From the Departments of Vascular Biology and
Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, California 92037 and the Graduate Program in
Immunology, the § Signal Transduction Program, Leonard and
Madlyn Abramson Family Cancer Research Institute, and the
¶ Department of Pathology and Laboratory Medicine, the University
of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
19104
Received for publication, November 26, 2000
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ABSTRACT |
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Platelet adhesion to fibrinogen through integrin
Integrins were originally identified as adhesion receptors for
extracellular matrix proteins
(ECM),1 but these Vav1 is an hematopoietic cell-specific guanine nucleotide exchange
factor for cdc42 and Rac (13). The GTP-bound forms of cdc42 and Rac
promote filopodial and lamellipodial extension, respectively (14).
SLP-76, the focus of the present work, is an hematopoietic
cell-specific adapter protein that contains an N-terminal SAM domain,
several potential tyrosine phosphorylation sites, a central
proline-rich region and a C-terminal SH2 domain (Fig.
1). Studies in lymphoid cells indicate
that three phosphotyrosines in SLP-76 (Y113, Y128, and Y145) are
required for interactions with the SH2 domains of Vav1 and Nck (15,
16). Of potential relevance to IIb
3 triggers actin
rearrangements and cell spreading. Mice deficient in the SLP-76 adapter
molecule bleed excessively, and their platelets spread poorly on
fibrinogen. Here we used human platelets and a Chinese hamster ovary
(CHO) cell expression system to better define the role of SLP-76 in
IIb
3 signaling. CHO cell adhesion to
fibrinogen required
IIb
3 and stimulated
tyrosine phosphorylation of SLP-76. SLP-76 phosphorylation required
coexpression of Syk tyrosine kinase and stimulated association of
SLP-76 with the adapter, Nck, and with the Rac exchange factor,
Vav1. SLP-76 expression increased lamellipodia formation induced by Syk
and Vav1 in adherent CHO cells (p < 0.001). Although
lamellipodia formation requires Rac, SLP-76 functioned downstream of
Rac by potentiating adhesion-dependent activation of PAK
kinase (p < 0.001), a Rac effector that associates with Nck. In platelets, adhesion to fibrinogen stimulated the association of SLP-76 with the SLAP-130 adapter and with VASP, a
SLAP-130 binding partner implicated in actin reorganization. Furthermore, SLAP-130 colocalized with VASP at the periphery of spread
platelets. Thus, SLP-76 functions to relay signals from
IIb
3 to effectors of cytoskeletal
reorganization. Therefore, deficient recruitment of specific adapters
and effectors to sites of adhesion may explain the integrin phenotype
of SLP-76
/
platelets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
transmembrane heterodimers also transmit information into the cell.
This process, referred to as "outside-in signaling," promotes
anchorage-dependent cellular responses including motility, growth, differentiation, and survival (1). Most integrin
and
cytoplasmic tails consist of 20-70 amino acid residues and are devoid
of enzymatic activity, implying that outside-in signals are transmitted
by integrin-associated proteins. Several transmembrane proteins can
interact with the extracellular domains of integrins, but attention has
focused on proteins that interact with integrin cytoplasmic tails (2,
3). These include
-actinin and talin, cytoskeletal proteins that
link integrins to actin filaments (4). Cell attachment to the ECM
causes lateral clustering of integrin heterodimers into oligomers and
evidence is accumulating that outside-in signaling is triggered, in
part, by the co-clustering of integrin-associated adapters, enzymes and
substrates into signaling complexes that localize within and modify the
actin cytoskeleton (1, 4, 5). This model is directly relevant to
integrin
IIb
3 signaling in blood platelets.
IIb
3 is a receptor for fibrinogen and von
Willebrand factor that mediates platelet aggregation and spreading on
vascular ECM (6). These responses are accompanied by major changes in platelet morphology and in organization of the actin cytoskeleton. For
example, platelet attachment to fibrinogen or von Willebrand factor is
accompanied by progressive actin polymerization, filopodial and
lamellipodial extension, and eventually full spreading (7-9). Recent
work has identified at least three sets of signaling responses triggered by ligand binding to
IIb
3 that
potentially mediate these changes. One, detectable within seconds of
cell attachment, involves tyrosine phosphorylation and activation of
the Syk protein tyrosine kinase (10). The others are detectable only
after 30-120 s and involve tyrosine phosphorylation and activation of
the FAK tyrosine kinase and tyrosine phosphorylation of the integrin
3 tail (11, 12). In sharp contrast to phosphorylation of
FAK and
3, Syk phosphorylation and activation are
unaffected by blockade of actin filament barbed ends by cytochalasin D,
suggesting that actin polymerization is not required. In fact, two of
the most prominent Syk substrates in platelets, Vav1 and SLP-76, may
function to promote actin polymerization downstream of
IIb
3.
