From COR Therapeutics, Inc., South San Francisco,
California 94080, the ¶ Section of Cellular and Molecular
Motility, Laboratory of Molecular Cardiology, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892-1762, and the
Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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Tyrosine phosphorylation of the
3 subunit of the major platelet integrin
IIb
3 has been shown to occur during
thrombin-induced platelet aggregation (1). We now show that a wide
variety of platelet stimuli induced
3 tyrosine
phosphorylation, but that this phosphorylation occurred only following
platelet aggregation. Several lines of evidence suggest that the
3 cytoplasmic domain tyrosine residues and/or their
phosphorylation function to mediate interactions between
3 integrins and cytoskeletal proteins. First, phospho-
3 was retained preferentially in a Triton X-100
insoluble cytoskeletal fraction of thrombin-aggregated platelets.
Second, in vitro experiments show that the cytoskeletal
protein, myosin, associated in a phosphotyrosine-dependent
manner with a diphosphorylated peptide corresponding to residues
740-762 of
3. Third, mutation of both tyrosines in the
3 cytoplasmic domain to phenylalanines markedly reduced
3-dependent fibrin clot retraction. Thus,
our data indicate that platelet aggregation is both necessary and sufficient for
3 tyrosine phosphorylation, and this
phosphorylation results in the physical linkage of
IIb
3 to the cytoskeleton. We hypothesize
that this linkage may involve direct binding of the phosphorylated
integrin to the contractile protein myosin in order to mediate
transmission of force to the fibrin clot during the process of clot
retraction.
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INTRODUCTION |
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Integrins are a family of heterodimeric transmembrane proteins
that link the extracellular matrix to the cytoskeletal/contractile apparatus within a cell (2). These cytoskeletal linkages are characteristically induced by integrin clustering that can occur by the
binding of multivalent or immobilized extracellular ligands, often
resulting in the assembly of "focal contacts" in cultured cells.
Several cytoskeletal proteins (e.g. talin, actin binding protein, -actinin) directly bind to integrin cytoplasmic domains (3-6), indicating that integrins may interact by multiple mechanisms for focal contact assembly. Focal contact assembly is often followed by
signal transduction events such as induction of gene transcription (7)
and prevention of apoptosis (8, 9), regulating a diversity of cellular
functions from embryonic development to hemostasis (10). Although
cytoskeletal and signaling proteins have been identified which bind
integrin cytoplasmic domains, major unsolved questions persist. For
example, what is the identity of the proteins responsible for the
initial interactions of integrins with the cytoskeletal structures? How
are these interactions regulated? In this regard, it is relevant to the
present study that focal contacts are major sites for protein tyrosine
phosphorylation, one of the earliest signaling events observed upon
integrin ligation (11).
On platelets, the interaction of the integrin
IIb
3 with its adhesive ligands,
fibrinogen, or von Willebrand factor leads to platelet aggregation and
association of
IIb
3 with the cytoskeleton (12, 13). Under normal conditions, platelet aggregation is the desired
response to external trauma, allowing for hemostasis. However,
inappropriate platelet aggregation does occur, as in ruptured
artherosclerotic plaques, resulting in the formation of occlusive
thrombi leading to myocardial infarction or thrombolytic stroke (14).
The importance of
IIb
3-mediated events in
both hemostasis and thrombosis is underscored in two ways. First,
patients who lack, or have mutated,
IIb
3,
a condition known as Glanzmann's thrombasthenia, have a bleeding
disorder that arises from the failure of the platelets to aggregate
(15). Second, clinical trials have shown that antagonists for
IIb
3 ligand binding are effective
antithrombotics (16).
IIb
3 is involved in both "inside-out"
and "outside-in" signaling pathways during platelet aggregation
(12). In order to bind soluble forms of its adhesive protein ligands,
IIb
3 on resting platelets has to undergo
a conformational change. This process, the consequence of
"inside-out"
IIb
3 signaling, occurs when agonists such as ADP or thrombin activate platelets. Binding of
fibrinogen and von Willebrand factor to
IIb
3 induces platelet aggregation and
IIb
3 clustering: the signals transduced
by this process are referred to as "outside-in" signaling events.
The cytoplasmic domains of the integrin are thought to play a critical role in these signaling events (17-20). In addition, platelet
aggregation induces the direct interaction of
IIb
3 with the cytoskeleton (21, 22). The
cytoskeletal proteins talin and
-actinin have been found to act
directly with
IIb
3 (4, 6). Along with
IIb
3, many other intracellular proteins,
including Src and FAK, redistribute to the cytoskeleton of aggregated
platelets (22, 23). In these ways, the integrin may play a direct role
not only in organizing the cytoskeleton but also in transducing signals to elicit cellular responses. Although it is clear that the cytoplasmic domains of
IIb
3 are involved in signal
transduction and cytoskeletal reorganization events, the precise
mechanisms regulating these processes remain to be discovered.
