From the Department of Pharmacology, Mansfield Road,
Oxford University, Oxford OX1 3QT, United Kingdom, the
¶ Department of Internal Medicine, Physiology and Biophysics and
the Interdisciplinary Immunology Program, University of Iowa College of
Medicine, Iowa City, Iowa 52242, the ** National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom, and
Roche Biosciences,
Palo Alto, California 94304
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ABSTRACT |
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Collagen-related peptide (CRP), a
collagen homologue, induces platelet activation through a tyrosine
kinase-dependent pathway, leading to sequential tyrosine
phosphorylation of Fc receptor (FcR) SLP-76 (SH21
domain-containing leukocyte protein of
76 kDa) was identified by association with the SH3 domain
of the Grb2 adapter protein in T cells and becomes
tyrosine-phosphorylated upon T cell receptor (TCR) stimulation (1).
SLP-76 has three potential tyrosine phosphorylation sites within its
amino terminus region: Tyr113, Tyr128, and
Tyr145. Tyr113 and Tyr128 have a
consensus binding site for the SH2 domain of Vav (DYESP) (2-5) and are
heavily tyrosine-phosphorylated following TCR engagement, whereas
Tyr145, which falls in the sequence DYEPP, is
phosphorylated to a lesser extent (6). SLP-76 also contains a central
proline-rich region that mediates the association with Grb2 (7) and a
carboxyl-terminal SH2 domain that binds to at least two
tyrosine-phosphorylated proteins, SLAP-130
(SLP-76-associated phosphoprotein
of 130 kDa) (8), a 62-kDa protein, and an uncharacterized
serine/threonine kinase after TCR engagement (7). SLP-76 is believed to
be an essential adapter protein in T cells. Overexpression of SLP-76 results in an enhancement of TCR-mediated induction of nuclear factor
of activated T cell and interleukin-2 promoter activity (3, 5-7, 9,
10). More recently, lack of expression of SLP-76 in Jurkat cells
demonstrated that SLP-76 is necessary for tyrosine phosphorylation of
phospholipase C- The three tyrosine phosphorylation sites, the proline-rich region, and
the SH2 domain of SLP-76 have all been shown to be important for the
regulation of T cell interleukin 2 production (10). The inducible
tyrosine phosphorylation of SLP-76 is mediated by ZAP-70 or Syk in COS
cells (9) and rat basophilic leukemia cells (14), respectively. The
mechanism by which SLP-76 is phosphorylated by ZAP-70 or Syk is not known.
We have previously reported the association of tyrosine-phosphorylated
SLP-76 with the SH3 domain of Grb2 in platelets in response to
stimulation of the low affinity IgG immunoreceptor Fc SLP-76 was recently reported to be a crucial adapter protein in
collagen-stimulated platelets, since aggregation and tyrosine phosphorylation of PLC- Antibodies and Reagents--
A CRP
(GCP*(GPP)10GCP*G; single amino acid code P* represents
hydroxyproline) was cross-linked via cysteine residues as described previously (23); CRP was kindly donated by Dr. M. Barnes (Cambridge, UK). Collagen (native collagen fibrils from equine tendons) was from
Nycomed (Munich, Germany). Fc Preparation and Stimulation of Platelets--
Human platelets
were isolated from blood taken on the day of the experiment as
described previously (27). Mouse platelets were prepared as described
previously (20). Stimulations were performed at 37 °C in the
presence of 1 mM EGTA and 10 µM indomethacin with continuous stirring at 1,200 rpm. Platelets were stimulated with 3 µg/ml CRP, 30 µg/ml collagen, or 1 unit/ml thrombin for 90 s.
Platelets were stimulated via Fc GST Precipitation, Immunoprecipitation, and
Immunoblotting--
Platelets were lysed with an equal volume of lysis
buffer (2% Nonidet P-40, 300 mM NaCl, 20 mM
Tris, 10 mM EDTA containing 2 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 µg/ml
pepstatin A, pH 7.3). Insoluble cell debris was removed by
centrifugation. Cell lysates were precleared with glutathione-agarose
or Protein A-Sepharose for GST precipitation and immunoprecipitation,
respectively. For some experiments, antibodies were covalently linked
to Protein A-Sepharose as described previously (28). For GST
precipitation, lysates were incubated with 5 µg of fusion protein
immobilized on agarose. Endogenous SLP-76 was immunoprecipitated using
4 µg of anti-SLP-76 mAb. The resulting protein complexes and
immunoprecipitates were resolved by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride
(PVDF) membranes. Immunoblotting was carried out as described
previously (24) with protein detection by enhanced chemiluminescence.
Sequential immunoprecipitation was performed following kinase assay as
described previously (29).
In Vitro Kinase Assay--
Protein immunoprecipitations or
precipitations were submitted to kinase assay as described (16).
Proteins were separated by SDS-PAGE and transferred to PVDF membranes.
Membranes were treated with 1 M KOH for 1 h at
55 °C to cleave serine/threonine phosphorylation and then subjected
to autoradiography and immunoblotting.
GST Fusion Proteins--
Wild type SLP-76 (1) and the triple
mutant SLP-76 YYY/FFF (5) were used as template for polymerase chain
reaction using the following primers: SLP-TyrF (5'-ATT GGA
TCC GGG GGT TGG TCG TCC TTT GAA-3') and SLP-TyrR (5'-ATT
CCC GGG GCT TCC TCG TCA TTG GAG GG-3'). The polymerase
chain reaction products were expressed in pGEX-2T (Amersham Pharmacia
Biotech, St. Albans, UK) as described previously (30).
