From INSERM U428, Faculté de Pharmacie, Université Paris-V, 75270 Paris, France
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ABSTRACT |
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The platelet receptor for the Fc domain of IgGs
(Fc The Cbl protein is the product of the c-cbl
protooncogene, the cellular homologue of the v-cbl oncogene
present in the Cas-NS-1 retrovirus, which induces pre-B cell lymphomas
and myeloid leukemias (1, 2). Cbl is found in a wide range of
hemopoietic cell lineages and some nonhemopoietic tissues such as lung,
brain, and testis (3). A deletion in the c-cbl sequence
(62% of the C-terminal domain) involving functional domains, such as
the leucine zipper motif and proline-rich region, converts this
protooncogene into the transforming one (4). Unlike the product of
v-cbl localized in both the cytoplasm and the nucleus, the
c-cbl product (p120cbl) is exclusively cytoplasmic
(5). A tumorigenic form of Cbl was detected in the 70Z/3 pre-B cell
lymphoma, in which Cbl tyrosine phosphorylation is increased as a
result of a deletion of 17 amino acids in the Cbl sequence (6). Cbl is
also heavily tyrosine-phosphorylated in v-src-transformed
hemopoietic cells (7).
Cbl becomes tyrosine-phosphorylated after cell stimulation through a
wide range of receptors including B and T cell receptors (8-12),
various growth factor receptors (13-21), integrins (22-24), and
receptors for the Fc domain of IgG (21, 25, 26). The primary structure
of Cbl shows no homology with any catalytic domain but contains a
number of tyrosine residues that can be phosphorylated and a
proline-rich region (2). Cbl has been shown to bind to a number of
signaling proteins, such as tyrosine kinases Src, Lyn, Fyn, Lck, Blk,
Syk, the lipid kinase phosphatidylinositol 3-kinase (PI
3-K),1 phospholipase C A regulator activity has recently been described for Cbl when
overexpressed in mast cells, in which it regulates p72syk
activity (36). Cbl is also proposed to regulate the T cell receptor-mediated Ras pathway activation via its association with Grb2
in T cells (37). In interleukin 4-treated B cells, Cbl is
tyrosine-phosphorylated and associated with p85/PI 3-K, and overexpression of Cbl enhances PI 3-K activity, mitogenic activity, and
cell survival (38). Taken together, the data suggest a role for Cbl in
multiple signaling pathways of different cell types.
In human platelets, Cbl has been identified and shown to be
constitutively associated with the Grb2 adaptor protein and
tyrosine-phosphorylated after thrombopoietin activation (27). Thus, Cbl
seems to be implicated in signal transduction after thrombopoietin
binding to c-mpl. However, the role of Cbl or its possible
involvement in platelet signal transduction mediated by other receptors
has not yet been documented. Platelet activation is mediated by a wide
variety of agonists, including thrombin, thromboxane A2, ADP, and
adhesion molecules such as von Willebrand factor and collagen. Some
antibodies directed against antigens on the platelet membrane
(e.g. the tetraspanin CD9, glycoprotein IV, and the integrin In the present study, we have investigated the involvement of Cbl in
platelet signaling after Fc Reagents--
Anti-Cbl polyclonal antibody was from Santa Cruz
Biotechnology (Santa Cruz, CA). Syb-1, a monoclonal IgG anti-CD9 (40), was kindly provided by Dr. C. Boucheix (INSERM U268). Anti-Fc Platelet Preparation--
Human platelets were isolated from
fresh platelet concentrates obtained from healthy donors who did not
taken aspirin for at least 1 week. The concentrates were centrifuged at
room temperature for 15 min at 130 × g to eliminate
other cell types and then subjected to a washing process as described
previously (41). Briefly, platelets were isolated on a metrizamide
gradient, collected, and resuspended in 10 mM HEPES buffer,
pH 7.4, containing 140 mM NaCl, 5 mM
NaHCO3, 0.5 mM MgCl2, 3 mM KCl, and 10 mM glucose. Platelet
concentration was adjusted to 109/ml for
immunoprecipitation studies or to 5.108/ml for studying the
total platelet lysates. CaCl2 (1 mM) was added
10 min before platelet stimulation.
