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INTRODUCTION |
Sphingosine 1-phosphate
(Sph-1-P)1 has been shown to
elicit a great variety of responses, including stimulation of cell
proliferation and survival, regulation of cell motility, and
cytoskeletal reorganization, in numerous cell types (1-4). Blood
platelets are unique in that they store Sph-1-P abundantly (possibly
due to the presence of highly active Sph kinase and lack of Sph-1-P
lyase) and release this bioactive lipid extracellularly upon
stimulation (5, 6). Hence, it is important to examine the effects of
Sph-1-P on vascular endothelial cell functions from the viewpoint of
platelet-endothelial cell interactions that may be involved in such
diverse activities as thrombosis, hemostasis, angiogenesis, and
atherosclerosis. We previously showed that Sph-1-P prevents apoptosis
induced by withdrawal of growth factors and stimulates DNA synthesis in
human umbilical endothelial cells (HUVECs) (7). Recently, Sph-1-P has
been reported to induce endothelial cell migration and morphogenesis into capillary-like networks and act as an angiogenic molecule (8-10).
Although originally postulated to function as an intracellular second
messenger (11), it is now established that Sph-1-P exerts many of its
effects as an extracellular mediator (1-4). Recently, plasma membrane
receptors for Sph-1-P have been identified; the endothelial
differentiation gene (EDG)-1, EDG-3, and EDG-5 (earlier known as AGR16
or H218) exhibit overlapping, as well as distinct, patterns of
expression in various tissues as Sph-1-P receptors (12-16). In HUVECs,
EDG-1 was shown to be abundantly expressed, whereas
EDG-3 was expressed at a lower level (8). Consistent with
the presence of its high affinity cell surface receptors on vascular
endothelial cells, nanomolar Sph-1-P reportedly induces a variety of
responses in these cells (7-10). As for intracellular signalings,
Sph-1-P induces Ca2+ mobilization, mitogen-activated
protein kinase activation, and Rho family small G protein activation
(8-10). Functionally, Sph-1-P stimulates endothelial cell
proliferation through a Gi-coupled receptor, probably EDG-1
(8, 9). Furthermore, Sph-1-P induces adherens junction assembly,
migration, capillary tube formation, and promotion of angiogenesis
(8-10). In this case, the signals mediated via EDG-1 (coupled with
Gi) and EDG-3 (coupled with G13 and
Gq) are both necessary, and Rho- and Rac-mediated
signalings are involved (8). Sph-1-P induction of angiogenesis through these EDG receptors may be related to involvement of activated platelets in this important process in vivo. However, the
overall signaling pathways leading to these cytoskeletal events have
not been elucidated. In the present study, we examined
Sph-1-P-stimulated signaling pathways related to HUVEC motility with a
special emphasis on Crk-associated substrate (Cas), a docking protein
involved in cytoskeletal reorganization (17).
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant Clostridium botulinum C3
exoenzyme was prepared as described previously (18) and kindly donated
by Dr. S. Narumiya (Department of Pharmacology, Kyoto University
Faculty of Medicine). Anti-Src monoclonal antibody (mAb) (clone
327) was kindly provided by Dr. G. Katoh (Yamanashi Medical University,
Yamanashi, Japan). Y-27632, a specific Rho kinase inhibitor (19), was a
gift from Welfide Corp. (Osaka, Japan).
The following materials were obtained from the indicated suppliers:
anti-focal adhesion kinase (FAK) mAb (clone 2A7, used for
immunoprecipitation), anti-Lyn polyclonal antibody, and
anti-phosphotyrosine mAb (4G10) (Upstate Biotechnology Inc., Lake
Placid, NY); anti-FAK mAb (clone 77, used for immunoblotting), anti-Cas
mAb, anti-Crk mAb, anti-paxillin mAb, and anti-phosphotyrosine mAb
(PY20) (Transduction Laboratories, Lexington, KY); tetramethylrhodamine
isothiocyanate (TRITC)-phalloidin, and D-erythro-Sph
(Sigma); Sph-1-P (Biomol, Plymouth Meeting, PA); anti-Lyn mAb (Wako
Pure Chemical Industries, Tokyo, Japan); anti-Fyn mAb (Santa Cruz
Biotechnology, Santa Cruz, CA); protein A-Sepharose 4B and
glutathione-Sepharose 4B (Amersham Pharmacia Biotech); fura2-AM
(Dojindo Laboratories, Kumamoto, Japan); PP2 (Calbiochem); pertussis
toxin (Kaken Pharmaceutical Co., Tokyo, Japan);
[
-32P]ATP (111 TBq/mmol) (Du Pont-New England Nuclear,
Boston, MA).