Measurements of Cytosolic Ca2+ Levels--
Mouse
platelets (108 cells/ml) were loaded with 1 µM Fura-2/AM for 30 min at 37 °C in RPMI 1640 medium
containing 1% fetal calf serum and resuspended at 2 × 108 cells/ml in Hepes-buffered salt solution (135 mM NaCl, 5 mM KCl, 10 mM Hepes, 1.2 mM CaCl2, 1.2 mM MgCl2)
containing 1% fetal calf serum. Stimulation and measurements were
performed at 37 °C. Cytosolic Ca2+ levels were measured
as described previously (31). The intracellular Ca2+
concentration was estimated using the equation described by Grynkiewicz et al. (32): (R SLP-76 Is Involved in Early Signaling Events Induced by
CRP--
We have previously reported that Fc
Further studies were performed with CRP, since this is a powerful
agonist for GPVI but is unable to bind to
SLP-76 Is Associated with Lyn and a 130-kDa Phosphoprotein
following Platelet Stimulation--
SLP-76 co-immunoprecipitated with
a tyrosine phosphoprotein of 130 kDa following stimulation by CRP,
collagen, and thrombin (Fig. 1A). Cross-linking of Fc
Immunoprecipitation of SLP-76 using an antibody covalently linked to
Protein A-Sepharose revealed the presence of a tyrosine-phosphorylated band of approximately 55 kDa (Fig. 1C) that was sometimes
resolved as a doublet. This band was hidden by the IgG heavy chain band under standard immunoprecipitation conditions. The 55-kDa band was
present in resting platelets, and its level of tyrosine phosphorylation increased with stimulation of platelets by CRP. It was identified as
the tyrosine kinase Lyn by immunoblotting (Fig. 1C). There was a small increase in association with SLP-76 following CRP stimulation, although this was less than the increase in tyrosine phosphorylation. Immunoblotting for Grb2 revealed the presence of a
similar level of the adapter protein co-immunoprecipitating with SLP-76
in resting and CRP-stimulated platelets (not shown). This is likely to
be mediated through the SH3 domain of Grb2 as previously shown in
Fc SLP-76 Is Associated with Lyn and SLAP-130 through Its SH2
Domain--
We used a fusion protein containing the SH2 domain of
SLP-76, GST-SLP-SH2, to locate the binding site of Lyn and the
component(s) of the 130-kDa protein. GST alone, used as control, did
not bind any tyrosine-phosphorylated proteins (Fig.
2). GST-SLP-SH2 precipitates two major
tyrosine-phosphorylated proteins of 55 and 130 kDa (Fig. 2) in resting
platelets. Stimulation of platelets by CRP strongly increased the
degree of tyrosine phosphorylation of 130-kDa protein that is
precipitated by GST-SLP-SH2, whereas the level of phosphorylation of
the 55-kDa protein underwent a small increase. Reprobing revealed Lyn
and SLAP-130 as components of these bands, respectively (Fig. 2). There
was a small increase in the association of Lyn and SLAP-130 in
CRP-stimulated lanes (Fig. 2). Association of SLAP-130 to SLP-76 under
basal conditions was also reported in T cells (8). Minor tyrosine-phosphorylated proteins of 90 and 75 kDa were also associated with GST-SLP-SH2 in CRP-stimulated platelets. The 75-kDa band was not
identified as Syk or Btk by immunoblotting, although either or both may
be below the detection sensitivity of the antibodies.
SLP-76 Tyrosine-phosphorylated in Vitro by a Member of the Src
Kinase Family--
Comparison of autoradiographs of kinase assays of
immunoprecipitated SLP-76 before and after KOH treatment indicated the
absence of serine/threonine kinase co-immunoprecipitating with SLP-76 in resting and CRP-stimulated platelets (not shown). Three major bands
of 130, 75, and 55 were phosphorylated in SLP-76 immunoprecipitates from resting platelets (Fig.
3A). The three bands of 130, 75, and 55 kDa correspond to the major tyrosine-phosphorylated bands observed in SLP-76 immunoprecipitates when immunoblotted for
phosphotyrosine (Fig. 3A). The level of tyrosine
phosphorylation of the 130-, 75-, and 55-kDa bands increased
dramatically in SLP-76 immunoprecipitates from CRP-stimulated
platelets. CRP stimulation also induced the appearance of three other
minor tyrosine-phosphorylated bands of 90, 60, and 38 kDa following
kinase assay. On a longer exposure, a doublet of 13/11.5 kDa could also
be seen in CRP-stimulated samples (not shown). The 90-kDa band was also
seen by anti-phosphotyrosine immunoblotting in Fig. 3A,
while the 60-kDa band could be seen in a longer exposure. The 75-kDa
band was identified as SLP-76 by immunoblotting. The remaining proteins
were identified through sequential immunoprecipitation. Following
in vitro kinase assay, the proteins co-immunoprecipitating
with SLP-76 were dissociated by boiling in the presence of 2% SDS.
Supernatant was diluted down to 0.1% SDS, and proteins from the
supernatant were immunoprecipitated with specific antibodies.
Components of the 13/11.5-, 55-, 60-, 90-, and 130-kDa radiolabeled
bands were identified as FcR
A kinase assay was also performed on proteins precipitated with
GST-SLP-SH2. This showed a similar profile of kinase activity as SLP-76
immunoprecipitates, except for the presence of a 38-kDa protein, an
uncharacterized doublet of 75 kDa and the lack of SLP-76 (Fig.
3B). The tyrosine-phosphorylated band of 38 kDa represented the fusion protein that was labeled during the process. Bands of 130, 75, and 53/56 kDa were tyrosine-phosphorylated under basal conditions.