Platelet Activation, Aggregation, and Release--
Platelets
were stimulated with either 10 µg/ml of antibodies or 1 unit/ml human
thrombin at 37 °C in an aggregometer (Coulter, Havertown, PA) with
constant stirring (1100 rpm). For Fc receptor pathway inhibition,
platelets were preincubated for 1 min at 37 °C with IV.3 (10 µg/ml) before addition of Syb antibody. For Fc receptor
cross-linking, platelets were preincubated for 1 min with IV.3 (10 µg/ml) and then stimulated by addition of RAM-F(ab)'2 (80 µg/ml) for various periods. To study the total platelet proteins, the
reactions were stopped by addition of 25% (v/v) of a buffer containing
10% SDS and 5 mM EDTA, and the samples were transferred to
ice for complete lysis. After 30 min, 25% (v/v) of 4 × concentrated Laemmli's buffer and 5% of 2-mercaptoethanol were added,
and the samples were subjected to Western blot analysis.
To study the platelet aggregation and release, the platelet-rich plasma
was adjusted to 5 × 108 platelets/ml and incubated
with 0.6 µM 14C-labeled serotonin (Amersham
Pharmacia Biotech) for 30 min at room temperature, and then the
platelets were isolated as described above. Imipramine was added to the
platelet-rich plasma 10 min before the agonist to prevent reuptake of
the serotonin during the experiment. Inhibition of PI 3-K was performed
by the preincubation of platelets with wortmannin (50 nM)
at 37 °C for 15 min before platelet activation. The aggregation was
determined by measuring changes in light transmission through a stirred
volume of platelets at 37 °C in aggregometer. The aggregation was
monitored for 5 min, and the reaction was stopped by transfer into 0.2 volume of ice-cold 0.1 M EDTA and immediate centrifugation
for 1 min at 12,000 × g. The
[14C]serotonin was measured in the supernatant by liquid
scintillation counting. Release was expressed as percent
[14C]serotonin liberated compared with the total
unstimulated platelet content.
Immunoprecipitation--
For immunoprecipitation (IP) studies,
platelet stimulation was stopped by the addition of one-third volume of
cold 3 × concentrated Nonidet P-40 lysis buffer containing 3%
(v/v) Nonidet P-40, 150 mM Tris, 450 mM NaCl,
15 µg/ml leupeptin, 15 µg/ml aprotinin, 3 mM EGTA, 3 mM Na3VO4, and 3 mM
phenylmethylsulfonyl fluoride. The mixture was transferred to ice for
30 min for complete lysis. Insoluble material was removed by
centrifugation for 10 min at 16,000 × g at 4 °C,
and the supernatant was incubated with antibodies against Cbl or
phosphotyrosine proteins (2.5 µg/ml) for 2 h at 4 °C. Immune
complexes were incubated with protein A-Sepharose beads (30 µl of
50% slurry) for 1 h at 4 °C and then isolated by brief
centrifugation. After washing three times with 0.5 ml of 1 × concentrated cold Nonidet P-40 lysis buffer (described above),
immunoprecipitates were resuspended in Laemmli's sample buffer
containing 5% 2-mercaptoethanol and analyzed by Western blot.
For the preclearing experiments, antibody concentrations used for
protein immunodepletion were raised to 5 µg/ml. The Nonidet P-40-soluble fraction was incubated with anti-Cbl, anti-phosphotyrosine (4G10), or control antibodies for 2 h, followed by addition of 40 µl of protein A-Sepharose (50% slurry). This step was reproduced after removing the immuncomplexes. Before addition of each antibody for
IP, an aliquot (50 µl) of the platelet lysate was conserved in
Laemmli's buffer at Western Blot Analysis--
Samples were boiled for 5 min, and
proteins were subjected to 10% SDS-PAGE. Separated proteins were
transferred electrophoretically to a nitrocellulose membrane (Bio-Rad
system). The membrane was incubated 1 h in a blocking buffer
containing 5% low dry milk, 2% Tween 20, 100 mM NaCl, and
20 mM Tris, pH 7.4. Specific antibodies against proteins of
interest were added for 2 h, followed by a 1-h incubation with
horseradish peroxidase-conjugated secondary antibody. An enhanced
chemiluminescence system was used for signal detection.
In some experiments, the membranes were stripped; the bound antibody
was removed by incubation in buffer containing 2% SDS, 62.5 mM Tris, pH 6.8, and 100 mM 2-mercaptoethanol
for 40 min at 60 °C. After extensive washing, the membrane was
reprobed with another antibody as described above.
GST Fusion Protein Studies--
Cultures of bacteria expressing
GST or GST fusion proteins (GST-SH3-(p85/PI 3-K), GST-(N or
C-terminal)-SH2-(p85/PI 3-K), and GST-p85 (full p85/PI 3-K)) were
grown, and GST fusion proteins were isolated as described previously
(42, 43). GST or GST fusion proteins bound to glutathione-Sepharose 4B
were incubated with platelet lysates (Nonidet P-40-soluble fraction)
for 2 h. After brief centrifugation the precipitates were washed
and treated as described for the IP studies.