Cell Culture and Preparation--
HUVECs were prepared as
described previously (7). They were grown and maintained in Dulbecco's
modified Eagle's medium supplemented with 10 ng/ml of recombinant
human basic fibroblast growth factor (PeproTech EC, London, UK), 20%
fetal calf serum (ICN Biomedicals, Aurora, OH), penicillin G (100 units/ml), and streptomycin sulfate (100 mg/ml), at 37 °C under an
atmosphere of 5% CO2 and 95% room air. HUVECs were not
used after the sixth passage. The cells were replated in either 10- or
3.5-cm dishes coated with 0.2% gelatin (Sigma) and were used when
confluent. When HUVECs were serum-starved, serum plus basic fibroblast
growth factor were removed from the medium 2 h before stimulation.
Human platelets were prepared as described previously (20).
Immunoblotting--
The proteins were resolved on an SDS-PAGE
and then electrophoretically transferred to PVDF membranes. The
membranes were blocked with 1% bovine serum albumin in
phosphate-buffered saline (PBS). After extensive washing with PBS
containing 0.1% Tween 80, the immunoblots were incubated with
anti-phosphotyrosine mAb (1 µg/ml PY20 plus 1 µg/ml
4G10), anti-Src mAb (1 µg/ml), anti-Fyn mAb (1 µg/ml), anti-Lyn
polyclonal antibody (2 µg/ml), anti-FAK mAb (0.25 µg/ml), anti-Crk
mAb (0.25 µg/ml), or anti-Cas mAb (0.25 µg/ml) for 2 h.
Antibody binding was detected using peroxidase-conjugated goat
anti-mouse IgG and visualized with ECL chemiluminescence reaction
reagents (Amersham Pharmacia Biotech). For reprobing with other
antibodies, the antibody bound on the PVDF membrane was removed with a
stripping buffer (2% SDS, 62.5 mM Tris/HCl (pH 6.8), 100 µM 2-mercaptoethanol) at 60 °C for 30 min. After washing twice with PBS containing 0.1% Tween 80, the membranes were
blocked with 1% bovine serum albumin and reprobed with the indicated antibody.
Immunoprecipitation and in Vitro Kinase Assay--
HUVECs were
lysed in a 1× ice-cold lysis buffer (50 mM Tris (pH 7.4),
1% Triton X-100, 1% Nonidet P-40, 20 mM NaF, 100 mM NaCl, 6 mM EDTA, 1 mM
Na3VO4, 0.5 mM phenylmethylsulfonyl
fluoride, and 25 µg/ml of leupeptin) with the aid of a cell scraper.
The lysates were stood on ice for 1 h with occasional mixing,
followed by centrifugation at 15,000 × g for 10 min.
The protein concentrations of the resultant supernatant were determined
by dye binding (21), and 0.5 mg of protein was used for
immunoprecipitation; some of the lysate supernatant was used as the
whole cell lysate. All subsequent immunoprecipitation steps were
carried out at 4 °C. The samples were precleared with protein
A-Sepharose 4B, and the resultant supernatants were incubated for
4 h with the antibody indicated. Protein A-Sepharose 4B was then
added and further incubated for 2 h. The resulting Sepharose beads
were washed with a 1× lysis buffer three times. The samples were then
separated into 2 aliquots. One was solubilized with an SDS sample
buffer and used for immunoblotting. When indicated, the other was
processed further for an in vitro kinase assay as follows.
The beads were washed once with a low salt buffer (10 mM
Tris (pH 7.2), 100 mM NaCl, and 5 mM
MnCl2) and then incubated with 35 µl of 20 mM
Hepes (pH 7.4) containing 5 mM MgCl2 and 12.5 µg of acid-treated enolase. The reaction was initiated by the
addition of 15 µl of kinase reaction buffer (300 mM
Hepes/NaOH (pH 8.0), 30 µM
Na3VO4, 150 mM MgCl2,
15 mM MnCl2, and 5 µM
[
-32P]ATP (0.75 µCi)). After 8 min at room
temperature, the reaction was terminated by the addition of 25 µl of
3× SDS sample buffer, and then the contents were boiled for 3 min. The
proteins were separated by an SDS-PAGE and electroblotted onto a PVDF
membrane. The membrane was treated with 1 M KOH at 60 °C
for 30 min and then 10% acetic acid at 20 °C for 15 min and dried.
The phosphorylated protein was quantified using a BAS-2000
PhosphorImager analyzer (Fuji Film, Tokyo, Japan).