The bands of 130 and 53/56 kDa correspond to SLAP-130 and Lyn,
respectively, and both proteins were found to undergo a further
increases in phosphorylation following stimulation (Fig. 3B). A weaker band of 60 kDa could be visualized by
immunoblotting for tyrosine phosphorylation on longer exposures in
CRP-stimulated platelets (Fig. 3B). This band has a similar
electrophoretic mobility to Fyn. The 75-kDa doublet displayed a similar
level of tyrosine phosphorylation in resting and CRP-stimulated platelets.
Immunoprecipitation of SLP-76, followed by kinase assay, was performed
on platelet lysates using a milder detergent, Brij 96 (compared with
Nonidet P-40). This revealed a marked increase in in vitro
tyrosine phosphorylation of FcR
SLP-76 co-immunoprecipitates with two members of the Src kinase family,
Lyn and Fyn, either or both of which could mediate the increase in
tyrosine phosphorylation observed in the in vitro kinase
assays. In order to investigate this, PP1, an inhibitor specific to Src
kinases (34), was added to the kinase assay. In the presence of 10 µM PP1, in vitro tyrosine phosphorylation of
SLP-76 and its co-immunoprecipitated proteins was abrogated under basal
and CRP-stimulated conditions in Nonidet P-40 or Brij 96 lysates (Fig.
5).
Tyrosine Phosphorylation of SLP-76 Is Abolished in Syk-deficient
Platelets--
The identity of the kinases underlying phosphorylation
of SLP-76 was investigated in knock-out mouse platelets.
Immunoprecipitation studies showed that SLP-76 is phosphorylated in
mouse platelets and that it associates with the same profile of
tyrosine-phosphorylated proteins as seen with human SLP-76 (not shown).
The increase in tyrosine phosphorylation of SLP-76 by CRP was not
altered in Lyn- or Fyn-deficient mouse platelets (Fig.
6, A and B).
In vitro phosphorylation of SLP-76, following
immunoprecipitation of these samples, was dramatically reduced in
Lyn-deficient platelets (Fig. 6A) but hardly altered in
Fyn-deficient platelets (Fig. 6B). This suggests that
in vitro tyrosine phosphorylation is mainly caused by Lyn but that this kinase is unlikely to be responsible for mediating in vivo phosphorylation of SLP-76 following stimulation by
CRP.
The role of Syk in SLP-76 tyrosine phosphorylation was carried out by
the study of platelets from Syk-deficient mice. In platelets of control
mice, SLP-76 exhibited an increase in tyrosine phosphorylation following CRP stimulation, which was abrogated in CRP-stimulated platelets from Syk-deficient mice (Fig.
7). An increase in tyrosine phosphorylation of the 130-kDa protein upon CRP activation could also
be seen with longer exposures, and this was also lost in the
Syk-depleted cells (data not shown).
SLP-76 possesses three N-terminal tyrosine phosphorylation sites,
Tyr113, Tyr128, and Tyr145. This
N-terminal region (amino acids 103-154) was expressed as a GST fusion
protein (GST-Tyr-WT) and was used as a substrate in kinase assays
performed on Lyn, Fyn, and Syk immunoprecipitates. A fusion protein
with the three tyrosine phosphorylation sites mutated to phenylalanine
(GST-3Tyr-Mut) was used as a control. Two proteins of 75 and 32 kDa
were tyrosine-phosphorylated in the Syk kinase assays, corresponding to
Syk and GST-Tyr-WT, respectively (Fig. 8A). A small increase
in Syk autophosphorylation and tyrosine phosphorylation of the fusion
protein was observed in stimulated conditions relative to basal level.
A similar level of tyrosine phosphorylation of the two proteins was
observed in the presence of PP1 (not shown). No tyrosine
phosphorylation of GST-3Tyr-Mut was detected in the Syk kinase assay
from resting and CRP-stimulated platelets (Fig. 8A). This
indicates that tyrosine phosphorylation of GST-Tyr-WT by Syk is
specific to one or more of Tyr113, Tyr128, and
Tyr145. In contrast, GST-Tyr-WT was weakly phosphorylated
by Lyn and Fyn, whereas both kinases underwent dramatic
autophosphorylation (Fig. 8, B
and C), which was abolished in the presence of PP1 (not
shown). GST-3Tyr-Mut was not tyrosine-phosphorylated in the Lyn and Fyn
kinase assays. This indicates that tyrosine phosphorylation of the
N-terminal tyrosine-rich region of SLP-76 is mediated by Syk with only
a very minor contribution from Lyn and Fyn.
Tyrosine Phosphorylation of both PLC-
The functional consequence of the reduction in PLC- Here we report that Fc Tyrosine phosphorylation of SLP-76 is one of the earliest of the events
following CRP stimulation and is sustained for up to 10 min. This
indicates that SLP-76 may be involved in initial events of the signal
transduction pathway induced by CRP. This is consistent with the fact
that tyrosine phosphorylation of SLP-76 was maintained in the presence
of the Ca2+ chelator
bis(O-aminophenoxy)-N,N,N',N'-tetraacetic
acid and a protein kinase C antagonist Ro 31-8220, a combination
designed to inhibit events downstream of PLC- SLP-76 was reported to be tyrosine-phosphorylated by ZAP-70 or Syk in T
cells and rat basophilic leukemia cells (9, 14), respectively.
Consistent with this, tyrosine phosphorylation of SLP-76 induced by CRP
was abrogated in Syk-deficient platelets. However, we were not able to
detect in vivo association between Syk and SLP-76 following
immunoprecipitation, suggesting an indirect, unstable, or weak
interaction between the two proteins.