Previously, we showed that tyrosine phosporylation of 3
occurs upon thrombin-induced platelet aggregation, indicating a
potential role for integrin cytoplasmic tyrosine residues in outside-in
IIb
3 signaling (1). In support of this
hypothesis, we observed that the signaling adaptor proteins SHC and
Grb2 interacted with peptides corresponding to the tyrosine
phosphorylated cytoplasmic domain of
3 (1). The present
study shows that tyrosine phosphorylation of
3 is a
unifying event of platelet aggregation and provides in vitro
evidence that tyrosine phosphorylation of this integrin subunit may
direct its binding to myosin, a specific element contained within the
platelet cytoskeleton.
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EXPERIMENTAL PROCEDURES |
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Reagents--
Aprotinin, apyrase, aspirin, prostaglandin
I2, phenylmethylsulfonyl fluoride, leupeptin, diisopropyl
fluorophosphate, sodium orthovanadate, sodium pyrophosphate, the
thromboxane A2 analog 9,11-dideoxy-11a,9a-epoxymethanoprostaglandin F2a (U46619), control mouse IgG, and Sigmacote were all purchased from Sigma. The platelet aggregation reagents ADP, epinephrine, and collagen were purchased from
Sigma Diagnostics. Thrombin receptor activating peptide
SFLLRN-NH2 (TRAP; Ref. 24) and the cyclic peptide
IIb
3 antagonist
Mpr-RGDWP-Pen-NH2 (25) were provided by COR Therapeutics,
Inc., Medicinal Chemistry Department. Human
-thrombin was purchased
from Hematologic Technologies, Inc., and human fibrinogen was from
Chromogenix. The
v
3 antibody LM609 was
generously provided by David Cheresh (Scripps Research Institute) (26).
The anti-LIBS6 monoclonal antibody has been described (27). Anti-human
3 monoclonal antibody C3a.19.5, which recognizes the
cytoplasmic tail of the
3 subunit, was described previously (1). Anti-myosin monoclonal antibody was from Immunotech, Inc. The anti-phosphotyrosine antibodies PY-20 and 4G10 were from Transduction Laboratories and Upstate Biotechnology, Inc.,
respectively. The horseradish peroxidase-conjugated secondary reagent
sheep anti-mouse Ig and horseradish peroxidase-conjugated streptavidin were from Amersham Pharmacia Biotech. Hank's buffered saline solution and Dulbecco's modified Eagle's medium were from Life Technologies, Inc. Goat anti-mouse fluorescein isothiocyanate-conjugated
F(ab')2 was from Jackson and Gamimmune N was from Miles.
Biotinylated integrin cytoplasmic domain peptides, synthesized by
SynPep Corporation using solid phase Fmoc (N-(9-fluorenyl)
methoxycarbonyl) chemistry, were dissolved at a concentration of 2 mg/ml in water and diluted as needed. Chymotrypsin and papain were from
Boehringer Mannheim. 4-20% gradient SDS-PAGE
1 gels were from Bio-Rad, and
ECL Hybond nitrocellulose was from Amersham Pharmacia Biotech. See-blue
molecular mass standards were purchased from Novex.
Platelet Preparation and Aggregation-- Blood from healthy volunteers was drawn on the day of use, and washed platelets were prepared as described previously (28) except 0.6 units/ml apyrase and 50 ng/ml prostaglandin I2 (final concentrations) were present in the collecting solution. Before stimulation, the platelets (~4-8 × 108/ml) were incubated for 1 h at 37 °C in Tyrodes-HEPES buffer (12 mM NaHCO3, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, 10 mM HEPES, pH 7.4, 1 mM CaCl2, 0.5 mM MgCl2) unless otherwise stated. Platelet samples of 0.5 ml were then stirred at 37 °C in a whole blood lumiaggregometer, and various agonists and conditions were examined. When platelet lysates were not prepared, 4× nonreducing Laemmli sample buffer containing vanadate (37 mM Tris, pH 6.8, 11.8% (v/v) glycerol, 2.36% (w/v) SDS, 2 mM sodium orthovanadate, and 0.002% (w/v) bromphenol blue (final concentration)) was added immediately after aggregation, and samples were boiled for 5 min.