GST Fusion Protein Production and Binding Studies--
The
plasmid that encoded the GST fusion protein containing the SH2 domain
of Crk was kindly provided by Dr. M. Matsuda (International Medical
Center, Tokyo, Japan). The fusion protein construct was transformed
into Escherichia coli for protein production. The protein
produced was purified on a glutathione-Sepharose column by affinity
chromatography, according to the manufacturer's recommendations (Amersham Pharmacia Biotech). For the binding experiments, the HUVEC
lysates were precleared with glutathione-Sepharose 4B, mixed with 20 µg of GST fusion protein, and incubated for 1 h at 4 °C on a
rotary shaker. Glutathione-Sepharose 4B beads were added to preabsorb
the protein complex. Following incubation for 1 h, the beads were
centrifuged and washed three times with a 1× lysis buffer. The bound
proteins were eluted with a 1× SDS sample buffer and boiled for 3 min.
The proteins were separated on an SDS-PAGE and immunoblotted, as
described above.
Actin Staining--
For actin staining, cells were fixed with
3% paraformaldehyde in PBS for 40 min and then permeabilized with
0.2% Triton X-100 for 8 min. Actin filaments were detected by staining
with 0.1 µg/ml TRITC-conjugated phalloidin. Actin staining was
observed and photographed using a confocal microscope.
Immunofluorescent Staining--
Fixed and permeabilized cells
were incubated with anti-paxillin mAb (2.5 µg/ml), anti-Cas mAb (25 µg/ml), or anti-Crk mAb (2.5 µg/ml) for 2 h at room
temperature, washed in PBS, and then incubated for 1 h with
FluoroLinkTM Cy3TM-labeled goat anti-mouse IgG
(Amersham Pharmacia Biotech). After incubation, the cells were washed
three times with PBS. Immunofluorescent staining was observed and
photographed using a confocal microscope.
Phagokinetic Assay Using Gold Sol-coated Plates--
Random cell
motility and phagocytotic activity were jointly estimated as the area
of phagokinetic tracks on gold sol particle-coated plates, as described
previously (22). Briefly, 3.5-cm dishes coated with 0.2% gelatin were
incubated with colloidal gold for 45 min and then washed two times with
PBS. HUVECs (2000 cells) in Dulbecco's modified Eagle's medium
containing 2% fetal calf serum were added to each dish. After 16 h at 37 °C, phagokinetic tracks were visualized using dark field
illumination with a confocal microscope. The area cleared of gold
particles was measured after photography, and the mean value for 20 cells was calculated in each experiment.
Migration Assay--
HUVEC migration was assessed by a modified
Boyden's chamber assay, i.e. in Transwell cell culture
chambers (Costar, Cambridge, MA) basically as described previously
(23). Polycarbonate filters with 8 µm pores, used to separate the
upper and lower chamber, were coated with 500 µg/ml of Matrigel
(Becton Dickinson, Bedford, MA). The coated filters were washed with a
serum-free medium and dried immediately. Then HUVECs were added to the
upper compartment of the chamber at a density of 1 × 105/100 µl of medium containing 0.1% bovine serum
albumin and incubated for 4 h at 37 °C. HUVECs were allowed to
migrate toward an indicated chemoattractant in the lower chamber. After
the reaction, the filters were fixed and stained with trypan blue.
After removal of nonmigrated cells by wiping with cotton swabs, cells
that had migrated through the filter to the lower surface were counted manually under a microscope in five predetermined fields at a magnification of × 200.
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RESULTS |
Expression of Cas in HUVECs and its Tyrosine Phosphorylation upon
Stimulation with Sph-1-P--
We first checked the expression of
cytoskeletal proteins such as Cas, FAK, talin, and vinculin in HUVECs.
The same amount of HUVEC lysate protein was resolved on an SDS-PAGE and
probed with each antibody. As expected, these cytoskeletal proteins
were confirmed to be expressed in HUVECs (Fig.
1A). One notable finding was
the abundant expression of Cas in HUVECs, although this was evaluated
only in comparison with platelets (Fig. 1A). To investigate the involvement of Cas in Sph-1-P signaling pathways, we examined the
effect of Sph-1-P on its tyrosine phosphorylation in HUVECs. As shown
in Fig. 1B, Sph-1-P induced tyrosine phosphorylation of this
cytoskeletal docking protein, reaching a maximal level 15-30 min after
stimulation. The time course of Cas tyrosine phosphorylation induced by
Sph-1-P was similar to that of FAK (Fig. 1B).

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Fig. 1.
Expression (A) and
Sph-1-P-induced tyrosine phosphorylation (B) of Cas
and FAK in HUVECs. A, the cell lysates obtained from
HUVECs and platelets were resolved on an SDS-PAGE and then
immunoblotted with the indicated antibody. The protein concentration of
each lysate was adjusted to 0.6 mg/ml. B, serum-starved
HUVECs were challenged with 1 µM Sph-1-P for various
durations. The cell lysates were immunoprecipitated (IP)
with anti-Cas antibody (left panel) or anti-FAK mAb
(right panel), resolved on an SDS-PAGE, and then
immunoblotted with anti-phosphotyrosine (PY) mAb
(upper panel) or the antibody used for immunoprecipitation
(lower panel). The data shown are representative of three
separate experiments.