SLP-76 contains three N-terminal tyrosine phosphorylation sites
including two consensus sequences (pYESP; where pY represents phosphotyrosine) for association to the SH2 domain of Vav. SLP-76 also
contains a proline-rich region that associates with the SH3 domain of
Grb2 and an SH2 domain that associates to tyrosine-phosphorylated proteins. Immunoprecipitation of SLP-76 revealed a marked association with the Src kinase Lyn and a phosphotyrosyl protein of 130 kDa. One
component of this 130-kDa phosphoprotein was identified as SLAP-130
through sequential immunoprecipitation following kinase assay. This
strategy also revealed a lower level of binding of other proteins with
SLP-76, namely Vav, Fyn, and FcR Kinase assays performed on immunoprecipitated SLP-76 from
CRP-stimulated platelets lysed with the mild detergent Brij 96 exhibited a stronger tyrosine phosphorylation of FcR In vitro tyrosine phosphorylation of SLP-76 was also
abrogated in the presence of PP1 in both Nonidet P-40 and Brij 96 lysates following CRP stimulation. Lck was reported to be able to
tyrosine-phosphorylate Tyr423/Tyr426 within the
SH2 domain of SLP-76, but this does not correlate with physiological
mapping of tyrosine phosphorylation sites (Tyr113,
Tyr128, and Tyr145) of SLP-76 following TCR
stimulation (9). Lyn and Fyn may also tyrosine-phosphorylate of SLP-76
on Tyr423/Tyr426, since the fusion protein for
the SH2 domain of SLP-76 becomes tyrosine-phosphorylated in
vitro as shown in Fig. 3. This was investigated further through
the study of Lyn- and Fyn-deficient platelets. Tyrosine phosphorylation
of SLP-76 in platelets from either knock-out mice was not altered when
compared with control mice. However, in vitro
phosphorylation was reduced in Lyn knock-out mice, suggesting this is
the predominant active kinase in the assays. Reduction of in
vitro tyrosine phosphorylation of SLP-76 but no change of in
vivo phosphorylation strengthens the idea that Lyn and Fyn are not
involved in phosphorylation of Tyr113, Tyr128,
and Tyr145. This was confirmed by very weak in
vitro phosphorylation of a fusion protein containing these three
tyrosine phosphorylation sites by immunoprecipitated Lyn or Fyn. In
contrast, Syk displayed a much greater ability to phosphorylate this
fusion protein in comparison with Lyn and Fyn, suggesting that Syk may
phosphorylate this region in vivo.
The inability to detect in vivo association of Syk and
SLP-76 suggests that other protein may bring the adapter molecule to the vicinity of the tyrosine kinase. One possibility is that this role
is fulfilled by the T cell adapter protein LAT, which undergoes tyrosine phosphorylation in platelets and forms a complex with Grb2 and
SLP-76 (15). A novel possibility is that SLP-76 is brought to Syk via
binding to either Fyn or Lyn associated with the FcR In this study, we show that tyrosine phosphorylation of
PLC- SLAP-130 associated with the SH2 domain of SLP-76 in platelets. The
function of SLAP-130 is not known. The relationship of the association
between the SH2 domain of SLP-76 and SLAP-130 was investigated in
SLP-76-deficient platelets. SLAP-130 was tyrosine-phosphorylated in
resting platelets, and this level of phosphorylation was unaltered in
SLP-76-deficient cells. The increase of tyrosine phosphorylation of
SLAP-130 induced by CRP-stimulation, however, was inhibited in the
absence of SLP-76. This suggests that tyrosine phosphorylation of
SLAP-130 is regulated through two pathways, one SLP-76-independent pathway corresponding to phosphorylation in resting platelets and a
SLP-76-dependent pathway involved in the increase of
phosphorylation following CRP stimulation. These observations place
SLAP-130 downstream of SLP-76 in CRP-induced signaling.
In conclusion, tyrosine phosphorylation of SLP-76 is downstream of
tyrosine phosphorylation of Syk in CRP-stimulated platelets and
upstream of phosphorylation of SLAP-130 and PLC--chain, Syk, and phospholipase
C-
2. Here we report that CRP and the platelet low
affinity immune receptor Fc
RIIA stimulate tyrosine phosphorylation
of the T cell adapter SLP-76, whereas the G protein-coupled receptor
agonist thrombin induces only minor tyrosine phosphorylation. This
suggests that SLP-76 has a specific role downstream of receptors that
signal via an immunoreceptor tyrosine-based activation motif.
Immunoprecipitation studies demonstrate association of SLP-76 with
SLAP-130, Vav, Fyn, Lyn, and the FcR
-chain in CRP-stimulated
platelets. Several of these proteins, including SLP-76, undergo
tyrosine phosphorylation in in vitro kinase assays
performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all
of these proteins in the in vitro kinase assay was
abrogated by the Src family kinase inhibitor PP1, suggesting that it is
mediated by either Fyn or Lyn. The physiological significance of this
is uncertain, however, since tyrosine phosphorylation of SLP-76
in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that
in vivo tyrosine phosphorylation of SLP-76 is downstream of
Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the
possibility that another protein is responsible for bringing SLP-76 to
Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR
-chain. Tyrosine phosphorylation of PLC-
2 and Ca2+
mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical
role in the regulation of the phospholipase. The increase in tyrosine
phosphorylation of SLAP-130 in response to CRP is also inhibited in
SLP-76-deficient platelets, placing it downstream of SLP-76. This work
identifies SLP-76 as an important adapter molecule that is regulated by
Syk and lies upstream of SLAP-130 and PLC-
2 in
CRP-stimulated platelets.