Platelet Lysate Preparation--
For two-dimensional gel
analysis of detergent-soluble and cytoskeletal fractions, platelets
were lysed immediately after aggregation by the addition of an equal
volume of ice-cold 2× Triton X-100 lysis buffer (1% (v/v) Triton
X-100, 100 mM NaCl, 20 mM Tris, pH 7.0, 2 mM EDTA, 2 mM ethyleneglycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 20 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 µM leupeptin, 4 mM sodium orthovanadate, 2 mM benzamidine, 0.27 mM diisopropyl
fluorophosphate, 5 mM sodium pyrophosphate (final
concentrations)). The lysate was then centrifuged for 6 min at
15,000 × g to remove the Triton X-100 insoluble
cytoskeletons formed during aggregation (22). The supernatant was
reserved and 100 µl of 2× RIPA buffer (see below) was added to the
pellet and sonicated for 20 min at room temperature in a Branson 5120 Sonicator to resolubilize the pellet. Nonreducing sample buffer (as
described above) was added to each of the samples (supernatant and
resolubilized pellet) and boiled for 5 min.
Determination of 3 Phosphorylation
Level--
Nonreduced-reduced two-dimensional gel electrophoresis was
performed to visualize the characteristic migration of
3
and assess its phosphorylation state as described previously (1, 29). The two-dimensional gels were transferred to nitrocellulose, and blots
were probed with anti-phosphotyrosine antibodies PY-20 and 4G10. The
blots were washed and incubated with horseradish peroxidase-conjugated sheep anti-mouse Ig and developed using the Enhanced Chemiluminescent (ECL) System. The level of phosphorylation was determined by
densitometry using Imagequant software on a Molecular Dynamics
densitometer. In each case, three to five different ECL exposures were
subjected to densitometry analysis to reduce the risk of erroneous
results from nonlinear signals. The blots were then stripped (according to ECL protocol, Amersham Pharmacia Biotech) and reprobed with the
3 antibody C3a.19.5 to determine
3
protein content for each sample. Phosphorylation results were
normalized for the total amount of
3 protein
present.
Preparation of Proteins-- Myosin was purified from human platelets as described, and purification yielded myosin heavy chain and light chains (30). Controlled proteolytic digests of platelet myosin with papain or chymotrypsin were performed as described (31) except that myosin was not phosphorylated prior to chymotryptic digestion. Papain was activated according to the instructions of the manufacturer. Digests were run on 4-20% SDS-PAGE and subjected to Coomassie Blue staining or transferred to nitrocellulose for ligand blotting.
Ligand Blot Analysis-- Platelet lysates or purified myosin were reduced, separated by SDS-PAGE, and transferred to nitrocellulose. The blots were wet briefly in HEPES blot buffer (HBB) (25 mM HEPES, 25 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol) at 4 °C. The transferred proteins were denatured by 6 M guanidine HCl in HBB for 10 min at 4 °C and renatured by 2-fold dilution of guanidine HCl (10-min incubations each with 3 M, 1.5 M, 0.75 M, 0.38 M, 0.19 m, and 0 M guanidine HCl in HBB). The blot was blocked in HBB containing 4% bovine serum albumin overnight at 4 °C and probed with 1 µM biotinylated peptide in HBB containing 0.5% bovine serum albumin for 3 h at room temperature. After washing in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4)/ 0.01% Nonidet P-40 three times at 4 °C, peptide-reactive bands were visualized by incubating the blots in horseradish peroxidase-conjugated streptavidin and employing ECL detection.
CHO Cell Generation and Flow
Cytometry--
3(Y747F, Y759F) was generated and stably
transfected into CHO cells as described (32). For flow cytometric
analysis, cells were detached with trypsin, washed in Dulbecco's
modified Eagle's medium + 25 mM HEPES once and resuspended
at 3 × 106 cells/ml in FACS buffer (Hanks buffered
saline solution with 3% heat-inactivated fetal bovine serum, 1%
bovine serum albumin, 1% normal goat serum, 0.1% Gamimmune N, 0.03%
sodium azide). The cells were then seeded at 200 µl/well, pelleted,
and incubated with 5 µg/ml primary antibodies LM609 or control mouse
IgG for 1 h at 4 °C. After two washes, the cells were incubated
with 1:200 goat anti-mouse fluorescein isothiocyanate-conjugated
F(ab')2 for 30 min at 4 °C. The cells were washed and
resuspended in FACS buffer, and the samples were analyzed by flow
cytometry on a FACSort (Becton Dickinson).
Clot Retraction Assays-- Clot retraction experiments were performed as described with minor modifications (33). In brief, cells were trypsinized, washed twice, and resuspended in Dulbecco's modified Eagle's medium + 25 mM HEPES. 0.5 ml of cell suspension containing 5 × 106 cells was mixed with 0.1 ml of fibronectin-depleted plasma in a 12 × 70-mm glass tube treated with Sigmacote. Fibrin clots were formed by adding 1 unit/ml thrombin and allowed to retract at 37 °C over a 2-3-h period. The extent of clot retraction was measured by removing and weighing the clot.