IIb
3
signaling, Nck is an adapter that can influence cytoskeletal events by
recruiting a Rac effector, the PAK1 serine-threonine kinase, to plasma
membrane adhesion sites (17). The proline-rich region of SLP-76
interacts with the SH3 domain of Gads, a Grb2-like adapter (18). The
SH2 domain of SLP-76 interacts with tyrosine-phosphorylated SLAP-130
(19, 20), an adapter that can bind in turn through an FP4 polyproline
motif to the EVH1 domain of VASP, an actin-binding protein implicated
in regulating actin polymerization within lamellipodia and focal
adhesions (4, 21, 22). Because all of these SLP-76 binding partners are
present in platelets, SLP-76 might be in a pivotal position to transmit
IIb
3 signals to the cytoskeleton. Consistent with this hypothesis, SLP-76-deficient mice exhibit a
bleeding diathesis and defects in platelet function, including reduced
spreading on fibrinogen. Retroviral reconstitution of SLP-76
/
bone marrow cells with SLP-76 corrects these
platelet defects (23).
View larger version (26K):
[in a new window]
Fig. 1.
Domain structure of SLP-76. Portions of
SLP-76 that interact directly with Vav1, Nck, Gads, and SLAP-130 are
indicated. The numbers orient the reader to the approximate
location of domains in the linear sequence. Arrows point to
potential effectors of Vav1, Nck, and SLAP-130 that are evaluated in
this study.
Based on these considerations, the present study was carried out to
determine more precisely the role of SLP-76 in outside-in IIb
3 signaling. Using a CHO cell model
system and human platelets, we show here that SLP-76 becomes
tyrosine-phosphorylated in fibrinogen-adherent cells in an
IIb
3 and Syk-dependent
manner. Furthermore, SLP-76 enhances lamellipodia formation triggered
by an outside-in signaling pathway that involves
IIb
3, Syk, Vav, and Rac. Whereas SLP-76 does not affect Rac activation, it does enhance
adhesion-dependent activation of PAK. Moreover, through its
interactions with SLAP-130, SLP-76 may help to localize VASP to
IIb
3 adhesion sites at the edges of
spreading platelets. Thus, SLP-76 functions to relay signals from
IIb
3 to actin.
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EXPERIMENTAL PROCEDURES |
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Reagents--
Monoclonal antibodies: anti-Syk (4D10) was from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-phosphotyrosine
(4G10 and PY20) from Upstate Biotechnology (Lake Placid, NY) and
Transduction Laboratories (Lexington, KY), respectively; anti-VASP from
ImmunoGlobe GmbH, Groostheim, Germany; anti-Vav, anti-FLAG M2 and
anti-Rac from Upstate Biotechnology; and anti-LIBS6 Fab, an integrin
3 activating antibody, was a gift from Mark Ginsberg
(Scripps). Rabbit polyclonal antibody to Nck (5547) was a gift from
David Schlaepfer (Scripps Research Institute). Polyclonal anti-PAK1 antibody was described previously (24). Sheep polyclonal antibodies to
SLP-76 and SLAP-130 were described previously (25). Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad
Laboratories (Hercules, CA). Cy-5-conjugated anti-mouse and anti-sheep
IgG were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA); fluorescein isothiocyanate-conjugated anti-mouse IgG was from
BIOSOURCE International Inc. (Camarillo, CA).
Rhodamine-phalloidin was from Molecular Probes (Eugene, OR), purified
human fibrinogen from Enzyme Research Laboratories Inc. (South Bend,
IN), bovine serum albumin and sodium orthovanadate from Fisher. Protein
A and Protein G-Sepharose were from Amersham Pharmacia Biotech
(Piscataway, NJ), glutathione-agarose and leupeptin from Sigma,
Pefabloc and aprotinin from Roche Molecular Biochemicals (Indianapolis,
IN), and LipofectAMINE from Life Technologies, Inc.
Cell Culture, Plasmids, and Transfections--
A5 CHO cells,
which stably express IIb
3, were
maintained in complete Dulbecco's modified Eagle's medium containing
10% fetal calf serum (26). The following mammalian expression plasmids were used: pEMCV/Syk and pEF/Myc-Vav1 (27); pSAP/CD8-
(a chimera containing the extracellular and transmembrane domain of CD8 fused to
the cytoplasmic domain of the
subunit of Fc
R1, Ref. 28); pcDNA3/HA-Rac1 (24); and pEF/FLAG-SLP-76 (wild-type and mutants) (15). Subconfluent cells in 100 mm dishes were transfected with up to 4 µg of plasmid DNA using LipofectAMINE according to the manufacturer's instructions. When necessary, pcDNA3 was added to
maintain equal amounts of DNA in all transfections. Twenty-four hours
later, the concentration of fetal calf serum was lowered from 10 to
0.5%, and cells were cultured for an additional 24 h before use
in functional assays.