INTRODUCTION
Top
Abstract
Introduction
References
1 (PLC-
1) and activation of the Ras pathway (11). Moreover, SLP-76 is required for normal thymocyte development, since SLP-76 knock-out mice lack peripheral T
cells (12, 13).
RIIA (15).
Increasing evidence suggests that the collagen receptor underlying the
major increase in tyrosine phosphorylation in platelets also signals
like an immune receptor. The collagen receptor is believed to comprise
a multimeric structure, containing the glycoprotein VI (GPVI), and the
Fc receptor (FcR)
-chain (16-19). Binding of collagen to GPVI
induces tyrosine phosphorylation of the immunoreceptor tyrosine-based
activation motif in the cytoplasmic tail of FcR
-chain (18), leading
to tyrosine phosphorylation of Syk and PLC-
2
(20).
2 in response to collagen is
abolished in SLP-76-deficient
platelets.2 In this study, we
have investigated the mechanism of tyrosine phosphorylation of SLP-76
and the function of SLP-76 in platelets following stimulation by a
collagen-related peptide (CRP), through the specific binding to GPVI
(22). CRP is a synthetic, triple helical peptide composed of
Gly-Pro-hydroxyproline repeats, cross-linked by cysteines at the N and
C termini (23). CRP activates platelets through the GPVI but, in
contrast to collagen, is unable to bind the integrin
2
1 (23, 24). It is a more powerful
agonist than collagen and exhibits less variation in response between individuals.
MATERIALS AND METHODS
RIIA-specific monoclonal antibody (mAb)
was purchased from Madarex Inc (Annandale, NJ). Sheep
F(ab')2 raised against mouse IgG (M-1522) and thrombin were
purchased from Sigma (Poole, UK). Fura-2/AM was from Molecular Probes,
Inc. (Eugene, OR). Anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology, Inc. (TCS Biologicals Ltd., Botolph Claydon, UK); polyclonal anti-Lyn antibody, Lyn(44), and polyclonal anti-Fyn antibody, Fyn(FYN3), were from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Anti-SLP-76 mAb and the GST-SLP-SH2 were described previously (1), and polyclonal anti-SLAP-130 rabbit antiserum was
described previously (8). Polyclonal anti-FcR
-chain rabbit antiserum was a gift from Dr. J. P. Kinet (Beth Israel Hospital, Boston, MA). PP1 (CP118,556) was kindly donated by Dr. J. Hanke (Pfizer
Central Research, CT). The Syk-deficient mice and murine anti-Syk
antiserum were described previously (25). The SLP-76 knock-out mice
were previously described (12). The Lyn knock-out mice were a gift from
Dr. A. Dunn (Ludwig Institute, Melbourne, Australia) (26). The Fyn
knock-out mice were purchased from Jackson Laboratories (Bar Harbor, ME).
RIIA using mAb IV.3 (1 µg/ml) for
1 min and then the cross-linker F(ab')2 (30 µg/ml) for
90 s.
Rmin)/(Rmax
R) × K, where K represents the value
Kd × Sf2/Sb2. The experimentally determined
value of K used in this study is 3.6 µM.
RESULTS
RIIA cross-linking
stimulates marked tyrosine phosphorylation of SLP-76 in human platelets (15), a result that is confirmed in the present study (Fig. 1A). Collagen was also
observed to stimulate marked tyrosine phosphorylation of SLP-76, in
agreement with the observation in mouse platelets,2 whereas
the G protein-coupled receptor agonist thrombin induced only a low
level of tyrosine phosphorylation of SLP-76 (Fig. 1A). CRP,
a synthetic peptide based on the triple-helical structure of collagen,
also stimulated tyrosine phosphorylation of SLP-76 (Fig.
1A). Reprobing the membrane for SLP-76 to check loading revealed that the anti-SLP-76 antibody has a better affinity for the
unphosphorylated form of SLP-76 (Fig. 1A).
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Fig. 1.
SLP-76 represents a major
tyrosine-phosphorylated protein in stimulated platelets.
A, platelets were incubated in Tyrode-Hepes buffer and
stimulated by the addition of 30 µg/ml collagen for 90 s, 3 µg/ml CRP for 90 s, anti-Fc RIIA mAb IV.3 (1 µg/ml) for 1 min followed by cross-linker F(ab')2 30 µg/ml for 90 s and 1 unit/ml thrombin for 90 s. Platelets were preincubated
with 10 µM Ro 31-8220 and 40 µM
bis(O-aminophenoxy)-N,N,N',N'-tetraacetic
acid 5 min prior to CRP stimulation. Stimulation was stopped by the
addition of an equal volume of Nonidet P-40 lysis buffer. SLP-76 was
immunoprecipitated from precleared lysate using the anti-SLP-76 mAb.
Immunoprecipitated proteins were separated by 10% SDS-PAGE,
electroblotted onto PVDF membranes, and detected by immunoblotting
using the anti-phosphotyrosine mAb 4G10. Membranes were stripped and
reprobed for SLP-76 using the anti-SLP-76 mAb (bottom).
B, platelets were stimulated with 3 µg/ml CRP for the time
indicated. Immunoprecipitated SLP-76 was analyzed on 10% SDS-PAGE and
immunoblotted using anti-phosphotyrosine mAb 4G10. Membranes were
stripped, and equal loading was checked by immunoblotting with the
anti-SLP-76 mAb (bottom). C, platelets were
stimulated with 3 µg/ml CRP for 90 s or left untreated. Proteins
were immunoprecipitated with an anti-SLP-76 mAb covalently linked to
Protein A-Sepharose, and proteins were separated on 10% SDS-PAGE and
immunoblotted using the anti-phosphotyrosine mAb 4G10. Membranes were
stripped and immunoblotted with the anti-Lyn polyclonal antibody
(middle) and using the anti-SLP-76 mAb
(bottom).
