From the INSERM EMI 00-19 Physiopathologie de l'Endothélium, UFR Pharmacie, Université de la Mediterranée, 13385 Marseille and the § Centre d'Immunologie INSERM-CNRS Marseille Luminy and Institut Universitaire de France, 13276 Marseille, France
Received for publication, August 4, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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CD146 (S-Endo 1 Ag or MUC18) is a transmembrane
glycoprotein expressed on endothelial cells on the whole vascular tree.
CD146 is located at the intercellular junction where it plays a role in
the cohesion of the endothelial monolayer. CD146 engagement initiates
an outside-in signaling pathway involving the protein tyrosine kinases
FYN and FAK as well as paxillin. Here we report that CD146 engagement
by its specific monoclonal antibody in human umbilical vein
endothelial cells induces a Ca2+ influx that is
sensitive to thapsigargin and EGTA treatment, indicating that CD146
engagement initiates a store-operated calcium mobilization. In
addition, biochemical and pharmacological analysis revealed that CD146
engagement initiates the tyrosine phosphorylation of phospholipase
C- CD146 (S-Endo 1 Ag), also referred to as Mel-CAM or MUC18, is an
integral membrane protein present on endothelial cells, regardless of
their anatomical site and the size of the vessels (1-3). CD146 expression is not restricted to the endothelium but is found on other
non-malignant and malignant cell types such as melanoma cells (4). In
these cells, the cell surface expression of CD146 is associated with
the metastatic properties of primary tumors (5, 6). CD146 belongs to
the Ig superfamily of cell adhesion molecules with a V-V-C2-C2-C2
structure in the extracellular portion, a single membrane-spanning
domain, and a relatively short cytoplasmic tail containing 61 amino
acids (7).
CD146 is involved in cell-cell adhesion through a heterophilic ligand
that still remains unknown (8). In melanoma cells, the homotypic
interaction of CD146 and its ligand contributes to cohesive
interactions among these cells (9). Similarly, the binding of CD146 to
its putative receptor on trophoblasts confers a stationary phenotype so
preventing trophoblastic migration/invasion within the myometrium (10).
Immunohistochemical studies have revealed that CD146 is localized at
the intercellular junction in endothelial cells (3). This localization
is consistent with a role of CD146 in the control of cohesive cell-cell
interactions.1
It is well known that cell adhesion molecules located at the
intercellular junction control the integrity of the endothelial monolayer (11). They promote adhesion through their extracellular domain, whereas the intracytoplasmic tail is implicated in the outside-in signaling pathway that is derived from their engagement (12-15). In endothelial cells, CD146 acts as a signal transduction molecule. CD146 initiates an outside-in signal cascade upon monoclonal antibody engagement. Whereas CD146 is not phosphorylated on tyrosine residues, its engagement promotes the recruitment of the Src family kinase p59fyn (FYN)2
as well as the tyrosine phosphorylation of a large panel of
intracellular proteins including p125FAK (FAK) and
paxillin, two proteins present in focal adhesion plaques (16).
Calcium is a central second messenger that mediates a large number of
cellular processes such as cell division, gene transcription, and/or
cell death (17, 18). Activation of multiple cell surface receptors
linked to PTK activation leads to increases in intracellular calcium
concentrations ([Ca2+]i) (19). A feature
of PTK-induced increase in [Ca2+]i involves a
two-step process characterized at first by a rapid, transient release
of Ca2+ stored in the endoplasmic reticulum (20). This
release of Ca2+ from intracellular stores occurs, at least
in part, via activation of phospholipase C (21, 22). PLC We show here that engagement of CD146 in HUVEC triggers a
store-dependent Ca2+ influx that requires the
tyrosine phosphorylation (Tyr(P)) of FYN and PLC Materials
Fluo3-AM, Pluronic F-127, and BAPTA-AM were from Molecular
Probes (Eugene OR). Thapsigargin, herbimycin, and ionomycin were from
Alexis Corp. (San Diego, CA); PP1 and U73122 were from Calbiochem.
Culture medium and culture reagents were from Life Technologies, Inc.