CHO Cell Binding to Fibrinogen--
A5 cell transfectants were
resuspended using trypsin-EDTA, washed twice with Dulbecco's modified
Eagle's medium, resuspended to 3 × 106 cells/ml in
Dulbecco's modified Eagle's medium, and incubated for 45 min with 20 µM cyclohexamide. To test the effects of binding of
soluble fibrinogen to IIb
3, cells were
incubated for an additional 15 min with 250 µg/ml fibrinogen, in the
presence or absence of 0.5 mM MnCl2 (to
activate integrins) or 10 µM Integrilin or 0.5 mM EDTA (to inhibit ligand binding to
IIb
3) (29). Cells were then washed with
phosphate-buffered saline, lysed for 10 min in ice-cold RIPA buffer
containing 1 mM sodium vanadate, 0.5 Mm leupeptin, 0.25 mg/ml Pefabloc, and 5 µg/ml aprotinin, clarified by sedimentation in
a microcentrifuge, and subjected to immunoprecipitation and Western
blotting (27, 28). For studies of cells adherent to fibrinogen,
bacterial tissue culture plates were precoated with 5 mg/ml bovine
serum albumin (BSA) or 100 µg/ml fibrinogen. After blocking for
2 h at room temperature with heat-denatured BSA, 4.5 × 106 cells in 1.5 ml were added to each plate and incubated
for 45 min at 37 °C in a CO2 incubator. After 60 min,
nonadherent cells from the BSA plates were sedimented at 14,000 rpm for
3 s in a microcentrifuge and lysed immediately in RIPA buffer.
Cells adherent to fibrinogen were rinsed twice in phosphate-buffered
saline, lysed on the plates directly, and then processed for
immunoprecipitation and Western blotting. When coprecipitation of two
or more proteins was being assessed, the cells were lysed in buffer
containing 0.5% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.4, and inhibitors.
Immunoprecipitation and Western Blotting-- Equal amounts of each lysate, typically 250-500 µg of protein in 500 µl, were immunoprecipitated with the indicated antibodies, subjected to electrophoresis on 7.5% SDS-polyacrylamide gels, and transferred onto nitrocellulose (27, 28). Membranes were blocked with 3% nonfat dry milk and probed with the indicated primary and horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected by enhanced chemiluminescence. Blots were scanned in a Hewlett-Packard ScanJet 5300C scanner, and labeled bands were quantified by calibrated densitometry using NIH Image software.
Rac GTPase and PAK Kinase Assays-- Endogenous PAK was immunoprecipitated from Nonidet P-40 lysates and PAK kinase activity was determined using an in gel kinase assay with myelin basic protein as substrate (24). Active Rac was monitored using a pull-down assay involving a recombinant fragment of PAK that binds specifically to Rac-GTP (24).
Confocal Microscopy-- Twenty-four hours after CHO cell transfection, cells were plated on coverslips coated with 100 µg/ml fibrinogen and incubated in the presence of 0.5% fetal calf serum for an additional 18 h at 37 °C in a CO2 incubator. Adherent cells were then fixed in 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with primary antibodies to transfected proteins, secondary antibodies conjugated with fluorescein isothiocyanate or Cy5, and rhodamine-phalloidin to stain F-actin. Lamellipodia formation in fibrinogen-adherent cells was quantified using an MRC 1024 Bio-Rad laser scanning confocal imaging system and expressed as the percentage of cells that contained one or more lamellipodium (27). Scoring was carried out in a blinded fashion by two independent observers, and at least 100 transfected cells were examined for every transfection combination in each experiment. Digital images were prepared using Adobe Photoshop, version 5.5.
Platelet Studies--
Fresh whole blood was obtained from normal
volunteers, anticoagulated with ACD, and washed platelets were prepared
(8). To enable soluble fibrinogen to bind to
IIb
3, platelets were resuspended to
3.5 × 108/ml and incubated for 5 min at 37 °C with
250 µg/ml fibrinogen in the presence of the
3
activating antibody, LIBS-6 Fab (150 µg/ml). As a control, parallel
samples were incubated in the presence of Ro 43-5054 (10 µM), a selective
IIb
3
antagonist that blocks fibrinogen binding (30). To study the effects of
adhesion to fibrinogen, platelets were incubated in fibrinogen-coated
or BSA-coated dishes in the presence of 2 units/ml apyrase for 30 min
(31). Then cells adherent to fibrinogen or suspended over BSA were
lysed in Nonidet P-40 buffer, the lysates were immunoprecipitated with antibodies to SLP-76 or SLAP-130, and Western blots were probed with
antibodies to phosphotyrosine, SLP-76, SLAP-130, or VASP (23). For
confocal microscopy, cells were plated onto fibrinogen-coated coverslips at 107/ml for 45 min at 37 °C in the presence
or absence of 200 nM phorbol myristate acetate (PMA) to
enhance spreading. Platelets were fixed, permeabilized, stained with
antibodies to SLAP-130 and VASP, and examined by confocal microscopy
(8).