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Effects of Pertussis Toxin and C3 Exoenzyme on Sph-1-P-induced Cas
Tyrosine Phosphorylation in HUVECs--
Bacterial toxins have been
shown to be useful in the elucidation of cellular responses elicited by
Sph-1-P. Sph-1-P-induced mitogen-activated protein kinase activation,
inhibition of adenylyl cyclase, and growth stimulation are reportedly
inhibited by pertussis toxin (8, 13, 24), which ADP-ribosylates and
inactivates Gi (25). In some systems, it was reported that
Sph-1-P-induced phospholipase C activation (and the resultant
intracellular Ca2+ mobilization) was sensitive to pertussis
toxin (8, 13, 24). On the other hand, cytoskeletal reorganization and
morphological changes induced by Sph-1-P are inhibited not by pertussis
toxin but by C3 exoenzyme (8, 16, 26), which ADP-ribosylates and
inactivates Rho (18).
In HUVECs, Cas tyrosine phosphorylation induced by Sph-1-P was
abolished by pretreatment with pertussis toxin (Fig.
2A, upper). On the other hand,
pretreatment with C3 exoenzyme failed to affect Sph-1-P-induced Cas
tyrosine phosphorylation (Fig. 2B, upper). In contrast,
Sph-1-P-induced FAK tyrosine phosphorylation was only marginally
inhibited by pertussis toxin (Fig. 2A, lower) but completely
inhibited by C3 exoenzyme (Fig. 2B, lower). We further
examined the effects of Y-27632, a specific Rho kinase inhibitor (19).
This compound did not inhibit Cas tyrosine phosphorylation induced by
Sph-1-P; it rather induced a slight Cas phosphorylation by itself and
enhanced the Sph-1-P effect (Fig. 2C, upper) for unknown
reason(s). Sph-1-P-induced FAK tyrosine phosphorylation was abolished
by Y-27632 (Fig. 2C, lower) as well as by C3 exoenzyme. These findings were unexpected in view of the previous findings that
Cas plays important role(s) in cytoskeletal reorganization through C3
exoenzyme-inhibitable Rho activation (27). Our data suggest that, in
HUVECs stimulated with Sph-1-P, cytoskeletal signalings may be
separable into Gi-mediated signaling pathways (involving
Cas) and Rho-mediated ones (involving FAK).

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Fig. 2.
Effects of pertussis toxin, C3 exoenzyme, or
Y-27632 on Cas tyrosine phosphorylation elicited by Sph-1-P.
HUVECs were pretreated without (control) or with 10 ng/ml of
pertussis toxin (PTX) for 12 h (A) or 5 µg/ml of C3 exoenzyme for 30 h (B) and then
serum-starved for 2 h. C, serum-starved HUVECs were
incubated without (control) or with 20 µM
Y-27632 for 30 min. The cells were then stimulated with 1 µM Sph-1-P for the indicated durations, and the tyrosine
phosphorylation of Cas (upper panel) and FAK (lower
panel) were examined, as described in the legend for Fig. 1. The
data shown are representative of four separate experiments.
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Involvement of Fyn in Cas Tyrosine Phosphorylation in HUVECs
Stimulated with Sph-1-P--
Since Src family tyrosine kinases
reportedly phosphorylate Cas in several systems (28-30), we tested
this possibility in Sph-1-P-stimulated HUVECs. In immunoprecipitation
studies using each Src family tyrosine kinase, Fyn, but not Src or Lyn,
was found to be constitutively associated with Cas in HUVECs (Fig.
3A). Fyn/Cas association was also observed when immunoprecipitates with anti-Cas antibody were probed with anti-Fyn antibody (data not shown). Furthermore, the kinase
activity of Fyn immunoprecipitates increased upon Sph-1-P stimulation;
in vitro kinase assays of Fyn immunoprecipitates revealed
stimulation-dependent 32Pi
incorporation into Fyn, enolase, and a 130-kDa protein (confirmed as
Cas with immunoblotting) (Fig. 3B). Fyn auto-phosphorylation clearly occurred before Cas phosphorylation (Fig. 3B). These
data suggest that HUVEC Cas is constitutively associated with Fyn, and
tyrosine-phosphorylated through Fyn activation, following Sph-1-P
stimulation. Since there were few bands other than Cas, Fyn, and
enolase (added as an exogenous substrate), in the in vitro
Fyn kinase assay (Fig. 3B), the Cas/Fyn interaction may be
highly specific in HUVECs stimulated with Sph-1-P.

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Fig. 3.
Fyn, but not Src or Lyn, constitutively
associates with Cas and phosphorylates Cas in HUVECs.