Chemical reagents were from Sigma. mAbs against CD146 (S Endo-1 and
7A4) were from Biocytex (Marseille, France), and goat anti-mouse
immunoglobulin F(ab')2 (GAM) was from Jackson Laboratories
(Palo Alto, CA), and mAbs against phosphotyrosine (PY20), FYN,
p130Cas, Pyk2, paxillin, and PLC Methods
Cell Culture--
HUVECs were isolated from umbilical cord veins
according to the method of Jaffe et al. (34). They were used
at subconfluency after one passage. Cell monolayers were starved for
3 h in serum-free RPMI containing 0.5% BSA. For drug treatment
experiments, HUVECs were pretreated with the drug for the indicated
time prior to CD146 engagement.
CD146 Engagement--
CD146 clustering was performed as
described previously (16). Briefly, quiescent HUVECs were incubated
with 10 µg/ml anti-CD146 F(ab')2 mAb in HBSS 30 min at
4 °C; after washing, cross-linking was then performed with 20 µg/ml goat anti-mouse IgG F(ab')2 (GAM), and the cells
were processed for calcium determinations. Control cells were incubated
with isotype-matched IgG1 and cross-linked with GAM.
Inhibitor Treatments--
Herbimycin, PP1, and U73122 were
dissolved in Me2SO and incubated with HUVECs in
serum-free HBSS, at the required concentration, 30 min prior to
engagement of CD146. When required, HUVECs were preincubated with
BAPTA-AM for 30 min at 37 °C and washed twice in HBSS, and
engagement of CD146 was performed as indicated above. To study the
effect of EGTA on the tyrosine phosphorylation of signaling proteins,
EGTA (5 mM) was added throughout the addition of GAM.
Measurements of [Ca2+]i--
Serum-starved
subconfluent HUVECs grown in 96-well plates were loaded with Fluo3-AM
(5 µM) and 0.2 mg/ml Pluronic F-127 by incubation in
loading buffer (HBSS supplemented with 10 mM HEPES, pH 7.4, 2 mM CaCl2, 1 mM MgCl2,
1% BSA) (35) for 30 min at 37 °C. Cells were then washed in loading
buffer and incubated for at least 15 min at room temperature, washed,
and incubated again in loading buffer. CD146 engagement was then
performed using anti-CD146 mAb at 4 °C as described (16). Cells were
then washed and incubated in Ca2+-free loading buffer to
which 2 mM CaCl2 was added when required, and
cross-linking with GAM was performed as described above. Intracellular Ca2+ levels were determined immediately upon addition of
GAM to the Fluo3-loaded cell monolayers at 37 °C using a
plaque-reader spectrofluorimeter (Cytofluor series 4000 Perspective
Biosystems). Levels of fluorescence from cells in individual wells were
monitored at an excitation wavelength of 485 nm and an emission
wavelength of 530 nm. The signal was calibrated by addition of 10 µM ionomycin containing Ca2+ (2 mM) to obtain maximal fluorescence
(Rmax). After a stable fluorescence at 530 nM (Rmax), the medium was changed to
one containing 5 mM EGTA in calcium-free medium with 10 µM ionomycin (Rmin). [Ca2+]i concentration was calculated using the
method of Grynkiewicz et al. (36) using the equation:
[Ca2+]i = Kd
(R Immunoprecipitation and
Immunoblotting--
Immunoprecipitation and immunoblotting were
performed as described previously (16). In brief, cell lysates
(300-600 µg of total proteins/sample, i.e. 0.3 to 1 × 107 cells/sample) were immunoprecipitated with 4 µg of
mAb for 3 h at 4 °C under constant agitation, followed by a 2-h
incubation with protein G-Sepharose beads. After washing, the recovered
proteins were subjected to SDS-PAGE and transferred to nitrocellulose
C+ membranes. After blocking, membranes were incubated in TBS-T 5% BSA
containing indicated antibodies (1 µg/ml) for 1 h at room temperature. Immunoreactive bands were visualized by chemiluminescence using horseradish peroxidase-conjugated anti-mouse IgG and ECL reagent.
When required, membranes were stripped and reblotted with the indicated antibodies.
Statistical Analysis--
Results are expressed as mean ± S.E. Calcium measurements were analyzed by the unpaired Student's
t test. Differences were considered significant
with p < 0.05.