The GST fusion proteins were subjected to a competition study. In this
case, the GST-p85 and GST-SH3 were preincubated 30 min with the
proline-rich peptides corresponding to residues 82-96 and 300-314 of
p85, which were previously shown to bind to the SH3 domain of the p85
itself (43), using an unrelated peptide as control (GSQVVRIVGGRD).
Preincubations were performed at 4 °C for 30 min with constant
stirring, added to platelet lysates, and treated as described above.
Cbl Tyrosine Phosphorylation during Platelet Activation--
To
examine whether activation of platelets through Fc
Platelet activation mediated through Fc
To determine the levels of Cbl tyrosine phosphorylation induced by the
former agonists, Cbl was immunoprecipitated from lysates of resting and
activated platelets. As shown in Fig. 3,
upper panel, Cbl was not significantly
tyrosine-phosphorylated in resting platelets. By contrast, a high
level of Cbl tyrosine phosphorylation was observed after Fc
To verify that the different levels of Cbl tyrosine phosphosphorylation
(depending on the agonist used) were not attributable to different
kinetics and to determine when Cbl phosphorylation occurred, we studied
the time course of Cbl tyrosine phosphorylation during platelet
stimulation. Strong Cbl tyrosine phosphorylation was already reached at
30 s after platelet activation induced by Fc Cbl Association with p85/PI 3-K in Activated Platelets--
As an
adaptor protein, tyrosine-phosphorylated Cbl was shown to associate
with various signaling proteins. Among them, Cbl was reported in
various cells to associate with PI 3-K (9, 28, 29, 45) and to enhance
PI 3-K activity (27, 38). Because PI 3-K plays an important role in
platelet function (46), we searched for an association between Cbl and
PI 3-K in platelets. Cbl was immunoprecipitated from resting, and
stimulated platelets and samples were analyzed with an anti-p85/PI 3-K
antibody. In resting platelets, p85/PI 3-K was hardly detectable in the
Cbl immunoprecipitates (Fig. 5). In
contrast, p85/PI 3-K co-immunoprecipitated with Cbl in platelets
activated through the Fc receptor. In thrombin-activated platelets,
Cbl/PI 3-K association was insignificant (Fig. 5a). The
amount of p85 protein present in the anti-Cbl IP of platelets activated
by Fc
To determine which domain of p85 was involved in Cbl/PI 3-K
association, we studied Cbl binding to bacterial GST fusion proteins corresponding to full p85 (GST-P85), the SH3 domain of p85 (GST-SH3), or the N- and C-terminal SH2 domains of p85. Incubation of the different GST fusion proteins with platelet lysates showed that none of
the two p85 SH2 domains (C- and N-terminal) bound Cbl (Fig.
6a). By contrast, an
association of p85 (full) or p85 SH3 domain with Cbl was observed in
both resting and activated platelet lysates. These data suggested an
interaction between the p85 SH3 domain and the Cbl proline-rich
region.
To determine whether Cbl/PI 3-K association was mediated by the p85 SH3
domain and the Cbl proline-rich region, we used competitive proline-rich peptides derived from p85 that were previously shown to
bind the SH3 domain of p85 itself (43). The two competitive peptides
strongly abolished Cbl association with GST-SH3 (p85) and GST-p85
(full). The inhibition was total when the two peptides were added
together (Fig. 6b), indicating that, in vitro,
the p85 SH3 domain mediates Cbl/p85 association through its interaction with the Cbl proline-rich region.