A, serum-starved HUVECs were stimulated without ( ) or with
(+) 1 µM Sph-1-P for 30 min. Immunoprecipitates
(IP) obtained with anti-Src mAb, anti-Fyn mAb, or anti-Lyn
mAb, along with the whole cell lysate (c) were applied to an
SDS-PAGE and immunoblotted with anti-Cas mAb (upper panel)
or the antibody used for immunoprecipitation (lower panel).
B, serum-starved HUVECs were challenged with 1 µM Sph-1-P for the indicated durations. An in
vitro kinase assay of Fyn immunoprecipitates was performed using
acid-treated enolase as an exogenous substrate. The locations of Cas,
Fyn, and enolase are indicated on the left, and
62 and 83 indicate the locations of the molecular
mass markers (in kDa). The data shown are representative of
three separate experiments.
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We further examined the involvement of Fyn activation in Cas tyrosine
phosphorylation with the use of PP2, a specific inhibitor of Src family
tyrosine kinase(s) (31). As expected, pretreatment with PP2 abolished
tyrosine phosphorylation of Cas (Fig.
4A), but not FAK (Fig.
4B), in Sph-1-P-stimulated HUVECs. Under these conditions,
auto-phosphorylation of Fyn was completely inhibited by PP2 (data not
shown). These data confirm that tyrosine phosphorylation of Cas, but
not FAK, is a downstream target of Fyn tyrosine kinase in HUVECs
stimulated with Sph-1-P.

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Fig. 4.
Effects of PP2 on tyrosine phosphorylation of
Cas and FAK in HUVECs. Serum-starved HUVECs were pretreated
without (control) or with 50 µM PP2 for 30 min
and then challenged with 1 µM Sph-1-P for the indicated
durations. Cas (A) and FAK (B) tyrosine
phosphorylation were examined as described in the legend for Fig. 1.
The data shown are representative of three separate experiments.
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As described above, Cas tyrosine phosphorylation was inhibited by
pertussis toxin (but not by C3 exoenzyme or Y-27632), and most probably
catalyzed by Fyn. Hence, we next investigated whether Fyn
auto-phosphorylation was regulated by Gi activation. In
HUVECs stimulated with Sph-1-P, Fyn (but not Src) auto-phosphorylation was observed in an in vitro kinase assay (Fig.
5). Pretreatment with pertussis toxin
inhibited the Sph-1-P-induced Fyn auto-phosphorylation (Fig.
5B). Furthermore, Fyn auto-phosphorylation was not affected by pretreatment with C3 exoenzyme or Y-27632 (data not shown). These
results further strengthen the idea that Sph-1-P induces Cas tyrosine
phosphorylation via Gi-mediated Fyn activation in HUVECs.

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Fig. 5.
Effects of pertussis toxin on Src or Fyn
activity induced by Sph-1-P in HUVECs. HUVECs were pretreated
without (control) or with 10 ng/ml pertussis toxin
(PTX) for 12 h and then serum-starved for 2 h. The
cells were stimulated with 1 µM Sph-1-P for the indicated
durations and then immunoprecipitated (IP) with anti-Src mAb
(A) or anti-Fyn mAb (B). An in vitro
kinase assay of these immunoprecipitates was performed using
acid-treated enolase as an exogenous substrate. The positions of Src,
Fyn, and enolase are indicated by arrows. The data shown are
representative of three separate experiments.
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Association of Cas with Crk in HUVECs Stimulated with
Sph-1-P--
Cas has a cluster of 15 potential SH2-binding motifs that
are thought to have a preferential affinity for the Crk-SH2 domain (17). In addition, the Crk-Cas complex reportedly has a role in
integrin-mediated cell migration in several systems (32, 33).
Accordingly, we examined whether Sph-1-P-induced phosphorylation of Cas
led to the formation of a complex between endogenous Crk and Cas in
HUVECs. When immunoprecipitates with anti-Crk mAb were immunoblotted
with anti-phosphotyrosine antibody, a 130-kDa tyrosine-phosphorylated protein was specifically detected in a manner dependent on Sph-1-P stimulation (Fig. 6A).
Immunoblotting with anti-Cas mAb revealed that this 130-kDa protein was
Cas; Cas weakly associated with Crk in resting HUVECs, and this
association was augmented after Sph-1-P stimulation (Fig.
6A). This stimulation-dependent Cas/Crk interaction was also detected using immunoprecipitates with anti-Cas polyclonal antibody, followed by anti-Crk immunoblotting (data not
shown). Furthermore, the SH2 domain of Crk was shown to bind to Cas in
HUVECs stimulated with Sph-1-P, and this interaction was disrupted by
pretreatment with pertussis toxin but not with C3 exoenzyme (Fig.
6B), as was the case with Cas tyrosine phosphorylation (Fig.
2). These data suggest that Crk, through its SH2 domain, interacts with
tyrosine-phosphorylated Cas in HUVECs stimulated with Sph-1-P.