CD146 Engagement-induced [Ca2+]i Increase in
HUVECs--
To determine whether the engagement of CD146 in HUVECs
triggers an increase in [Ca2+]i, cells loaded
with 5 µM Fluo3-AM were pretreated with anti-CD146 mAb
F(ab')2 fragment (10 µg/ml) at 4 °C for 20 min and
equilibrated at 37 °C for 180 s, and
[Ca2+]i was measured after cross-linking with GAM
F(ab')2 antibody (20 µg/ml). In the presence of 2 mM extracellular calcium, the engagement of CD146 resulted
in an increase in [Ca2+]i characterized by an
initial peak followed by a phase showing a slow decrease (Fig.
1). The initial increase peaked from
basal levels of 139.4 ± 20.6 to 912.9 ± 188.3 nM (mean values ± S.E., p < 0.001, n = 15) within 30 s upon GAM addition. This [Ca2+]i rise was followed by a gradual decline in
[Ca2+]i levels which at the end of the incubation
had not yet returned to base-line levels. [Ca2+]i
rise was dose-dependent, and 10 µg/ml anti-CD146
generated a maximal [Ca2+]i response (data not
shown). No change in [Ca2+]i was observed when an
isotype-matched antibody was used instead of anti-CD146 mAb indicating
that intracytoplasmic calcium changes are specific for the engagement
of CD146.
Role of Intra- and Extracellular Sources of Calcium in Response to
CD146 Engagement--
To determine which Ca2+ sources
contribute to the intracytoplasmic calcium flux in response to CD146
engagement, internal Ca2+ stores were depleted by 1 µM thapsigargin (TG), an inhibitor of endoplasmic
Ca2+ pumps (38). Addition of 1 µM TG to
HUVECs preincubated with anti-CD146 mAb induced a transient increase in
[Ca2+]i (1643.6 ± 334.5 in TG-treated cells
versus 128.7 ± 21.5 nM in control cells,
n = 4) which peaked in 20 s. After
[Ca2+]i has returned down to base-line values
within 180 s, GAM was added to the monolayer. Preincubation of
HUVEC with TG (1 µM) completely blocked
[Ca2+]i increase after addition of GAM as
compared with [Ca2+]i increase obtained in cells
without TG treatment (Fig. 2A,
n = 4). These results indicated that the initial rise
of [Ca2+]i in response to CD146 engagement was
dependent on intracellular store depletion.
Cross-linking of CD146 was then performed in the presence or absence of
EGTA (5 mM). EGTA had no effect on the initial transient increase but strikingly decreased the [Ca2+]i in
the second phase from 832.5 ± 106.3 to 332.9 ± 45.8 nM (p < 0.002, n = 4)
(Fig. 2B). The role of extracellular calcium was confirmed
in a subsequent experiment where CD146 cross-linking was performed in
the absence of extracellular calcium. As shown in Fig. 2C,
only the first transient rise in [Ca2+]i was
observed. After the peak has returned close to the basal level,
subsequent addition of 2 mM Ca2+ to the
extracellular medium restored the second slow phase of [Ca2+]i (Fig. 2C). Taken together,
these data indicate that the second phase of slow decrease was on the
dependence of extracellular calcium sources. These results suggest that
CD146 engagement stimulates Ca2+ influx into the cells that
depends both on Ca2+ release from internal stores and
Ca2+ entry from the extracellular milieu.
Involvement of Protein Tyrosine Kinases (PTK) in Calcium Flux
Induced by CD146 Engagement--
For receptors coupled to PTK, calcium
mobilization requires the activation of PTK and the recruitment of
PLC
CD146 engagement also induced the Tyr(P) of PLC Calcium-dependent Recruitment of Pyk2 and
p130Cas by CD146 Engagement--
CD146 engagement leads to
the Tyr(P) of FAK, a PTK present in focal adhesion (15). PYK2, another
PTK related to FAK, is activated by an increase in intracellular
calcium concentration (reviewed in Ref. 41). To determine whether PYK2
was involved in the signaling pathway mediated by CD146 engagement,
anti-Pyk2 immunoprecipitates were immunoblotted with anti-Tyr(P) PY20
and anti-Pyk2. GAM cross-linking performed for 15 min induced the Tyr(P) of a band of ~100 kDa identified as Pyk2 (Fig.