2
1 (23, 24). Tyrosine phosphorylation of
SLP-76 by CRP occurred within 10 s and reached a maximum at
60 s, being sustained for up to 10 min (Fig. 1B). The
time course showing tyrosine phosphorylation of total platelet protein
indicated that a major band of 75 kDa displayed the same tyrosine
phosphorylation pattern as SLP-76 (not shown). SLP-76 was identified as
a component of this band. We have previously shown that Syk is also a
component of this band. Pretreatment of platelets with the
Ca2+ chelator
bis(O-aminophenoxy)-N,N,N',N'-tetraacetic
acid and the protein kinase C antagonist Ro 31-8220 to inhibit the
action of the second messengers produced by PLC-
2
indicated that tyrosine phosphorylation of SLP-76 is independent of
PLC-
2 activation (Fig. 1A).
RIIA
only induces a small increase in phosphorylation of this 130-kDa
phosphoprotein. SLAP-130, which associates with the SH2 domain of
SLP-76 following TCR engagement (8), and PLC-
2, the only
PLC-
isoform to be tyrosine-phosphorylated in CRP and
collagen-stimulated platelets (24, 33), have a similar electrophoretic
mobility to this tyrosine-phosphorylated protein of 130 kDa. However,
subsequent immunoblotting with SLAP-130- or
PLC-
2-specific antibodies failed to identify
conclusively the 130-kDa co-precipitated protein, possibly because the
level of protein was below that of the sensitivity of detection. The presence of SLAP-130 in the 130-kDa band was subsequently shown by
sequential immunoprecipitations as described below.
RIIA-stimulated platelets (15).
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Fig. 2.
Lyn and SLAP-130 associate to the SH2 domain
of SLP-76. Platelets were stimulated with 3 µg/ml CRP for
90 s. The precleared lysate was incubated with 5 µg of
GST-SLP-SH2 or GST alone (control). Precipitated proteins were resolved
on 10% SDS-PAGE and immunoblotted using the anti-phosphotyrosine mAb
4G10. Membranes were stripped and immunoblotted using the anti-Lyn
polyclonal antibody (middle panel) and using the
anti-SLAP-130 antiserum (bottom panel).
-chain (Fig.
4A), Lyn (Fig. 4B),
Fyn (Fig. 4C), Vav (Fig. 4D), and SLAP-130 (Fig.
4E), respectively. A more prominent association with Fyn was
seen in some studies as illustrated in Fig.
5. The weak band corresponding to Vav is
likely to reflect both a low level of binding and the fact that it
serves as a poor substrate in the in vitro kinase assay.
Neither Syk nor PLC-
2 were detected in these studies,
suggesting that they do not associate with SLP-76 or that
PLC-
2 is not a substrate in the in vitro
kinase assay.
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Fig. 3.
In vitro tyrosine kinase activity
is detected in SLP-76 immunoprecipitates and GST-SLP-SH2
precipitates. A, SLP-76 was immunoprecipitated from
resting and stimulated platelets with 3 µg/ml CRP for 90 s.
B, 5 µg of GST-SLP-SH2 were used for protein precipitation
from lysate of resting or CRP-stimulated platelets. Immunoprecipitated
and precipitated proteins were submitted to in vitro kinase
assay and separated on 10-18% gradient SDS-PAGE and transferred to
PVDF membranes. Membranes were immunoblotted using the
anti-phosphotyrosine mAb 4G10. Membranes were then incubated in 1 M KOH for 1 h at 55 °C before
autoradiography.
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Fig. 4.
FcR -chain,
Lyn, Fyn, Vav, and SLAP-130 co-immunoprecipitate with SLP-76 following
CRP stimulation. SLP-76 was immunoprecipitated as described in the
legend of Fig. 3. Following an in vitro kinase assay,
immunoprecipitated proteins were parted in lysis buffer containing 2%
SDS. Proteins were heated, and subsequently the SDS concentration of
the supernatant was reduced to 0.1% by the addition of Nonidet P-40
lysis buffer. Lysates were used for immunoprecipitation of FcR
-chain (A), Lyn (B), Fyn (C), Vav
(D), and SLAP-130 (E). Proteins were analyzed on
10% SDS-PAGE, expect for FcR
-chain immunoprecipitation, which was
resolved on 10-18% gradient SDS-PAGE. SDS-polyacrylamide gels were
dried and exposed to autoradiography.
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Fig. 5.
Effect of PP1 on in vitro
kinase assay performed on SLP-76 immunoprecipitated from resting
or CRP-stimulated platelets lysed in Nonidet P-40- or Brij
96-containing buffer. Stimulation of platelets with Tyrode-Hepes
buffer or 3 µg/ml CRP was stopped after 90 s by the addition of
Nonidet P-40 lysis buffer or Brij 96 lysis buffer. SLP-76 was
immunoprecipitated and submitted to an in vitro kinase
assay. When indicated, kinase assay was performed in the presence of 10 µM PP1. Proteins were resolved on 10-18% gradient
SDS-PAGE and transferred to PVDF membranes. Membranes were treated with
1 M KOH for 1 h at 55 °C and submitted to
autoradiography (upper panel). Equal loading was
checked by immunoblotting the membranes using the anti-SLP-76 mAb
(bottom panel).