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Fig. 6.
Formation of the Cas-Crk complex in HUVECs
stimulated with Sph-1-P. A, serum-starved HUVECs were
challenged with 1 µM Sph-1-P for various durations. The
cell lysates were immunoprecipitated with anti-Crk mAb, resolved on an
SDS-PAGE, and then immunoblotted with anti-phosphotyrosine
(PY) mAb, anti-Cas mAb, or anti-Crk mAb. B,
HUVECs were pretreated without (control) or with 10 ng/ml of
pertussis toxin (PTX) for 12 h or C3 exoenzyme for
30 h and then serum-starved for 2 h. After stimulation with 1 µM Sph-1-P for the indicated durations, the cell lysates
were mixed with GST fusion protein containing SH2 domain of Crk and
further incubated with glutathione-Sepharose 4B. Proteins associated
with GST fusion protein were subjected to an SDS-PAGE and probed with
anti-Cas mAb (upper panel). The loading amount of GST fusion
protein in each gel lane was confirmed to be equal by Coomassie
Brilliant Blue (CBB) staining (lower panel). The
data shown are representative of three separate experiments.
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Localization of Both Cas and Crk to Membrane Ruffles, but Not to
Focal Adhesion, in Sph-1-P-stimulated HUVECs--
Targeting of
signaling proteins to specific or appropriate regions of the cell is
important for regulation of various cellular functions, especially
cytoskeletal reorganization (34). Previous studies demonstrated that
most Cas exists in the cytosol in resting states and shifts to membrane
ruffles (32) or focal adhesions (35) during the initial attachment of
cells to matrix proteins. To analyze the correlation between the
intracellular localization of Cas/Crk and the formation of stress
fibers or focal adhesions in HUVECs stimulated with Sph-1-P, the cells
were examined using (immuno)fluorescent staining and confocal
microscopy. Sph-1-P induced HUVEC stress fiber formation (F-actin
bundling) (Fig. 7A) and an
increase in paxillin staining, indicative of focal adhesion assembly
(Fig. 7B). These cytoskeletal responses induced by Sph-1-P
were completely inhibited by Y-27632 but not by PP2 (Fig. 7,
A and B). On the other hand, Cas was mainly
localized in the cytoplasm in resting states and translocated to the
cell periphery after Sph-1-P stimulation (Fig. 7C).
Simultaneous phalloidin staining studies showed that Cas translocation
was observed in the cells assembling a mesh work of actin filaments at
the cell periphery (Fig. 7A). In addition, not only Cas but
also Crk was found to translocate to cortical actin in a manner
dependent on Sph-1-P stimulation (Fig. 7D). Furthermore,
Sph-1-P-elicited translocation of Cas and Crk was completely abolished
by PP2, but not by Y-27632 (Fig. 7, C and D).
These data suggest that HUVEC stimulation with Sph-1-P results in
translocation of Cas and Crk (which form a complex upon stimulation) to
the cell periphery (membrane ruffles) through Gi (Fyn)
mediation and that these responses are independent of stress fiber and
focal adhesion formation, which are mediated by Rho.

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Fig. 7.
Translocation of both Cas and Crk to membrane
ruffles, but not to focal adhesion, in Sph-1-P-stimulated HUVECs.
Serum-starved HUVECs were pretreated without or with 50 µM PP2 or 20 µM Y-27632 for 30 min. The
cells were then challenged without or with 1 µM Sph-1-P
for 30 min, fixed, and incubated with TRITC-phalloidin (A),
anti-paxillin mAb (B), anti-Cas mAb (C), or
anti-Crk mAb (D). Actin staining and immunofluorescent
staining were performed as described under "Experimental
Procedures." The data shown are representative of four separate
experiments.
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Requirement of Both Rho- and Gi-mediated Pathways for
Sph-1-P-enhanced HUVEC Motility--
As described above, in HUVECs,
Cas is likely to be tyrosine-phosphorylated by Fyn (which undergoes
activation through a Gi-mediated pathway), whereas FAK
tyrosine phosphorylation (and the resultant cytoskeletal
reorganization) is mediated mainly by a Rho-mediated pathway. Since Cas
and FAK have been implicated in the regulation of cell motility in
various systems (30, 32), we investigated the involvement of these
pathways in Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic
assay using gold sol-coated plates (for chemokinesis) and a Boyden's
chamber assay (for migration). Consistent with recent studies (9, 10),
Sph-1-P potently stimulated HUVEC chemokinesis (Fig.
8A) and migration (Fig.
8B). Pertussis toxin or PP2 completely inhibited these
Sph-1-P-elicited responses (Fig. 8, A and B).