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RESULTS |
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IIb
3 Signals to SLP-76--
In
platelets, fibrinogen binding to
IIb
3
leads to rapid tyrosine phosphorylation of SLP-76 (23). To study the
mechanisms and consequences of integrin signaling to SLP-76, this
adapter was transiently expressed in A5 CHO cells, which stably express
IIb
3. To determine whether
IIb
3 signals to SLP-76 in CHO cells as in
platelets, A5 cells were first incubated with soluble fibrinogen in the
presence of 0.5 mM MnCl2, an extrinsic
activator of integrins (32). Despite successful ectopic expression of
SLP-76, no tyrosine phosphorylation of SLP-76 was observed. In
contrast, if SLP-76 was cotransfected with Syk, there was now a marked
increase in tyrosine phosphorylation of SLP-76 when fibrinogen binding
was induced by MnCl2 (Fig.
2A). This response as well as
the tyrosine phosphorylation of Syk itself were inhibited by compounds
that block fibrinogen binding to
IIb
3,
including the selective
IIb
3 antagonist,
Integrilin, and the divalent cation chelator, EDTA. In the experiment
shown in Fig. 2A, fibrinogen binding was carried out for 15 min, but similar results were obtained at 30 s, the earliest time
point tested. Identical results were obtained if fibrinogen binding was
induced by a
3-specific-activating antibody, anti-LIBS6
Fab, instead of MnCl2 (not shown). In addition, tyrosine phosphorylation of SLP-76 was observed when cells were plated on
immobilized fibrinogen, even in the presence of 10 µM
cytochalasin D, an inhibitor of actin polymerization (Fig.
2B). As previously observed (27), cytochalasin D blocked
IIb
3-dependent tyrosine phosphorylation and activation of FAK but not Syk. These results indicate that SLP-76 is situated downstream of
IIb
3 and Syk in a signaling pathway that
becomes activated by fibrinogen binding, independent of actin
polymerization.
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In antigen-stimulated T cells, SLP-76 phosphorylation is dependent on
signals generated through T cell receptor subunits that bear
ITAM motifs, and it is abolished by phenylalanine substitution of SLP-76 tyrosines 113, 128, and 145 (SLP-76-(3YF), see Ref. 15).
To evaluate whether these tyrosine residues are involved in
IIb
3 signaling, SLP-76-(3Y
F) was
expressed with Syk in A5 CHO cells. Despite levels of expression
similar to wild-type SLP-76, adhesion-dependent tyrosine
phosphorylation of SLP-76-(3Y
F) was reduced by an average of 66%
(three experiments). Under the same conditions,
tyrosine-phosphorylation of SLP-76-(3Y
F) induced by overexpression
of an ITAM-bearing CD8
chimera was virtually abolished (Fig.
2C). In addition, adhesion-dependent tyrosine phosphorylation was still observed with a SLP-76 truncation mutant consisting of the N-terminal half of the molecule (amino acids 1-266)
but not with SLP-76-(1-266)-(3Y
F) or with a mutant consisting of
SLP-76 residues 157-533 (Fig. 2D). Thus, SLP-76 tyrosine
residues 113, 128, and/or 145 are major targets of
Syk-dependent phosphorylation during outside-in
IIb
3 signaling. However, in contrast to
ITAM-based signaling, one or more additional tyrosines in SLP-76 are
phosphorylated in response to integrin ligation.
SLP-76 Enhances Lamellipodia Formation during
IIb
3-dependent Cell
Adhesion--
SLP-76-deficient mouse platelets exhibit reduced
spreading on fibrinogen (23). One early event during spreading is
lamellipodia formation, a response that involves
IIb
3, Syk, Vav1 and Rac (27, 33). Because
SLP-76 is situated downstream of
IIb
3 and
Syk, we asked whether SLP-76 expression affected the formation of
lamellipodia. Twenty-four hours after transfection of Syk, Vav1, and
SLP-76, A5 cells were plated on fibrinogen-coated coverslips for
another 18 h and then examined by confocal microscopy.
Identification of cells expressing the recombinant proteins was
accomplished by three color fluorescence analysis, as shown in Fig.
3A. As observed previously
(27), the percentage of fibrinogen-adherent CHO cells that exhibited
one or more lamellipodium was greatly increased by coexpression of Syk
and Vav1, and this response was dependent on the amount of Vav1
expressed (Fig. 3B). Even in the presence of Syk alone
however, SLP-76 caused a modest but consistent increase in lamellipodia
formation (p < 0.03), and the enhancing effect of
SLP-76 was maintained in the presence of Vav1 (p < 0.003, Fig. 3B).