-chain (Fig. 5) in CRP stimulated
samples. The intensity of the band corresponding to Lyn in Brij 96 immunoprecipitation is lower than with Nonidet P-40, in agreement with
the decrease of Lyn as shown by immunoblotting with anti-Lyn antibody
(not shown). A similar result is seen for Fyn. The reduction in the
level of these two kinases is in contrast to the increase in tyrosine
phosphorylation of the FcR
-chain, suggesting that the latter is a
consequence of a greater amount of protein in the immunoprecipitate.
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Fig. 6.
In vivo tyrosine phosphorylation
of SLP-76 was not altered in Lyn- or Fyn-deficient platelets, but
in vitro tyrosine phosphorylation of SLP-76 was
reduced in Lyn-deficient platelets. Platelets from control or Lyn
/
(A) or Fyn
/
(B) mice were stimulated
with CRP for 90 s. The reaction was stopped by the addition of
Nonidet P-40 lysis buffer. SLP-76 was immunoprecipitated from these
lysates and submitted to in vitro kinase assay. Proteins
were resolved on 10% SDS-PAGE and immunoblotted using the
anti-phosphotyrosine mAb 4G10 (A and B,
upper part). Membranes were stripped and reprobed
using the anti-SLP-76 mAb (A and B,
middle part). Autoradiographs of the membranes
are shown in the bottom part of A and
B.
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Fig. 7.
Tyrosine phosphorylation of endogenous SLP-76
is dependent on Syk activity. SLP-76 was immunoprecipitated from
control wild-type or Syk-deficient platelets that were resting or were
stimulated with 3 µg/ml CRP for 90 s. Immunoprecipitated
proteins were separated on 10-18% gradient SDS-PAGE and immunoblotted
using the anti-phosphotyrosine mAb 4G10 (upper
panel). Membranes were stripped and reprobed using the
anti-SLP-76 mAb (bottom panel).
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Fig. 8.
Syk, but not Lyn and Fyn, in vitro
phosphorylates Tyr113, Tyr128, and
Tyr145 from SLP-76. Syk (A), Lyn
(B), and Fyn (C) were immunoprecipitated under basal or CRP-stimulated conditions
and subjected to an in vitro kinase assay. 5 µg of
GST-Tyr-WT, GST-3Tyr-Mut, or GST alone were added in the kinase assay.
Kinase reactions were analyzed on 10% SDS-PAGE and electroblotted on
PVDF membranes. Ser/Thr phosphorylation was removed by treating the
membranes with 1 M KOH at 55 °C for 1 h. The
upper part of each panel shows an
autoradiograph of the kinase assay. Membranes were immunoblotted using
anti-Syk (A, middle part), anti-Lyn
(B, middle part), and anti-Fyn
(C, middle part) polyclonal
antibodies. Fusion protein equal loading was checked by immunoblotting
with anti-GST mAb (A-C, lower
part).
2 and SLAP-130
Is Lost in SLP-76-deficient Mouse Platelets--
The role of SLP-76 in
tyrosine phosphorylation of PLC-
2 and SLAP-130 was
investigated in SLP-76-deficient platelets. A similar profile of
tyrosine phosphorylation was observed in basal and CRP-stimulated
samples from wild-type (+/+) and heterozygous (+/
) platelets, whereas
there was a marked decrease in phosphorylation of proteins of 130 and
75 kDa in the SLP-76-deficient (
/
) cells (not shown). The reduction
in the 75-kDa protein is likely to be due to the absence of SLP-76,
which migrates in this area. The remaining increase in phosphorylation
of this band is likely to be due to phosphorylation of Syk, which
co-migrates with SLP-76. This was confirmed by immunoprecipitation of
the kinase (Fig. 9A). The
130-kDa band migrates in the region of SLAP-130 and
PLC-
2. Phosphorylation of PLC-
2 was
dramatically reduced in response to CRP in SLP-76-deficient platelets
(Fig. 9B), although a residual increase could be seen on a
longer exposure (not shown). SLAP-130 was tyrosine-phosphorylated in
resting platelets, and the level of phosphorylation increased following
stimulation by CRP. The level of phosphorylation of SLAP-130 under
basal conditions was not altered in the SLP-76-deficient platelets,
whereas the increase induced by CRP was abolished (Fig.
9C).
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Fig. 9.
Tyrosine phosphorylation of
PLC- 2 and SLAP-130 is inhibited
following CRP stimulation of SLP-76-deficient platelets. Syk
(A), PLC-
2 (B), or SLAP-130
(C) were immunoprecipitated from resting or CRP-stimulated
control (SLP-76 +/
) and SLP-76-deficient (SLP-76
/
) mice
platelets. Proteins were separated on 10% SDS-PAGE, electroblotted to
PVDF, and immunoblotted using the anti-phosphotyrosine mAb 4G10
(A-C, upper part). Membranes were
stripped and reprobed using the anti-Syk rabbit antiserum
(A, bottom part), the
anti-PLC-
2 polyclonal antibody (B,
bottom part), and the anti-SLAP-130 rabbit
antiserum (C, bottom part).
2
phosphorylation was monitored through measurement of intracellular
Ca2+ in Fura-2/AM-loaded platelets. CRP was unable to
elevate Ca2+ in SLP-76-deficient platelets (Fig.
10A) in contrast to the
robust response in control cells. Ca2+ mobilization in
response to the G protein-coupled receptor agonist thrombin was
unaltered (Fig. 10B) in SLP-76-deficient platelets, suggesting that SLP-76 plays a crucial function downstream of immunoreceptor tyrosine-based activation motif-containing receptor.
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Fig. 10.