Inhibition of PI 3-K Activity Abolished the Platelet Responses
after Fc The adaptor protein Cbl has been identified in a variety of cells,
including platelets, but its involvement in platelet signaling remains
uncharacterized. The present work was devoted to studying the
involvement of Cbl in signal transduction after platelet activation induced by Fc Cbl was not significantly tyrosine-phosphorylated in resting platelets,
but it became phosphorylated during platelet activation depending on
the agonist used. After activation by Fc Because Cbl association with p85/PI 3-K has been suggested to increase
PI 3-K activity in a number of cell systems (14, 27, 38), we examined
the association of Cbl with PI 3-K in platelets. We found that Cbl/PI
3-K association was negligible in resting and thrombin-activated
platelets. In contrast, Cbl was strongly associated with PI 3-K after
Fc We could not exclude, however, that in vivo Cbl/PI 3-K
association may require tyrosine phosphorylation of Cbl. Indeed, our experiments favor a relationship between these two events. In Nb2
cells, constitutive Cbl/PI 3-K association is mediated by the Cbl
proline-rich region and the p85 SH3 domain, whereas increased Cbl/p85
association was proposed to occur through both the p85 SH2 and SH3
domains after cell activation and Cbl tyrosine phosphorylation (27). In
fact, both SH2 and SH3 domains of p85 interact with Cbl in other cells
(19, 22, 33, 50). Interestingly, Soltoff and Cantley (19) suggested
that engagement of the p85 SH2 domain exposes the SH3 domain, which can
then further interact with Cbl and increase the affinity of p85 for
Cbl. The authors proposed that Cbl could act as an adaptor protein that
recruits PI 3-K in the epidermal growth factor-mediated activation of
PC12 cells (19). Moreover, Cbl has a Tyr-X-X-Met
motif, which could associate with a p85 SH2 domain if phosphorylated on
tyrosine (51). Alternatively, the tyrosine phosphorylation of Cbl could
be necessary for its relocalization near PI 3-K. In that respect,
Tanaka et al. (25) showed that in epidermal growth
factor-activated macrophages and fibroblasts, Cbl tyrosine
phosphorylation may be accompanied by its subcellular translocation. A
last hypothesis could be that in resting platelets, the Cbl
proline-rich region may not be in a conformation that allows its
association with the p85 SH3 domain. After platelet activation and Cbl
phosphorylation, a conformational change in Cbl would render possible
the association between the two proteins. By analogy, in stimulated
fibroblasts, a phosphotyrosine-dependent conformational change of Cbl was proposed to transiently expose the Cbl N-terminal region, permitting interaction with platelet-derived growth factor receptor To determine whether Cbl tyrosine phosphorylation and association with
PI 3-K occurred before PI 3-K activation, we used wortmannin to inhibit
PI 3-K activity. We found that wortmannin had no effect on Cbl
phosphorylation or on Cbl/PI 3-K association induced by different
agonists, which indicates that the two events occurred upstream of the
lipid kinase activation (data not shown). However, a noncovalent
association between PI 3-K and Fc That the level of Cbl/p85 association was stronger after Fc receptor
engagement than after thrombin addition suggests a differential role
for PI 3-K in the signaling induced by the two types of platelet activation. In the presence of wortmannin, platelet aggregation mediated through Fc In conclusion, Cbl was strongly tyrosine-phosphorylated during
FcRIIa) triggers intracellular signaling through protein tyrosine
phosphorylations leading to platelet aggregation. In this study, we
focused on the adaptor protein p120cbl (Cbl), which became
tyrosine-phosphorylated after platelet activation induced by
antibodies. Cbl phosphorylation was dependent on Fc receptor
engagement. An association of Cbl with the p85 subunit of
phosphatidylinositol 3-kinase (PI 3-K) occurred in parallel with Cbl
tyrosine phosphorylation. We showed by in vitro experiments that Cbl/p85 association was mediated by the Src homology 3 domain of
p85/PI 3-K and the proline-rich region of Cbl. Inhibition of PI 3-K
activity by wortmannin led to the blockade of both platelet aggregation
and serotonin release mediated by Fc
RIIa engagement, whereas it only
partly inhibited those induced by thrombin. Thus, PI 3-K may play a
crucial role in the initiation of platelet responses after Fc
RIIa
engagement. Our results suggest that Cbl is involved in platelet signal
transduction by the recruitment of PI 3-K to the Fc
RIIa pathway,
possibly by increasing PI 3-K activity.
INTRODUCTION
Top
Abstract
Introduction
References
,
and the adaptor proteins Grb2 and Vav (9-11, 22, 27-33). Cbl
phosphorylation on serine residues has also been reported in phorbol
ester-activated T cells, allowing its interaction with 14.3.3 protein
(34). Finally, Cbl contains a phosphotyrosine binding domain in the
N-terminal region that directly binds to phosphorylated ZAP-70 in
activated T cells (35).
IIb-
3) are also able to induce platelet
activation. In most cases, the activation induced by IgGs is dependent
on the binding of their Fc domain on the specific receptor, Fc
RIIa
(39).