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Because tyrosine-phosphorylated SLP-76 can bind directly to the SH2
domain of Vav1, we wondered if this interaction was necessary for the
SLP-76 effect on lamellipodia. In the SLP-76-(3YF) mutant, the
tyrosines needed for interaction with Vav1 (or Nck) are not present
(15, 16). Indeed, unlike the case for wild-type SLP-76, Vav1, and Nck
were not observed in SLP-76-(3Y
F) immunoprecipitates from
fibrinogen-adherent A5 cells (Fig. 4).
Despite this, SLP-76-(3Y
F) was fully able to enhance lamellipodia
formation induced by Syk and Vav1 (Fig. 3B). Moreover, a
SLP-76 deletion mutant (
224-244) that lacks the polyproline binding
site for Gads and a mutant (R448K) that abrogates the SLP-76 SH2
interaction with SLAP-130 (34) were similarly able to enhance
lamellipodia formation (Fig. 3B). On the other hand, SLP-76
truncation mutants containing either the N-terminal half () or
the C-terminal half () of the molecule failed to support
lamellipodia formation (Fig. 3B). Taken together, these
results indicate that one function of SLP-76 is to enhance lamellipodia
formation during
IIb
3 signaling.
Furthermore, at least when ectopically expressed, the loss of any
single known adapter function of SLP-76 does not prevent this
effect.
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Downstream Effectors of SLP-76 in
IIb
3 Signaling--
Lamellipodial
extension involves actin polymerization in localized regions of the
cortical cytoskeleton. Because SLP-76 is a multivalent adapter, it has
the potential to influence the subcellular localization or function of
several proteins that have been implicated in regulating cytoskeletal
dynamics. We focused attention on three such proteins, Rac, PAK, and
VASP. Rac promotes actin polymerization and lamellipodial extension in
many cell types, including platelets and A5 CHO cells (27, 35, 36). In
platelets, Rac may be activated to its GTP-bound form by Vav1 (37).
Because the Rac exchange activity of Vav1 increases following its
tyrosine phosphorylation (13, 38, 39), we examined whether SLP-76
expression influenced tyrosine phosphorylation of Vav1 or levels of
Rac-GTP. In A5 cells cotransfected with Syk and Vav1, tyrosine
phosphorylation of Vav1 increased 4-fold 30-45 min after cell adhesion
to fibrinogen, and cotransfection of SLP-76 had no effect on this
response (not shown). Moreover, although levels of Rac-GTP increased
10-fold upon adhesion of Syk/Vav1 transfectants to fibrinogen, SLP-76 did not alter this response (Fig. 5).
Thus, although SLP-76 enhances a Rac-dependent
lamellipodial response triggered by
IIb
3,
it does not do so by activating Rac. This suggests that SLP-76
functions downstream of Rac and/or independent of it.
|
Because wild-type SLP-76 interacted with Nck in fibrinogen-adherent A5
cells (Fig. 4), we asked whether SLP-76 expression increased the
activity of PAK, a Nck-binding partner and Rac effector (17). In the
presence of Syk and Vav1, adhesion of A5 cells to fibrinogen stimulated
more than a 3-fold increase in PAK activity (p < 0.01). However, in the presence of SLP-76,
adhesion-dependent PAK activity increased more than 10-fold
(p < 0.01) (Fig. 6,
A and C). On the other hand, expression of
SLP-76-(3YF) had no effect on PAK activity, consistent with a
requirement for an interaction between SLP-76 and Nck for this response
to take place (Fig. 6, B and C). Thus, one effect
of wild-type SLP-76 in
IIb
3 signaling is
to increase the activity of PAK. However, the results with the
SLP-76-(3Y
F) mutant indicate that this effect is not essential for
enhancement of lamellipodia formation, at least when SLP-76-(3Y
F) is
ectopically expressed (Fig. 3B).
|
VASP is a prominent phosphoprotein in platelets (40), and it may
interact directly with SLAP-130 in lymphoid cells (22). Therefore, we
asked if there was any relationship between SLP-76, SLAP-130, and VASP
during outside-in IIb
3 signaling in
platelets. Human platelets were incubated with soluble fibrinogen in
the presence or absence of the LIBS-6 Fab
3-activating
antibody. Then Nonidet P-40 cell lysates were immunoprecipitated with
antibodies to SLP-76 or SLAP-130. Western blots of SLP-76
immunoprecipitates revealed a prominent 130 kDa tyrosine-phosphorylated
band that comigrated with SLAP-130 (Fig.