Furthermore, treatment with C3 exoenzyme (Fig. 8, A and
B) or Y-27632 (data not shown) also resulted in inhibition
of Sph-1-P-enhanced cell motility. These data suggest that
Sph-1-P-enhanced HUVEC motility requires coordinated signalings from
Gi and Rho activation.

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Fig. 8.
Sph-1-P-enhanced HUVEC motility. HUVECs
were pretreated without or with pertussis toxin (PTX), C3
exoenzyme, or PP2 as described in the legend for Fig. 2. A,
HUVEC chemokinesis was measured with a phagokinetic assay (using gold
sol-coated plates). HUVECs were incubated in the absence or presence of
1 µM Sph-1-P, and after 16 h, the area cleared of
gold particles for each cell was measured. Columns and
error bars represent the mean ± S.D.
(n = 3). B, HUVEC migration was assessed
with a modified Boyden's chamber assay. Sph-1-P (100 nM)
was placed in the lower chamber (or not added), and HUVECs were allowed
to migrate. Columns and error bars represent the
mean ± S.D. (n = 3).
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Effects of Suramin on Sph-1-P-induced Cytoskeletal
Responses--
To discriminate definitively between the signaling
pathways mediated by the HUVEC Sph-1-P receptors EDG-1 and EDG-3, we
used the polycyclic anionic compound suramin, which selectively
antagonizes EDG-3 (36). Of Sph-1-P-elicited cytoskeletal responses, the Rho-mediated formation of stress fibers and focal adhesions were specifically inhibited by pretreatment of the cells with suramin (Fig.
9, A and B). On the
other hand, the Gi-mediated Cas/Crk translocation to the
cell periphery was not affected by this EDG-3 antagonist (Fig. 9,
A and D). These data suggest that EDG-3 plays an
important role in cytoskeletal responses mediated by Rho but not
Gi.

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Fig. 9.
Effects of suramin on stress fiber and
focal adhesion formation and Cas/Crk translocation in
Sph-1-P-stimulated HUVECs. Serum-starved HUVECs were pretreated
without or with 500 µM suramin for 5 min. The cells were
then challenged without or with 1 µM Sph-1-P for 30 min,
fixed, and incubated with TRITC-phalloidin (A),
anti-paxillin mAb (B), anti-Cas mAb (C), or
anti-Crk mAb (D). Actin staining and immunofluorescent
staining were performed as described under "Experimental
Procedures." The data shown are representative of three separate
experiments.
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DISCUSSION |
Sph-1-P-induced HUVEC Cas Tyrosine Phosphorylation through
Mediation of Gi and Fyn but Not Rho--
Cas was
originally identified as a tyrosine-phosphorylated protein of 130 kDa
in cells transformed by v-Src or v-Crk (17). Cas can be phosphorylated
through a variety of pathways, including integrin, G protein-coupled
receptors, and receptor-type tyrosine kinases (28, 35, 37). Cas is
thought to be a component of focal adhesion and contributes to
cytoskeletal reorganization through assembly of actin filaments (30).
Cas becomes tyrosine-phosphorylated with kinetics similar to FAK in
response to integrin-mediated cell adhesion (30) and, in some cells, is
found to be actually phosphorylated by FAK (38). However, the mechanism
by which Cas is phosphorylated and how this docking protein is
functionally involved remain to be clarified in vascular endothelial
cells, especially when they are stimulated with the ligand for G
protein-coupled receptors.
In this study, we first confirmed the abundant expression of Cas in
HUVECs, and then we examined the mechanism by which Cas is
phosphorylated and its possible involvement in cytoskeletal reorganization and motility upon stimulation with Sph-1-P; Sph-1-P acts
on its G protein-coupled receptors expressed on HUVECs (see Introduction). Consistent with previous studies (26, 39), we confirmed
that inactivation of Rho by C3 exoenzyme or inhibition of Rho kinase by
Y-27632 resulted in inhibition of Sph-1-P-induced FAK tyrosine
phosphorylation, which was examined in parallel with Cas
phosphorylation. The inhibition of FAK phosphorylation was accompanied
by elimination of stress fiber formation and focal adhesion assembly.