6A, upper panel, lane b) that
was not observed in the absence of CD146 engagement. The Tyr(P) of Pyk2
was abolished by PP1 (10 µM) (lane c), by the intracellular calcium chelator BAPTA-AM (25 µM,
lane d), and by EGTA (5 mM, lane e).
This phosphorylation was observed despite the absence of a molecular
association between CD146 and Pyk2 (data not shown). These data
indicate that Pyk2 is phosphorylated on tyrosine residues following
CD146 engagement by a process requiring both the activation of an Src
family PTK and calcium mobilization from intra- and extracellular
stores.
The adapter protein p130Cas localizes to focal adhesion
points and interacts with Pyk2 (32, 33). The role of Pyk2 as a
transducing molecule toward proteins of the focal adhesion plaques was
then investigated in response to CD146 engagement. Similar to
experiments with Pyk2, CD146 cross-linking with GAM for 15 min induced
the Tyr(P) of a band with a molecular mass of
Pyk2 has been shown to associate with paxillin (42). Paxillin is
tyrosine-phosphorylated upon CD146 engagement (16). Therefore, we
investigated whether paxillin and also p130Cas associate
with Pyk2 in HUVECs upon CD146 triggering. As shown in Fig.
7, anti-Pyk2 immunoprecipitates performed
on anti-CD146-stimulated HUVEC lysates contained a 130-kDa band
reactive with anti-p130Cas mAb as well as a 70-kDa band
reactive with anti-paxillin mAb (lanes b). Of note,
constitutive association of Pyk2 and paxillin but not of Pyk2 and
p130Cas was observed in unstimulated HUVECs (lanes
a).
Time Course of Tyr(P) of FYN, PLC Previous data have indicated that CD146 initiates an outside-in
signal transduction pathway that involves the recruitment of the Src
PTK FYN and leads to the Tyr(P) of FAK and paxillin, two proteins
present in the focal adhesion plaques (16). We demonstrate here that in
endothelial cells, CD146 engagement promotes an increase in
[Ca2+]i both by Ca2+ release from
TG-sensitive Ca2+ stores and entry of extracellular
Ca2+. This process requires the activation of FYN and
PLC The Ca2+ influx initiated by CD146 engagement in HUVECs is
representative of the well known SOCE. Indeed, the initial rapid Ca2+ mobilization depends upon TG-sensitive stores, whereas
the long lasting decrease depends on the extracellular
Ca2+. This SOCE is mediated by activation of FYN and PLC It should be noted that the mechanism leading to SOCE is not well
elucidated. Some recent data suggest that Ca2+ influx
results from a secretory pathway induced by a close interaction between
ER calcium stores and the plasma membrane via an involvement of the
actin cytoskeleton (47, 48). The secretion requires intracellular
fusion events mediated by SNAP-25, a membrane protein belonging to the
SNAP receptor proteins (49).
The mobilization of calcium ions in addition to the recruitment of FYN
play an active role in the outside-in signaling pathway mediated by
CD146. Indeed, CD146 engagement leads to Tyr(P) of Pyk2, the
calcium-dependent tyrosine kinase related to FAK (reviewed in Ref. 41), by a calcium-dependent mechanism. FYN is also
involved in the activation of Pyk2. It is known that several Pyk2
tyrosine residues (Tyr-402, Tyr-579, and Tyr-580 within the catalytic
domain and Tyr-881 within the carboxyl terminus domain) create binding sites for the SH2 of the Src-like PTK (50, 51). Pyk2 also associates
with and is phosphorylated by FYN during stimulation of T cell antigen
receptor (52). Nevertheless, in the case of CD146 engagement, FYN
exerts an indirect effect on Pyk2 because no direct association between
Fyn and Pyk2 could be found (data not shown). Moreover, Pyk2 Tyr(P)
occurs more later than that of FYN, indicating that Pyk2 acts
downstream of FYN.