RIIa engagement. Two models of platelet
activation involving the Fc receptor were used: the cross-linking of
Fc
RIIa and bridging of the CD9 antigen with Fc
RIIa by an
activating monoclonal antibody (mAb) (anti-CD9, Syb). Our results
demonstrate that after Fc
RIIa engagement, Cbl was heavily
tyrosine-phosphorylated. In parallel with Cbl phosphorylation, we found
that Cbl was associated with p85/PI 3-K. Moreover, the use of
wortmannin, an inhibitor of PI 3-K, abolished platelet aggregation and
release induced by antibodies, underlining the crucial role of PI 3-K
during immunological activation. The results suggest an important role
for Cbl in Fc
RIIa-mediated platelet activation, possibly through
the regulation of PI 3-K activity.
EXPERIMENTAL PROCEDURES
RII mAb
IV.3 was from MEDAREX Inc. (West Lebanon, NH). Irrelevant monoclonal
and polyclonal antibodies and rabbit polyclonal F(ab)'2 anti-mAb (RAM) were from Immunotech (Marseille, France). Anti-p85/PI 3-K antisera was from Upstate Biotechnology Inc. (Lake Placid, NY).
Sheep anti-mAb horseradish peroxidase-labeled antibody was from
Amersham Pharmacia Biotech. Goat anti-rabbit horseradish peroxidase-labeled antibody was from Bio-Rad. Anti-phosphotyrosine monoclonal antibodies PY20 and 4G10 were from Transduction Laboratories (Lexington, KY) and Upstate Biotechnology, respectively. Human thrombin
was from Diagnostica-Stago (Asnière, France), Metrizamide was
from Eurobio (Les Ulis, France), and protein A-Sepharose, leupeptin,
aprotinin, phenylmethylsulfonyl fluoride, wortmannin, and
isopropyl-
-thiogalactopyranoside were from Sigma. Bacterias expressing p85/PI 3-K glutathione S-transferase (GST) fusion
proteins were a kind gift from Prof. L. Cantley (Beth Israel Hospital, Boston, MA), and Dr. S. Fischer (INSERM U363, Paris, France).
20 °C. Both immunoprecipitates and aliquot samples were subjected to Western blot analysis.
RESULTS
RIIa involved Cbl,
we activated platelets by Fc receptor cross-linking, using IV.3
(anti-Fc receptor) in the presence of RAM-F(ab)'2 or by an
anti-CD9 antibody (Syb) known to induce platelet activation through an
Fc
RIIa-dependent pathway. We also compared these results with those observed after thrombin stimulation.
RIIa or the thrombin receptor
induced an increase in the level of phosphotyrosine proteins (PYs). We
focused our attention on a band at ~120 kDa, which was
tyrosine-phosphorylated after 2 min of platelet activation (Fig.
1, upper panel). To see
whether this band could correspond to p120cbl, the
nitrocellulose membrane was reprobed with anti-Cbl antibody. This
experiment confirmed the presence of Cbl in platelets, which is
localized within the 120-kDa band (Fig. 1, lower panel).
This result was further confirmed by preclearing experiments. Lysates of Syb-activated platelets were subjected twice to anti-PY or anti-Cbl
immunoprecipitations. Many PYs were absent after immunodepletion of
total PY, including the tyrosine-phosphorylated 120-kDa band (Fig.
2a, upper panel, lanes
4 and 6). A fraction of Cbl remained in the total
lysate subjected to anti-PY immunodepletion, which probably corresponds
to unphosphorylated Cbl (Fig. 2a, lower panel, lanes
4 and 6). In platelet lysates depleted of Cbl, the
tyrosine-phosphorylated band at ~120 kDa remained unchanged (Fig.
2a, upper panel, lanes 2 and 5),
indicating the presence of additional proteins in the 120-kDa band.
Anti-Cbl immunoblotting of the same membrane confirmed the total
depletion of Cbl in the lysate (Fig. 2a, lower panel, lane
5). Furthermore, Cbl was present in the anti-PY immunoprecipitates of Syb-activated platelets (Fig. 2b, lanes 3 and
6). Altogether, these data confirmed the presence of Cbl
within the 120-kDa band and indicated that in Syb-activated platelets,
the 120-kDa phosphotyrosine band corresponds to several PYs including
Cbl.
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Fig. 1.
Identification of Cbl among
tyrosine-phosphorylated proteins during platelet activation.