It is likely that Sph-1-P-induced activation of Rho/Rho kinase (through
G12/13), leading to phosphorylation (inactivation) of
myosin light chain phosphatase (39, 40), and the resultant myosin
light chain phosphorylation (39, 40) were inhibited by treatment with
C3 exoenzyme or Y-27632. Contrary to our initial expectations, however,
Cas was phosphorylated in a manner distinct from FAK in HUVECs; the
association of FAK with Cas was barely observed in immunoprecipitation
studies (data not shown). Sph-1-P-induced Cas tyrosine phosphorylation
was accompanied by Cas interaction with Crk and localization to
membrane ruffles (but not focal adhesion). Importantly, all these Cas
reactions elicited by Sph-1-P were abolished by pertussis toxin but not C3 exoenzyme or Y27632. Although Cas has been shown to be localized to
focal adhesions and dependent on Rho signaling pathways in several cell
types (27, 39), Cas functional responses independent of these have also
been reported (32, 41). Cas is markedly concentrated in membrane
ruffles and has an important role during cell migration (32, 41). Cas
accumulates at the leading edge of the lamellipodia, and assembly of a
Cas-Crk complex and Rac activation are necessary for membrane ruffling,
independent of actin-myosin contraction (32, 41). It is likely that Cas
tyrosine phosphorylation and Cas-Crk complex formation in membrane
ruffles, following Gi activation, are related to Rac
(rather than Rho) activation in Sph-1-P-stimulated HUVECs.
Another important finding in our study was that Cas may be
phosphorylated by Fyn in HUVECs stimulated with Sph-1-P. It has been
reported that Src family tyrosine kinase(s) phosphorylate Cas in
several systems (28-30) and that the C-terminal region of Cas can
associate with several Src kinases in vitro (28-30). We found that in HUVECs, Fyn, but not Src or Lyn, associated with Cas, and
Cas tyrosine phosphorylation was inhibited by the specific Src family
tyrosine kinase inhibitor PP2. In addition, we confirmed that Fyn
actually phosphorylated Cas in an in vitro kinase assay. Recently, Okuda et al. (42) reported that in HUVECs exposed to shear stress, Src phosphorylates Cas. They used the clone 28 mAb,
which recognizes the activated form of Src family kinases (43), to
examine endogenous Src activity, but this mAb has also been found to
recognize activated Fyn (as well as Src) (44).
Involvement of Gi-mediated Cas Pathways in HUVEC
Motility Enhanced by Sph-1-P in Cooperation with Rho-mediated
Pathways--
In this study, we found that Sph-1-P-enhanced HUVEC
motility was inhibited by pertussis toxin or PP2, as is the case with Cas tyrosine phosphorylation and the resultant formation and
translocation (to membrane ruffles) of the Cas-Crk complex. Recent
observation of Cas(
/
) fibroblasts suggested that Cas plays a key
role in mediating cell migration (45). It has also been reported that formation of a Cas-Crk adapter protein complex serves as a
molecular switch facilitating a Rac-dependent cell
migration response in the extracellular matrix (32). By taking these
recent findings into consideration, it is likely that
Gi-mediated Cas tyrosine phosphorylation and its related
responses are necessary for enhanced cell motility in HUVECs stimulated
with Sph-1-P.
On the other hand, Sph-1-P-induced HUVEC motility was also abolished by
C3 exoenzyme or Y-27632, suggesting the requirement of Rho/Rho kinase
pathway. Although a rapid reorganization of actin to the cell edge is
important for initiation of migration for a cell to move, it must also
organize actin into a functional actin-myosin motor unit capable of
generating contractile force resulting from Rho activation (46).
Indeed, Rho activation is required for the crawling movements of NIH3T3
cells on a flat surface (47), for hepatocyte growth factor-induced
motility of mouse keratinocytes (48), and for lysophosphatidic
acid-induced invasion of hepatoma cells (49). Migratory defects have
also been reported in cells lacking FAK (50); FAK appears to modulate focal adhesion turnover (46, 50). Also in endothelial cell motility
enhanced by Sph-1-P (which we examined in this study), the C3
exoenzyme-sensitive Rho mediation and accompanying FAK activation seem
to play important role(s) in cooperation with Gi-mediated
Cas/Crk pathways.
HUVECs reportedly express the Sph-1-P receptors EDG-1 and EDG-3, the
former being coupled predominantly with Gi and the latter with G13 and Gq (8, 51, 52). It has been
postulated that stimulation of both the Rac pathway via EDG-1 and the
Rho pathway via EDG-3 are necessary for cell morphogenesis and adherens
junction assembly (8). In this context, Cas-related responses following Gi (Fyn) activation may originate from EDG-1, whereas Rho
(and FAK)-related responses originate from EDG-3. To discriminate
definitively between these pathways, we employed the selective EDG-3
antagonist suramin (36). As expected, suramin was found to inhibit the Rho-mediated formation of stress fibers and focal adhesion but not the
Gi-mediated response. Synergism between the two independent pathways from EDG-1 and EDG-3 seems crucial in Sph-1-P-elicited signalings in vascular endothelial cells, especially those related to
cytoskeletal reorganization. Since Sph-1-P, released from activated platelets and interacting with endothelial cells, is likely to play
important roles under conditions in which critical platelet-endothelial cell interactions (including thrombosis, hemostasis, angiogenesis, and
atherosclerosis) occur, thorough elucidation of EDG signaling pathways
in vascular endothelial cells will lead to a greater understanding of
vascular events or diseases.