7A). Fibrinogen binding to the platelets in response to LIBS-6 Fab caused an increase in tyrosine phosphorylation of this band as well as of SLP-76, and this response was inhibited by RO 43-5054, a selective
IIb
3 antagonist (30). Similar results
were obtained if lysates were immunoprecipitated with SLAP-130 instead
of SLP-76 (Fig. 7B). In both cases, the binding of soluble
fibrinogen to the platelets caused little or no increase in the amount
of SLAP-130 that coimmunoprecipitated with SLP-76, suggesting that the
main effect of
IIb
3 ligation under these
conditions was on tyrosine phosphorylation of these adapters (Fig. 7,
A and B). Thus, SLP-76 and SLAP-130 interact in
platelets and their states of tyrosine phosphorylation are modulated by
outside-in signaling through
IIb
3.
|
For SLAP-130 and VASP to play a coordinate role in
IIb
3-dependent platelet
spreading, it would be predicted that they interact with each other and
localize within lamellae at the platelet periphery. To assess the
subcellular localization of SLAP-130 and VASP, platelets were allowed
to attach to fibrinogen-coated coverslips for 45 min in the presence or
absence of PMA, the latter added to enhance actin-rich lamellae and
spreading (8). Under these conditions, a portion of total cellular
SLAP-130 and VASP colocalized to a rim at the edges of the spread
platelets (Fig. 8A). SLP-76
localization could not be evaluated because our antibodies are not
suitable for immunofluorescence. To determine whether SLAP-130 and
SLP-76 form a physical complex with VASP, platelets were allowed to
adhere to fibrinogen for 30 min, and Nonidet P-40 lysates were
immunoprecipitated with an antibody to SLAP-130 or SLP-76. VASP could
be detected in SLAP-130 and SLP-76 immunoprecipitates from
fibrinogen-adherent platelets, but there was substantially less VASP in
immunoprecipitates from nonadherent platelets (Fig. 8B).
These results indicate that platelet adhesion to fibrinogen promotes
formation of a multimolecular complex involving SLP-76, SLAP-130, and
VASP. They suggest that one function of SLP-76 in
IIb
3 signaling may be to recruit SLAP-130 and VASP to adhesion sites at the edges of spreading platelets.
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DISCUSSION |
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The goal of this study was to determine how SLP-76 functions
during IIb
3 signaling to influence cell
shape and cytoskeletal organization. The study was motivated by recent
observations that normal human and mouse platelets show increased
tyrosine phosphorylation of SLP-76 in response to fibrinogen binding to
IIb
3, suggesting that SLP-76 is involved
in outside-in signaling through this integrin. Furthermore, mouse
platelets deficient in SLP-76 spread poorly on fibrinogen and display
reduced overall tyrosine phosphorylation, and both abnormalities are
corrected by in vivo reconstitution of platelets with SLP-76
(23). Here we used a CHO cell model system and human platelets to
evaluate the adapter functions of SLP-76 in the context of outside-in
IIb
3 signaling. The main conclusions are
as follows. 1) Ligation of
IIb
3, whether
with soluble or immobilized fibrinogen, induces rapid tyrosine
phosphorylation of SLP-76 but only in the presence of Syk. 2)
Integrin-dependent SLP-76 phosphorylation involves
tyrosines 113, 128, 145 as well as other tyrosine residues in the
protein. 3) Phosphorylation of SLP-76 does not require actin
polymerization. Rather, SLP-76 expression enhances an actin-driven
response, lamellipodia formation, that is triggered by a pathway
involving
IIb
3, Syk, Vav1, and Rac. 4)
Whereas SLP-76 interacts with Vav1, enhancement of lamellipodia formation by SLP-76 is not because of increased tyrosine
phosphorylation of Vav1 or activation of Rac. Instead, it may be
because of interactions of SLP-76 with other proteins, including Nck
and SLAP-130. For example, interaction of SLP-76 with Nck may account
for the enhanced adhesion-dependent activation of PAK
observed in SLP-76 transfectants. Furthermore, interaction of SLP-76
with SLAP-130 may promote localization of SLAP-130 and VASP to the
periphery of fibrinogen-adherent platelets. Thus, SLP-76 functions to
relay signals between
IIb
3 and effectors that influence the actin cytoskeleton. In addition to defining a key
pathway linking
IIb
3 to changes in
F-actin, these results provide a molecular explanation for the integrin
phenotype of SLP-76-deficient platelets.
Integrin Signaling to SLP-76--
SLP-76 is one of numerous
proteins that become tyrosine phosphorylated in response to ligand
binding to IIb
3. These can be grouped
based on their temporal profiles and whether actin polymerization is
required (6). For example, Syk and Vav1 become phosphorylated within
seconds of fibrinogen binding to
IIb
3 in
platelets or A5 CHO cells, and this response is independent of actin
polymerization or cell aggregation (10, 27, 37). In contrast, FAK,
Hic-5, SHIP, and the integrin
3 cytoplasmic tail become
phosphorylated later, only after actin polymerization and cell
aggregation or spreading have occurred (11, 41-45). The rapid tyrosine
phosphorylation of SLP-76 after fibrinogen binding to platelets or A5
CHO cells and its resistance to cytochalasin D places SLP-76 in the
group with Syk and Vav1. Indeed, the present study establishes that
IIb
3-dependent SLP-76
phosphorylation requires Syk. Moreover, SLP-76 functions with Syk and
Vav1 to enhance lamellipodia formation. Thus, SLP-76 is situated
downstream of
IIb
3 in an early phase of
outside-in signaling.