The signaling pathway initiated by CD146 engagement includes the Tyr(P)
of FAK and its substrate paxillin (16). p130Cas belongs to
the group of proteins that associate with FAK and is involved in cell
migration (33). p130Cas tyrosine phosphorylation in
response to CD146 engagement strengthens the relationship between CD146
and focal adhesion points by a way that is dependent on FYN
recruitment. Indeed, p130Cas Tyr(P) depends on an Src PTK
activation as demonstrated by PP1 inhibition and on calcium influx as
evidenced by its partial inhibition by the calcium chelators. Src PTKs
have been implicated in the Tyr(P) of p130Cas mediated by
integrins or shear stress (53, 54). Nevertheless Pyk2 does not
associate with p130Cas in anti-CD146-treated HUVECs. The
molecular structures implicated in the association of Pyk2 and
p130Cas upon CD146 engagement are not known, but the SH3
domain of p130Cas and the two proline-rich sequences of
Pyk2 have been involved in the formation of such a complex (55).
p130Cas is a ligand of FAK by its SH3 domain (56) and
increases cell migration promoted by FAK (57). Taken together, the fact
that p130Cas plays an important role in the organization of
the cytoskeletal framework (58, 59) and that Pyk2 forms a constitutive
complex with paxillin (42, 60) expands the concept that CD146 is in relation with the actin cytoskeleton. These results are consistent with
data indicating that a part of CD146 is recovered in the Triton
X-100-insoluble fraction and that CD146 colocalizes with F-actin (1).
Nevertheless a direct effect of CD146 engagement on the actin
reorganization has never been observed (data not shown).
In summary, the present study thus demonstrates that engagement of
CD146 induces a complex signaling pathway that includes Ca2+ influx as a second messenger involved in cytoskeleton
dynamics. FYN plays a crucial function in the initiation of CD146
signal transduction. Upon engagement, FYN is recruited and
phosphorylated by CD146. FYN allows the activation of PLC CD146 engagement leads to the Tyr(P) of FAK and paxillin and their
association on the one hand (16) and Tyr(P) of Pyk2 and its association
with paxillin on the other hand. It is known that FAK and Pyk2 are
differentially regulated by cell adhesion and by soluble factors or
develop distinct signal transduction events. Whereas FAK mainly
localizes to focal contact sites, Pyk2 exhibits a punctate perinuclear
distribution in FAK The localization of CD146 at the intercellular junction suggests that
CD146 might mediate cell-cell and cell-extracellular matrix
interactions that initiate an outside-in signaling cascade after its
engagement with its ligand. Further studies are needed to determine the
in vivo physiological relevance of the signaling pathways
linked to CD146.
, Pyk2, and p130Cas. Pharmacological inhibition of
Ca2+ flux with
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in
Ca2+ is required for Pyk2 and p130Cas tyrosine
phosphorylation. Moreover, a complex association was observed between
Pyk2, p130Cas, and paxillin. These results indicate that
CD146 is coupled to a FYN-dependent pathway that triggers
Ca2+ flux via phospholipase C-
activation leading
subsequently to the tyrosine phosphorylation of downstream targets such
as Pyk2, p130Cas, FAK, and paxillin. In addition to its
role in cell-cell adhesion, CD146 is a signaling molecule involved in
the dynamics of actin cytoskeleton rearrangement.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activation
results in increased production of inositol 1,4,5-triphosphate and
emptying of inositol 1,4,5-triphosphate receptor-gated Ca2+
stores. Depletion of intracellular Ca2+ stores then induces
sustained extracellular calcium influx via store-operated calcium entry
(SOCE) (23). Numerous endothelial cell functions are regulated by
elevation in [Ca2+]i levels such as the secretion
of endothelial cell granules, regulation of endothelial permeability,
adhesion, and transmigration of circulating cells as well as
rearrangement of actin cytoskeleton (24-28).
. In addition, CD146
engagement induces the Tyr(P) of the PTK Pyk2 (29-31), and the adaptor
protein p130Cas (32, 33), by a process involving
Ca2+ mobilization. Thus, these data confirm and expand the
concept that besides its function as adhesive protein, CD146 is also a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were from Transduction
Laboratories (Lexington, KY).