Washed platelets were left untreated or were treated by different
agonists for 2 min under constant stirring. At the end of the
stimulation period, samples were solubilized and resolved on 10%
SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with a
mixture of 4G10 and PY20 anti-phosphotyrosine antibodies (upper
panel). Lower panel, same nitrocellulose membrane
stripped and immunoblotted with anti-Cbl antibody. IV.3,
anti-Fc RII antibody (10 µg/ml); RAM, F(ab)'2 rabbit
anti-mouse antibody (80 µg/ml); Syb, anti-CD9 antibody (10 µg/ml); thrombin (1 unit/ml). Results are representative of five
experiments.
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Fig. 2.
Depletion of Cbl and phosphotyrosine proteins
in platelet lysates. Washed platelets were activated with Syb
antibody (10 µg/ml). After 2 min of stimulation, the cells were lysed
with Nonidet P-40 buffer, and the lysates were immunoprecipitated twice
with either anti-Cbl antibody or anti-PY antibodies (5 µg/ml).
a, aliquots of each lysate were collected and conserved at
20 °C before and after each immunoprecipitation. Whole platelet
lysates were subjected to SDS-PAGE and immunoblotting with anti-PY
(upper panel) or anti-Cbl antibodies (lower
panel). Platelet lysates: lane 1, before any IP;
lane 2, after the first IP with anti-Cbl antibody;
lane 3, after control IP with nonimmune antibody; lane
4, after control IP with anti-PY; lane 5, after the
second IP with anti-Cbl; lane 6, after the second IP with
anti-PY. b, immunoprecipitates of the first and second
control IPs (lanes 1 and 4), first and second
anti-Cbl IPs (lanes 2 and 5), and first and
second anti-PY IPs (lanes 3 and 6) were
solubilized and resolved on SDS-PAGE, transferred to a nitrocellulose
membrane, and blotted with anti-Cbl. Results are representative of two
experiments.
RIIa
cross-linking. Cbl was also strongly tyrosine-phosphorylated in
Syb-activated platelets, although to a lesser level than after
Fc
RIIa cross-linking. Cbl was only minimally phosphorylated in
thrombin-activated platelets (Fig. 3a). Densitometer
scanning of the autoradiographs of five experiments confirmed this
difference (Fig. 3b). Reprobing the membrane with anti-Cbl
antibody (Fig. 3, lower panel) confirmed that the increase in tyrosine phosphorylation of Cbl was not the consequence of a change
in the recovery of the protein. The addition of IV.3 alone did not
induce any tyrosine phosphorylation. Preincubation of IV.3 to block the
binding of Syb Fc domain to Fc
RIIa totally inhibited Syb-induced
tyrosine phosphorylation of Cbl. The results demonstrate that Cbl was
heavily tyrosine-phosphorylated after Fc
RIIa engagement and suggest
a Cbl involvement in Fc
RIIa-mediated platelet signaling.
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Fig. 3.
Cbl tyrosine phosphorylation in activated
platelets. a, Washed platelets were incubated for 2 min with
the indicated agonist (as described in Fig. 1). Cells were lysed with
Nonidet P-40 buffer and immunoprecipitated with anti-Cbl antibody.
After resolution of the immunoprecipitated proteins using 10%
SDS-PAGE, proteins were transferred to nitrocellulose and immunoblotted
with either anti-phosphotyrosine antibodies 4G10 plus PY20 (upper
panel) or anti-Cbl antibody (lower panel). Results are
representative of five experiments. b, mean intensity of Cbl
tyrosine phosphorylation in five experiments was evaluated by scanning
the autoradiographs. A.U, arbitrary unit.
RIIa cross-linking,
with a plateau obtained between 1 and 2 min and a decrease to lower
levels thereafter (Fig. 4, upper panel). Syb-induced Cbl tyrosine phosphorylation kinetics was similar to that observed after Fc
RIIa cross-linking (Fig. 4, middle panel), with or without a lag depending on the donor
used. Indeed, the Fc
RIIa His-Arg-131 polymorphism has been shown to play a crucial role in the ability of Fc
RIIa to bind the Fc domain of mAb-IgG1 and, consequently, in the cell activation induced by these
antibodies (44). We and others have shown that platelets from
homozygous His donors respond more slowly than platelets from
homozygous Arg donors to anti-CD9 antibodies, whereas the lag phase of
platelets from heterozygous donors is intermediate (41, 44). Thrombin
induced a weak and slow Cbl tyrosine phosphorylation, which peaked
between 2 and 5 min after platelet stimulation and decreased thereafter
(Fig. 4, lower panel). The data suggest that Cbl was
tyrosine-phosphorylated in the early stages of platelet activation and
could participate in the first events triggered by Fc receptor
engagement. During thrombin activation, Cbl would be involved to a
lesser extent and in later stages after platelet activation.