Studies with SLP-76-(3YF), which lacks tyrosines 113, 128, and 145, clarify the importance of these residues in integrin signaling and
suggest possible differences in SLP-76 function downstream of integrins
and immune response receptors. These tyrosines are necessary for the
binding of SLP-76 to the SH2 domains of Vav1 and Nck (Refs. 16, 46 and
Figs. 1 and 4). In contrast to SLP-76 phosphorylation in response to T
cell receptor ligation (46) or to overexpression of an ITAM-bearing
CD8/
chimera in CHO cells, we observed residual tyrosine
phosphorylation of SLP-76-(3Y
F) in response to fibrinogen binding to
IIb
3 (Figs. 2C and 4). This
indicates that one or more additional tyrosines in SLP-76 are targeted
by a tyrosine kinase, possibly Syk, during
IIb
3 signaling. However, the identity and
function of these tyrosines remain to be determined.
In most T cells, a T cell-specific Src family kinase, Lck, and the Syk
homologue, ZAP-70, are required for SLP-76 phosphorylation. In
platelets and A5 CHO cells, Syk is the relevant ZAP-70 family member,
and its optimal activation is dependent on one or more Src family
kinases other than Lck (28, 47). Thus, differences in SLP-76
phosphorylation downstream of ITAM receptors and
IIb
3 might be caused by, in part,
differences in substrate specificities or subcellular localization of
related, but not identical, protein tyrosine kinases. However, this
cannot explain the differences we observed in SLP-76 phosphorylation
downstream of
IIb
3 and CD8/
in CHO
cells because both membrane receptors utilized Syk in the same cellular
background. Murine platelets express
IIb
3 and the ITAM-bearing collagen receptor, GPVI/Fc
, and both are functionally linked to SLP-76 (47). Because retroviruses can be used to
reconstitute SLP-76 in hematopoietic cells of SLP-76
/
mice (23), it should be possible to determine the functional importance
of specific SLP-76 tyrosine residues downstream of
IIb
3 and GPVI/Fc
by introducing SLP-76
tyrosine mutants into SLP-76
/
platelets.
Integrin Signaling from SLP-76 to the Actin Cytoskeleton--
The
spreading defect in fibrinogen-adherent SLP-76/
mouse
platelets (23), and the involvement of SLP-76 in actin cap formation in
antigen-activated T cells (16), suggest that one function for SLP-76 in
platelets may be to relay
IIb
3 signals to
the cytoskeleton. This idea is supported by the current findings that SLP-76 phosphorylation occurred rapidly following A5 CHO cell adhesion
to fibrinogen, and this response was independent of actin polymerization (Fig. 2B). Furthermore, SLP-76 expression in
CHO cells enhanced lamellipodia formation, an event dependent on
localized actin polymerization (Fig. 3B). Although caution
is warranted in extrapolating results from studies of ectopically
expressed SLP-76 in CHO cells to the function of SLP-76 in platelets,
these results indicate that SLP-76 has the potential to participate in
signaling from
IIb
3 to the platelet
cytoskeleton, and they provide an explanation for the spreading defect
in SLP-76
/
platelets
How does SLP-76 enhance lamellipodia formation? One possibility we
considered is that when tyrosine-phosphorylated SLP-76 binds to Vav1,
it may promote either the guanine nucleotide exchange activity of Vav1
or its proximity to effectors, such as Rac. SLP-76 coimmunoprecipitates
with Vav1 in activated T cells (16) and in collagen-stimulated
platelets (47), implying that it may be close enough to Vav1 to
directly influence its function. In addition, Vav1 can activate Rac and
promote lamellipodia formation downstream of
IIb
3 (Fig. 3B, Ref. 27).
Despite this circumstantial evidence, however, we found that expression
of SLP-76 in A5 cells affected neither the
integrin-dependent tyrosine phosphorylation of Vav1, which
regulates Vav1 exchange activity (13, 38, 39), nor the
adhesion-dependent activation of Rac (Fig. 5A).
Moreover, lamellipodia formation was still enhanced by SLP-76-(3Y
F),
despite the fact that it was unable to coimmunoprecipitate with Vav1
(Figs. 3B and 4). Therefore, enhancement of lamellipodia
formation by SLP-76 must involve pathways to the cytoskeleton that are
downstream of and/or independent of Rac. Studies of PAK and VASP
suggest that both possibilities may be correct.