Rmin)/(Rmax
R) with Kd = 400 nM (37). In some experiments, HUVECs were pretreated at 37 °C before cross-linking for 5 min with 1 µM
thapsigargin for measurement of calcium release from intracellular
stores. The effect of extracellular calcium was analyzed in presence of
5 mM EGTA in the loading buffer. Calcium influx was then
assayed as required. All pharmacological and monoclonal antibody
treatments did not alter the cell viability assessed both by treatment
with 10 mM ionomycin to trigger calcium influx and by
trypan blue dye exclusion demonstrating that the cells were still viable.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Intracellular Ca2+ mobilization
after CD146 engagement in HUVECs. HUVECs loaded with Fluo3-AM (5 µM) were incubated with anti-CD146 mAb
F(ab')2 fragment or isotype-matched IgG1 15mn at 4 °C.
After equilibration, cross-linking was performed with GAM
F(ab')2 as described under "Experimental Procedures,"
and Ca2+ release was immediately assayed. The
arrow indicates the addition of GAM (20 µg/ml). ,
anti-CD146 (10 µg/ml); *, isotype-matched IgG1 mAb (control, 10 µg/ml). Each trace is representative of 15 experiments
performed in triplicate.
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Fig. 2.
Effect of calcium chelators on calcium influx
induced by CD146 engagement in HUVEC. HUVECs loaded as described
in Fig. 1 were treated with anti-CD146 mAb F(ab')2 fragment
and incubated with 1 µM TG in calcium-containing medium
(A). Changes in [Ca2+]i were monitored
for 180 s. After GAM cross-linking (arrow),
intracellular calcium modifications were further assayed for 360 s. CD146 engagement without ( ) and with (*) TG treatment is shown.
B, 5 mM EGTA in calcium-containing medium.
[Ca2+]i was assayed for 360 s after
cross-linking with GAM (arrow) in the presence of EGTA.
CD146 engagement without (
) EGTA and with (*) EGTA is shown.
C, with GAM (20 µg/ml) in the absence of exogenous calcium
for 300 s (GAM-Ca2+). 2 mM
extracellular calcium was then added (+Ca2+),
and calcium influx was monitored for an additional 200 s. The
curves are representative of four experiments performed in
triplicate.
to the plasma membrane (20). Previous data have indicated that
upon engagement, CD146 recruits the PTK FYN (16). To define the roles
of FYN in the mobilization of Ca2+ linked to CD146
engagement, cell lysates were immunoprecipitated using anti-FYN mAb,
and the immunoprecipitates were analyzed by immunoblotting using
anti-Tyr(P) mAb (PY20). As shown in Fig. 3 (upper panel), Tyr(P) of FYN
was observed after CD146 engagement (lane b) but was not
observed after treatment of HUVECs with an isotype-matched IgG1 mAb
(lane a). The Tyr(P) of FYN was totally abolished by
preincubation of HUVECs with herbimycin (2 µM, lane c) and PP1 (10 µM, lane d), a Src family
kinase inhibitor selective for the kinases FYN and Lck (39).
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Fig. 3.
Effect of CD146 engagement on tyrosine
phosphorylation of FYN in HUVECs. Engagement of CD146 was
performed as described under "Experimental Procedures." Lysates
(500 µg) were immunoprecipitated using anti-FYN mAb, separated on
5-12% gradient SDS-PAGE, and immunoblotted. Upper panel,
with PY20 anti-Tyr(P). Lane a, IgG1
isotype-matched control; lane b, CD146 engagement;
lane c, CD146 engagement in the presence of 2 µM herbimycin; and lane d, 10 µM
PP1. The position of FYN is indicated by an arrow. Blots
were then stripped and reblotted with anti-FYN (lower
panel).
(Fig.
4, upper panel, lane b) which
was not observed in control cells (lane a). PLC
phosphorylation was abolished by preincubation of HUVECs with PP1 (10 µM) (lane c). Preincubation of cells with
BAPTA-AM, a chelator of intracellular calcium, or EGTA did not
inhibited the Tyr(P) of PLC
(lanes d and
e). These results suggest that the Tyr(P) of
PLC
induced by CD146 engagement is mediated by FYN activation.