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Fig. 4.
Kinetics of Cbl tyrosine phosphorylation
during platelet activation. Washed platelets were activated for
30 s to 10 min by cross-linking Fc RIIa using mAb IV.3 (10 µg/ml) plus F(ab)'2 RAM (80 µg/ml), by Syb (10 µg/ml), or by
thrombin (1 unit/ml). Platelets were then lysed by Nonidet P-40
containing buffer, and Cbl was immunoprecipitated. The
immunoprecipitates were subjected to SDS-PAGE, followed by
anti-phosphotyrosine immunoblotting. Results are representative of
three experiments.
RIIa cross-linking or by Syb reached a plateau between 30 s and 2 min of platelet stimulation and subsequently decreased back to
resting level (Fig. 5b). Notably, the time course of Cbl/p85
association paralleled that of Cbl phosphorylation (Fig. 5, compare
with Figs. 3 and 4), suggesting that Cbl/PI 3-K association was
dependent on Cbl tyrosine phosphorylation.
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Fig. 5.
Cbl association with p85/PI 3-K. The
nitrocellulose membranes corresponding to Figs. 3 and 4 were reprobed
with anti-p85/PI 3-K antibody. a, IP anti-Cbl from platelets
incubated 2 min with IV.3 (10 µg/ml), IV.3 plus F(ab)'2 RAM (80 µg/ml), Syb (10 µg/ml), or thrombin (1 unit/ml). b, IP
anti-Cbl from platelets activated for 30 s to 10 min by IV.3 plus
RAM, Syb, or thrombin. Results are representative of four
experiments.
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Fig. 6.
Binding of Cbl to GST fusion proteins of
p85/PI 3-K. a, GST or GST fusion proteins containing full
p85, p85 SH3 domain, or p85 N-terminal SH2 domain or C-terminal SH2
domain coupled to agarose beads were added to lysates of both resting
and Syb-activated platelets (10 µg/ml). The samples were resolved on
SDS-PAGE and immunoblotted with anti-Cbl antibody. b,
GST-p85 (full) and GST-SH3 (p85) were incubated 30 min with 0.1, 0.5, or 1 p85 proline-rich peptides (PRP1, 80-96;
PRP2, 300-314 amino acids of p85 sequence) or 1 mM unrelated peptide (control P). The mixtures
were then added to lysates of resting and activated platelets and
incubated for an additional 1 h. The samples were resolved by
SDS-PAGE and immunoblotted with anti-Cbl antibody. Results are
representative of three experiments.
RIIa Engagement--
To determine whether PI 3-K plays a
role in platelet activation mediated by the Fc receptor, we studied the
effect of wortmannin (50 nM), an inhibitor of PI 3-K
activity, on platelet aggregation and serotonin release. Platelet
aggregation induced by Fc
RIIa cross-linking or by Syb was strongly
inhibited by wortmannin (100 and 88%, respectively; Fig.
7). By contrast, platelet aggregation induced by thrombin was poorly inhibited in the presence of wortmannin and became reversible. Serotonin release induced by Fc
RIIa
engagement was greatly inhibited by preincubation of platelets with
wortmannin. Indeed, 85% inhibition of serotonin release was observed
in platelets activated by cross-linking, and 70% inhibition was
observed after activation by Syb. Wortmannin had no significant effect
on thrombin-induced serotonin release (Fig. 7). The data suggest a key
role for PI 3-K in Fc receptor-mediated platelet activation. If
tyrosine-phosphorylated Cbl enhances PI 3-K activity, Cbl would also
play an important role in platelet activation.
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Fig. 7.
Wortmannin inhibits platelet responses
induced by Fc RIIa engagement.
[14C]Serotonin-labeled platelets were preincubated with
vehicle or with 50 nM wortmannin (wt) for 15 min
at 37 °C and activated in the aggregometer by cross-linking
Fc
RII, Syb, or thrombin. Aggregation was measured, and the released
[14C]serotonin was counted and expressed as percentage of
total serotonin. Results are representative of three experiments.
DISCUSSION
RIIa cross-linking or Syb antibody (anti-CD9), which activates platelets via Fc
RIIa. We demonstrated strong and rapid tyrosine phosphorylation of Cbl in platelets activated through Fc
RIIa. In addition, we showed that after platelet activation, p85/PI 3-K association with Cbl correlates with the intensity of Cbl
tyrosine phosphorylation. Furthermore, we showed that the PI 3-K
inhibitor wortmannin abolished antibody-mediated platelet responses,
indicating a crucial role for PI 3-K in antibody-induced platelet activation.