PAK is a Rac effector that has been implicated in regulating actin
polymerization, and it is targeted to integrin-based adhesion sites by
binding to the central SH3 domain of Nck (17, 48). In this context,
adhesion of A5 CHO cells to fibrinogen stimulated the binding of Nck to
tyrosine-phosphorylated SLP-76 (Fig. 4). This was associated with an
increase in the kinase activity of PAK that was over and above the
activity observed in fibrinogen-adherent cells in the absence of SLP-76
(Fig. 6). Thus, one plausible scenario is that fibrinogen binding to
IIb
3 triggers activation of Syk followed
by tyrosine phosphorylation of SLP-76 and Vav1 in proximity to the
integrin. Then whereas Vav1 activates Rac, SLP-76 recruits Nck-PAK to
the adhesion site where PAK becomes activated by Rac, resulting in
lamellipodia formation. However, this cannot explain how
SLP-76-(3Y
F) enhanced lamellipodia formation in A5 CHO cells, because this mutant neither bound to Nck nor increased PAK activation (Figs. 4 and 6, B and C). Interestingly, mutation
of other single adapter sites in SLP-76 for Gads or SLAP-130 also
failed to prevent lamellipodial enhancement (Fig. 3B). We
speculate that when a single adapter region in SLP-76 is mutated, the
remaining regions may be able to compensate and promote cytoskeletal
changes, particularly if the mutant protein is expressed at high
levels, as in the CHO cell system. This interpretation is in keeping
with the observation that deletion of larger portions of SLP-76
eliminated its ability to enhance lamellipodia formation (Fig.
3B). It may be useful to determine whether portions of
SLP-76 not yet assigned an adapter function, including the SAM
domain or phosphotyrosines other than those at positions 113, 128, and
145, are involved in signal relay from
IIb
3 to actin.
VASP is an actin-bundling protein that regulates actin dynamics within
lamellipodia, possibly by bringing the Arp2/3 actin polymerization
machinery and profilin into proximity within integrin-based focal
complexes (4, 49-52). VASP has the potential to interact directly with
SLAP-130 in lymphocytes (22), where tyrosine-phosphorylated SLAP-130
interacts with SLP-76 (20, 53). Because VASP and SLAP-130 are expressed
in platelets, this suggested to us a possible link between
IIb
3, SLP-76, SLAP-130, and VASP. Indeed,
we found that the adhesion of human platelets to fibrinogen was
associated with 1) tyrosine phosphorylation of a pool of SLAP-130 that
coimmunoprecipitated with SLP-76 (Fig. 7), 2) coprecipitation of
SLAP-130 and SLP-76 with VASP (Fig. 8B), and 3)
colocalization SLAP-130 and VASP at the platelet periphery (Fig.
8A). Interestingly, VASP
/
murine platelets
exhibit increased fibrinogen binding in response to agonists, further
suggesting a close functional relationship between VASP and
IIb
3 (54, 55). Thus, further
investigation is warranted into the role of a SLP-76·SLAP-130·VASP
complex in
IIb
3 signaling to the
platelet cytoskeleton.
These studies, when taken together with recent results obtained with
platelets from SLP-76 knockout mice (23), establish that SLP-76 relays
signals from IIb
3 to actin by virtue of
the regulated interaction of SLP-76 with multiple downstream effectors that modify the assembly and localization of actin filaments. This
model does not require that a given SLP-76 molecule interact simultaneously with all possible binding partners in the relay, because
many SLP-76 molecules could be drawn into a large signaling organelle
nucleated by
IIb
3 within focal complexes
at sites of platelet adhesion. In this context, antibody-mediated
clustering of integrin
4
1 in lymphocytes
stimulates tyrosine phosphorylation of SLP-76 and SLAP-130, and
overexpression of SLAP-130 enhances lymphocyte migration through
fibronectin-coated filters (56). Thus, delineation of the function of
SLP-76 in platelet
IIb
3 signaling may
have wider implications for integrin signaling in other hematopoietic cells.
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ACKNOWLEDGEMENTS |
---|
We thank David Phillips (Cor Therapeutics, Inc., South San Francisco, CA) for providing Integrilin, Beat Steiner (Roche, Basel, Switzerland) for RO 43-5054, David Schlaepfer (Scripps) for the anti-Nck antibody, and Mark Ginsberg (Scripps) for the LIBS6 Fab.
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FOOTNOTES |
---|
* These studies were supported by research grants from the National Institutes of Health (to M. A. S., G. A. K., S. J. S) and postdoctoral fellowships from the Deutsche Forschungsgemeinschaft (to A. O.) and the Human Frontiers Science Program (to M. A. P).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: Dept. of Vascular Biology, The Scripps Research Inst., 10550 North Torrey Pines Rd., VB-5, La Jolla, CA 92037. Tel.: 858-784-7148; Fax: 858-784-7422; E-mail: shattil@scripps.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M010639200
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ABBREVIATIONS |
---|
The abbreviations used are: ECM, extracellular matrix; BSA, bovine serum albumin; PMA, phorbol myristate acetate; RIPA, radioimmune precipitation assay buffer; CHO, Chinese hamster ovary cells; FAK, focal adhesion kinase.
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