However, no direct association between CD146 and PLC
was initiated
by CD146 engagement (data not shown). The involvement of FYN and PLC
in [Ca2+]i mediated by CD146 engagement was
confirmed by using pharmacological inhibitors. Pretreatment of HUVECs
for 30 min with 2 µM herbimycin (Fig.
5A) or 10 µM PP1
(Fig. 5B) prior to CD146 engagement extinguished
[Ca2+]i increase induced by CD146 cross-linking.
Similarly, preincubation of HUVECs 30 min with 10 µM
U73122, an inhibitor of PLC
(40), abolished the rise in
[Ca2+]i in response to CD146 engagement (Fig.
5C). Taken together, these results are consistent with a
scheme according to which CD146 engagement induces the recruitment and
activation of FYN, leading to the Tyr(P) of PLC
, which in turn
participates to the increase in [Ca2+]i.
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Fig. 4.
Effect of CD146 engagement on the tyrosine
phosphorylation of PLC in HUVECs. After
engagement of CD146, lysates (300 µg) were immunoprecipitated using
anti-PLC
mAb, separated on a 7.5% SDS-PAGE, and immunoblotted
(IB) using PY20 anti-Tyr(P) mAb as indicated (upper
panel). Lane a, IgG1 isotype-matched
control; lane b, CD146 engagement; lane c, CD146
engagement in presence the of 10 µM PP1; lane
d, 25 µM BAPTA-AM; lane e, 5 mM EGTA. Blots were then stripped and reblotted with
anti-PLC
(lower panel). The position of PLC
is
indicated by an arrow.
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Fig. 5.
Effect of kinase inhibitors on calcium influx
induced by CD146 engagement in HUVEC. CD146 engagement was
performed as described under "Experimental Procedures." Calcium
release in HUVECs loaded as described Fig. 1 was assayed for 360 s
after GAM cross-linking ( ). Prior to CD146 engagement, cells were
incubated 30 min with inhibitors (*). A, herbimycin
(Herb, 2 µM); B, 10 µM PP1; and C, 10 µM U73122. The
curves are representative of four experiments performed in
triplicate.
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Fig. 6.
Effect of CD146 engagement on the tyrosine
phosphorylations of Pyk2 and p130Cas in HUVEC.
Engagement of CD146 was performed as described under "Experimental
Procedures." After immunoprecipitation, the proteins were separated
on a 7.5% SDS-PAGE. Tyrosine phosphorylations of Pyk2 (A)
or p130Cas (B) were studied by
immunoprecipitation with anti-Pyk2 (600 µg of cell lysate) and
anti-p130Cas (300 µg of cell lysate), respectively, and
immunoblotting (IB) using PY20 anti-Tyr(P) mAb. Upper
panel, lane a, IgG1 isotype-matched control;
lane b, CD146 engagement; lane c, CD146
engagement in the presence of 10 µM PP1; lane
d, 25 µM BAPTA; lane e,
5 mM EGTA. Blots were then stripped and reblotted with
anti-Pyk2 mAb (A, lower panel) or anti-p130Cas
(B, lower panel). The respective positions of Pyk2 and
p130Cas are indicated by an arrow.
130 kDa
corresponding to p130Cas (Fig. 6B, upper panel, lane
b). No phosphorylation was observed in HUVECs treated with a
control isotype-matched (IgG1) mAb (lane a). The Tyr(P) of
p130Cas was abrogated by pretreatment of HUVECs with PP1
(10 µM, lane c) and was reduced by BAPTA-AM
(25 µM) or EGTA (5 mM) (lanes d and e). Nevertheless, CD146 did not associate with
p130Cas (data not shown).
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Fig. 7.
Molecular interactions between PYK2,
p130Cas, and paxillin. CD146 engagement was performed
as described under "Experimental Procedures." Cell lysates (300 µg) were immunoprecipitated with anti-Pyk2 and immunoblotted
(IB) with anti-p130Cas or anti-paxillin.
Lanes a, IgG1 isotype-matched control;
lane b, CD146 engagement. The position of the
different proteins is indicated by an arrow.