Ca2+ mobilization is inhibited
in SLP-76-deficient platelets stimulated by CRP. Mouse platelets
were loaded with Fura-2/AM dye. Platelets were left unstimulated for 2 min and then stimulated with 3 µg/ml CRP (A) or 100 nM thrombin (B). Calculation of the cytosolic
Ca2+ concentration from the fluorescence ratio (340/380 nm)
was performed by the use of a calibration curve with Ca2+
standards. The arrowheads indicate the addition of
agonist.
DISCUSSION
RIIA, CRP, and collagen stimulate
dramatic tyrosine phosphorylation of SLP-76 in contrast to the G protein-coupled receptor agonist thrombin. This suggests that this
adapter protein has a specific role in immunoreceptor tyrosine-based activation motif-mediated signaling. This is consistent with the absence of other reports of phosphorylation of SLP-76 by G
protein-coupled receptor agonists.
2,
following CRP stimulation.
-chain. SLP-76 has been reported to
co-immunoprecipitate with SLAP-130 and an uncharacterized protein of 62 kDa, as well as a serine/threonine kinase in rat basophilic leukemia
cells (14) and T cells (7, 8). No evidence for association of the
latter two proteins was found in the present study. However, SLAP-130,
Lyn, and Fyn were found to interact with the SH2 domain of SLP-76
expressed as a fusion protein. It is unclear whether the association of FcR
-chain also occurs through this region, because radiolabeled incomplete GST fusion protein products co-migrated in this region of
the gel. Vav did not associate with the SH2 domain of SLP-76, consistent with an interaction occurring between the SH2 domain of Vav
and the N-terminal tyrosine-phosphorylated sites as shown in stimulated
T cells (2-5).
-chain than in
Nonidet P-40 lysates following CRP stimulation. This is in contrast
with the lower level of co-immunoprecipitating Lyn and Fyn found in Brij 96 lysates. The interaction with the FcR
-chain could be direct
or indirect, since Fyn and Lyn were recently reported to associate with
FcR
-chain irrespective of stimulation by collagen (35). In the
latter case, the increase in the level of FcR
-chain in Brij 96 suggests either that the interaction is more stable in Brij 96 compared
with Nonidet P-40 or that there is a selective solubilization of a pool
of Lyn and/or Fyn associated with FcR
-chain. In Nonidet P-40,
SLP-76 could co-immunoprecipitate at least two pools of Src tyrosine
kinases, a pool associated with FcR
-chain and a second pool
possibly associated with downstream events. Lyn and Fyn are likely to
tyrosine-phosphorylate FcR
-chain in the in vitro kinase
assay, since the addition of PP1, the Src kinase inhibitor, abolished
tyrosine phosphorylation.
-chain. A
direct role of Lyn and Fyn in this way cannot be ruled out through the
analysis of Lyn and Fyn knock-out platelets because of possible
redundancy between members of the Src kinase family.
2 and Ca2+ mobilization are almost fully
inhibited in response to CRP. This is consistent with the observations
of Clements et al.2 that aggregation and
tyrosine phosphorylation of PLC-
2 in SLP-76-deficient platelets by collagen is abrogated. A similar result was observed with
TCR-mediated tyrosine phosphorylation of PLC-
1 in T
cells lacking SLP-76 (11). Tyrosine phosphorylation of
PLC-
1 and PLC-
2 has also been shown to be
downstream of tyrosine phosphorylation of Blnk in B cells (36).
Tyrosine phosphorylation of Blnk following B cell stimulation is
thought to recruit PLC-
isoforms to Blnk, enabling phosphorylation
by Syk, co-localized with Blnk. A similar model could apply for
tyrosine phosphorylation of PLC-
2 in CRP-stimulated platelets. Residual tyrosine phosphorylation of PLC-
2
could be seen in CRP-stimulated SLP-76-deficient platelets, indicating the existence of a second, minor pathway leading to tyrosine
phosphorylation of PLC-
2. This cannot be attributed to
Blnk, since this is not expressed in
platelets.3 This might occur
through a direct interaction between Syk and PLC-
2,
similar to the interaction seen in B-cells (21, 37) or indirectly
through LAT as suggested for residual phosphorylation of
PLC-
1 in T cells (11).
2.
SLP-76 co-immunoprecipitates with the tyrosine kinases Lyn and Fyn, and this interaction may be important for upstream as well as for downstream events. This work confirms SLP-76 as an important link between Syk activation and PLC-
2 regulation, although
further work is required to establish a complete understanding of this pathway, including the role of SLAP-130.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. J. P. Kinet and M. Barnes for the supply of materials. We also thank Lynn Quek for comments on the manuscript and helpful discussion.
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FOOTNOTES |
---|
* This work was supported by the Wellcome Trust and British Heart Foundation.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.
§ A Wellcome Prize Student. To whom correspondence should be addressed: Dept. of Pharmacology, Mansfield Rd., Oxford OX1 3QT, UK. Tel.: 44-1865-271590; Fax: 44-1865-271853; E-mail: barbara.gross{at}users.jesus.ox.ac.uk.
§§ A British Heart Foundation Senior Research Fellow.
2 Clements, J. L., Lee, J. Ran, Gross, B., Yang, B., Olson, J. D., Sandra, A., Watson, S., Lentz, S. R., and Koretzky, G. A., (1999) J. Clin. Invest. 103, 19-25
3 I. Hers and S. Watson, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
SH2, Src homology 2;
CRP, collagen-related peptide;
GST, gluthatione
S-transferase;
FcR, Fc receptor;
PLC-, phospholipase
C-
;
TCR, T cell receptor;
PAGE, polyacrylamide gel electrophoresis;
mAb, monoclonal antibody;
PVDF, polyvinylidene difluoride;
GPVI, glycoprotein VI.
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REFERENCES |
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