VEGFR-2 is a critical residue required for
VEGFR-2-mediated PLC
1 activation, IP3 production, and
intracellular calcium release in PAE cells. There are several possible
explanations for the discrepancies between our present results and the
results of previous studies. Many of the previous studies used
wild-type systems, including endothelial cell lines, most of which
endogenously express VEGFR-1, VEGFR-2, neuropilin-1, and neuropilin-2.
VEGF binds to all of these receptors. Thus, it is highly
possible that activation of these receptors and their signal
transduction relays are influenced by the presence of these receptors
likely due to establishment of receptor homo- and heterodimerization in
endothelial cells (8, 9, 12). The chimeric model of VEGFR-2 employed in
our study is highly selective, and contribution of other VEGFRs to
PLC
1 activation is unlikely. Another possibility is that murine VEGFR-2, in which the chimeric receptor CKR was created, unexpectedly differs from human VEGFR-2.
Our results also demonstrate that the N-SH2 domain of PLC
1
alone is not able to associate significantly with CKR. Only a modest
association of CKR with the C-SH2 domain was detected. In contrast,
when the both N- and C-SH2 domains were fused to GST, a strong
association between CKR and the GST fusion proteins containing the N-
and C-SH2 domains was detected. These results suggest that the presence
of the N-SH2 domain of PLC
1 alone is not sufficient to mediate
association of PLC
1 with VEGFR-2. In contrast, the C-SH2 domain is,
in part, able to associate with VEGFR-2 without the N-SH2 domain.
However, the presence of the N-SH2 domain greatly facilitated the
ability of the C-SH2 domain to interact with VEGFR-2, suggesting that
the C- and N-SH2 domains of PLC
1 cooperatively associate with
VEGFR-2. Also our results demonstrate that the presence of tyrosine
1006 (but not tyrosine 1173) is required for association of PLC
1
with VEGFR-2.
Stimulation of many RTKs, including the platelet-derived growth factor
receptor, fibroblast growth factor receptor, hepatocyte growth
factor receptor, and VEGFR-2, has been shown to activate PLC
1 (3,
14-19). Nevertheless, the significance of PLC
1 in the RTK-initiated
cellular responses such as cell proliferation and differentiation still
is not clear. In some cases such as the platelet-derived growth factor
receptor, activation of PLC
1 is suggested to be involved in cell
proliferation (20). Unlike these observations, many recent elegant
studies on the biological importance of PLC
1 demonstrated a negative
role for PLC
1 in cell proliferation induced by RTKs. For instance,
fibroblast cells obtained from PLC
1 knockout mice are normal in
their ability to proliferate in response to epidermal growth factor and
other growth stimulation (21, 32). Overexpression of PLC
1 in
fibroblast cells also appears to have no effect on platelet-derived
growth factor- and basic fibroblast growth factor-dependent
cell proliferation (22). A point mutation of the fibroblast growth
factor receptor that abolishes PLC
1 association with the receptor
also shows no negative effect on the ability of the fibroblast growth
factor receptor to stimulate cell proliferation (23, 24). Moreover, the
transforming potentials of the epidermal growth factor receptor and
nerve growth factor receptor/Trk have been shown to inversely correlate
with their ability to associate with PLC
1 (25). Altogether, these
studies suggest that activation of PLC
1 is dispensable or
compensatory for cell proliferation.
How can mutation of tyrosine 1006 of VEGFR-2 and the lack of
PLC
1 activation by this receptor enhance the mitogenic effect of
VEGFR-2 in endothelial cells? One possibility is that the recruitment of PLC
1 by VEGFR-2 through tyrosine 1006 promotes endothelial cell
tubulogenesis and differentiation, thus preventing cells from
proliferation. This possibility is supported by the observation that
mutation of tyrosine 1006 to phenylalanine not only did not decrease
the ability of the VEGFR-2 chimera CKR to stimulate cell proliferation,
but rather caused the receptor to stimulate cell proliferation even
better than wild-type CKR. This is consistent with the elimination of
the ability of CKR to stimulate cell differentiation.
We have recently shown that activation of CKR stimulates endothelial
cell proliferation in a PI3K-dependent manner and that tyrosines 799 and 1173 of VEGFR-2 are required for both PI3K activation and cell proliferation (4). The opposing effects of PLC
1 and PI3K on
cellular functions reported in the literature are consistent with our
proposed model of VEGFR-2-mediated cell proliferation and
differentiation. The role of PI3K in RTK-mediated mitogenic responses
has been documented extensively (26). In contrast, activation of
PLC
1 has been described as a negative feedback regulator of cell
proliferation induced by RTKs (25, 27). Moreover, recent studies
suggest that PLC
1 activation is directly linked to cell
differentiation (28, 29), suggesting that PLC
1 activation may even
encounter cell proliferation by promoting cell differentiation.
Thus, based on our previous (4) and current data, we propose that
VEGFR-2-mediated signal transduction relay (in particular, the decision
whether endothelial cells proliferate or differentiate) depends on
reciprocal activation of PI3K and PLC
1 by VEGFR-2. Consistent with
this idea, tyrosine 799 and 1173 mutations of CKR enhance its ability
to stimulate cell differentiation, whereas these mutations abolish the
mitogenic ability of CKR (4). Interestingly, F1006/CKR displayed an
inverse phenotype in its ability to stimulate cell differentiation
versus cell proliferation. Coordinated endothelial cell
proliferation and differentiation are essential prerequisites for
angiogenesis. Our study demonstrates that VEGFR-2 initiates these
opposing cellular events by recruiting and activating PLC
1. Activation of PLC
1 is inversely correlated with the ability of VEGFR-2 to stimulate cell proliferation. Activation of PLC
1 by VEGFR-2 promotes tubulogenesis and differentiation. Furthermore, studies are required to determine the underlying mechanisms involved in
PLC
1-mediated endothelial cell differentiation.