RIIa cross-linking, Cbl was
strongly and rapidly tyrosine-phosphorylated. To a lesser extent, Syb
induced a similar Cbl phosphorylation to that obtained after Fc
RIIa
cross-linking. The former difference in the Cbl phosphorylation was
probably attributable to distinct modes of platelet activation induced
by Syb and Fc
RIIa cross-linking, as suggested by others (47). That
specific binding of Syb antibody to its antigen (CD9), in the presence
of IV.3 (anti-Fc
R), did not induce Cbl phosphorylation indicates
that Cbl tyrosine phosphorylation occurred after Fc
RIIa engagement.
These results suggest that, unlike thrombin, which induced a faint and
slow Cbl tyrosine phosphorylation, Fc receptor engagement strongly
involves Cbl at the first steps of platelet signaling. Another protein
involved in the first steps of Fc
RIIa-mediated signal transduction
is the tyrosine kinase Syk (48). The latter could be a potential
candidate to phosphorylate Cbl in platelets, because it was previously
demonstrated to participate in Cbl phosphorylation in activated T cells
(49). We could not, however, detect any Cbl association with Syk after
platelet activation. Cbl tyrosine phosphorylation was transient,
suggesting an action of tyrosine phosphatase(s) on phosphorylated Cbl.
This is supported by the fact that in the presence of the protein
tyrosine phosphatase inhibitor phenylarsine oxide, Cbl phosphorylation
remained stable for up to 10 min of platelet activation (data not shown).
RIIa-mediated platelet activation. In in vitro
experiments, using GST fusion proteins, we did not find any association
between the N-SH2 or C-SH2 domains of p85 and Cbl in resting or
Syb-activated platelets. In contrast, full p85 and the p85 SH3 domain
precipitated Cbl from resting and Syb-activated platelet lysates. It is
thus most likely that Cbl and p85 associate via the SH3 domain of p85
and the proline-rich region of Cbl.
(52).
RIIa has been previously shown
(53). Therefore, tyrosine-phosphorylated Cbl would play a role in
Fc
RIIa-mediated platelet signaling by linking PI 3-K with the Fc
receptor pathway, possibly by enhancing PI 3-K activity.
RIIa was abolished. In contrast, and as
previously demonstrated with thrombin receptor activating peptide (54, 55), platelet aggregation induced by thrombin was only partly inhibited
and became reversible. These data indicate that PI 3-K participates in
the control of platelet aggregation, especially that which occurs after
Fc receptor engagement. The crucial role of PI 3-K in
Fc
RIIa-mediated platelet activation was also confirmed by the
demonstration that wortmannin strongly inhibited antibody-induced serotonin release from dense granules but only weakly inhibited serotonin release induced by thrombin. These results demonstrated that
in platelets, PI 3-K activation was required to initiate platelet
responses after Fc
RIIa engagement. Thus, if Cbl increases PI 3-K
activity as previously proposed, Cbl would also play a crucial role in
platelet activation mediated through Fc
RIIa .
RIIa-mediated platelet activation, and levels of Cbl tyrosine phosphorylation paralleled levels of Cbl/PI 3-K association. Because PI
3-K activity appeared crucial in platelet responses dependent on
Fc
RIIa engagement, we suggest that Cbl participates in signal transduction mediated through the Fc receptor by enhancing PI 3-K
activity. Thus, Cbl could be one of the key adaptor and regulator proteins in this system.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. Bryckaert and G. Chang for critical reading of the manuscript and S. Aitsiali for editorial help.
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FOOTNOTES |
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* 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.
Supported by Diagnostica-Stago (Asnière, France).
§ Supported by Ministère de la Recherche et de la Technologie, France.
¶ To whom correspondence should be addressed: INSERM U428, Faculté de Pharmacie, 4 ave. de l'Observatoire, F-75006, Paris, France. Tel.: 33-1-53-73-96-19; Fax: 33-1-44-07-17-72; E-mail: bachelot{at}pharmacie.univ-paris5.fr.
The abbreviations used are:
PI 3-K, phosphatidylinositol 3-kinase; FcRIIa, platelet receptor for the Fc
domain of IgGs; mAb, monoclonal antibody; RAM, rabbit polyclonal
F(ab)'2 anti-mAb; GST, glutathione
S-transferase; IP, immunoprecipitation; PAGE, polyacrylamide
gel electrophoresis; SH, Src homology; PY, phosphotyrosine.
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REFERENCES |
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