, Pyk2, and
p130Cas--
The time course of the Tyr(P) of FYN, PLC
,
Pyk2, and p130Cas upon CD146 engagement was then
investigated. Cell lysates were immunoprecipitated with anti-Tyr(P) mAb
and immunoblotted with the indicated mAbs. CD146 engagement rapidly
stimulates the Tyr(P) of FYN and PLC
(Fig.
8) which reached their maximum 2 and 5 min, respectively, after GAM cross-linking. It should be noted that Tyr(P) of Fyn was detected as soon as 15 s upon CD146 engagement (data not shown). Maximal Tyr(P) of Pyk2 and p130Cas were
observed after 20 min. The Tyr(P) of Pyk2 was transient, whereas that
of p130Cas remained sustained after 30 min. These results
indicate that CD146 engagement induces an outside-in signal pathway
involving at first FYN and PLC
followed by the phosphorylation of
Pyk2 and p130Cas.
View larger version (36K):
[in a new window]
Fig. 8.
Time course of tyrosine phosphorylation of
FYN, PLC , Pyk2, and p130Cas in
response to CD146 engagement. Cross-linking by GAM was performed
for the indicated times. Lysates (300 µg for PLC
and
p130Cas and 600 µg for FYN and Pyk2) were
immunoprecipitated with anti-Tyr(P) PY20 and immunoblotted with
anti-FYN, anti-PLC
, anti-Pyk2, and anti-p130Cas. The
position of the different proteins is indicated by an
arrow.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Moreover, Ca2+ appears to serve as a second
messenger by coupling Ca2+ release to the Tyr(P) of the
related FAK kinase, Pyk, and the adapter protein, p130Cas,
and favoring their association.
(20), a key enzyme involved in calcium traffic (19). CD146 recruitment of FYN initiates the Tyr(P) of PLC
, as evidenced by the inhibition of PLC
Tyr(P) by PP1, and is consistent with the time course. These
data of FYN and PLC
phosphorylation are in agreement with previous
reports indicating that FYN is involved in calcium mobilization by
initiating the activation of PLC
(44-46).
which in
turns can hydrolyze membrane phosphoinositides. The binding of inositol 1,4,5-triphosphate to its receptor would induce the release of Ca2+ from internal stores and initiate a
store-dependent entry of extracellular Ca2+. In
addition, FYN regulates the Tyr(P) of targets of CD146 localized in the
focal adhesion plaques by a Ca2+-dependent
mechanism. FYN initiates the phosphorylation of Pyk2 which in turns
associates with p130Cas and paxillin and will allow their
localization in focal adhesion plaques.
/
embryo fibroblasts.
Only a small fraction of Pyk2 is found in focal adhesion points (43,
61, 62). We can hypothesize that upon CD146 engagement, distinct
signaling pathways might be developed. The cellular context might
dictate which nonreceptor PTK is activated.
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ACKNOWLEDGEMENTS |
---|
We thank Andrée Boyer for technical assistance and René Giordana for skillful help in preparing endothelial cells. We are grateful to Biocytex Company for providing 7A4 mAb and S-Endo 1 F(ab')2 fragment.
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FOOTNOTES |
---|
* This work was supported by Ministère de l'Education Nationale Grant UPRES EA.2195 and INSERM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: EMI 00-19, Laboratoire
d'Hématologie-Immunologie, UFR Pharmacie, 27 Bd. Jean Moulin,
13385 Marseille Cedex 5, France. Tel.: 33 4 91 83 56 00; Fax: 33 4 91 83 56 02; E-mail: anfosso@pharmacie.univ-mrs.fr.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M007065200
1 N. Bardin, F. Anfosso, E. Cramer, F. Sabatier, J. Sampol, and F. Dignat-George, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: FYN, p59fyn; FAK, p125FAK; HUVECs, human umbilical vein endothelial cells; SOCE, store-operated calcium entry; [Ca2+]i, intracellular calcium; PTK, protein tyrosine kinase; GAM, goat anti-mouse immunoglobulin; anti-Tyr(P), mouse monoclonal antibody against phosphotyrosine; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester; mAb, monoclonal antibody; PLC, phospholipase C; BSA, bovine serum albumin; TG, thapsigargin.
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