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RESULTS |
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3 Tyrosine Phosphorylation Is a General Consequence
of Platelet Aggregation--
We previously reported that aggregation
of platelets by thrombin in a stirred suspension induced a marked
increase in the tyrosine phosphorylation of the
3
subunit of
IIb
3 (1). To determine whether
this effect was thrombin-specific, we examined
3
tyrosine phosphorylation in response to various agonists. Adding ADP or
ADP + epinephrine to a stirred suspension of platelets in the presence
of added fibrinogen induced platelet aggregation and an increase in
3 phosphorylation, similar to that seen in thrombin-aggregated platelets (Table I).
In contrast, when ADP was added in the absence of added fibrinogen or
was added without stirring, no platelet aggregation occurred and no
increase in
3 tyrosine phosphorylation was seen. ADP
added in this manner did, however, induce platelet stimulation since
other substrates were tyrosine phosphorylated and fibrinogen binding on
unstirred preparations occurred (data not shown). An illustration of
the increase in
3 tyrosine phosphorylation upon
thrombin- or ADP-induced platelet aggregation is shown in Fig.
1A. Thus, ADP and ADP + epinephrine induced tyrosine phosphorylation of
3 in an
aggregation-dependent manner.
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Tyrosine Phosphorylated 3 Preferentially
Redistributes to the Cytoskeleton in Aggregated Platelets--
To gain
insight into the functional significance of tyrosine phosphorylation of
3, we assessed its effects on the partitioning of the
receptor with the cytoskeleton, a process known to occur upon platelet
aggregation (21, 22). Platelets were aggregated by the addition of
thrombin and lysed with Triton X-100 lysis buffer. Under the
solubilization conditions described under "Experimental Procedures," approximately 34% (p = 0.002) of the
total
3 protein associated with the cytoskeletal
fraction upon aggregation, in agreement with earlier studies (21, 22).
Notably, previous work has established that only about 5% of the
IIb
3 is found in the Triton
X-100-insoluble residue of unstimulated platelets (21). Densitometry of
anti-phosphotyrosine immunoblots indicated that approximately 72% of
the tyrosine-phosphorylated
3 redistributes to the
cytoskeletal fraction (Fig. 2). Thus,
tyrosine-phosphorylated
3 is more than twice as likely
to become associated with the cytoskeleton (p = 0.018),
which may indicate a pivotal role for this
3
modification in linking a ligand-occupied receptor on the surface of
aggregated platelets to the cytoskeletal/contractile apparatus
within.
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Doubly Phosphorylated Cytoplasmic Domain 3 Peptide
Binds a 200-kDa Protein Identified as Platelet Myosin--
The above
observation, that tyrosine-phosphorylated
3
preferentially partitions with the cytoskeleton, highlighted the
possibility that phosphorylation of
3 could be involved
in mediating interactions between
3 and elements within
the cytoskeleton. We have found, as have others (3, 4, 6), that the
poor detergent solubility of many of the components of the cytoskeleton
makes it technically difficult to observe interactions between
cytoskeletal and other proteins in vivo. Thus, to address
this issue, we employed an in vitro ligand blotting approach
in an attempt to identify candidate proteins that, by binding to
tyrosine-phosphorylated cytoplasmic domain of
3, could
direct association of
IIb
3 to the
cytoskeleton. Proteins from platelet lysates were separated by
SDS-PAGE, transferred to nitrocellulose and renatured on the blot.
Synthetic peptides corresponding to the cytoplasmic domain of
3, which contained biotin at their amino termini, were
used to probe the nitrocellulose blot
(Fig. 3A). The direct binding
of peptide to renatured proteins was visualized by the addition of
streptavidin-horseradish peroxidase and detected by chemiluminescence.
As illustrated in Fig. 3B, the
3 peptide
corresponding to residues 740-762 in the
3 cytoplasmic domain bound to a 200-kDa protein in ligand blot analysis of platelet lysates. Binding was detected only when both
3 tyrosine
residues (Tyr-747 and Tyr-759) were phosphorylated; the
nonphosphorylated
3 peptide and singly phosphorylated
peptides failed to bind the 200-kDa protein under the conditions of
this assay (data not shown). To test the specificity of binding of the
phosphorylated cytoplasmic domain of
3 to this 200-kDa
protein, a second, doubly phosphorylated
3 peptide
containing a naturally occurring single point mutation of
3 found in a patient with Glanzmann's thrombasthenia,
was used (36).
IIb
3 harboring this
mutation does not support platelet aggregation (36) and is defective in
signaling (37). Despite having two phosphorylated tyrosine residues,
the S752P Glanzmann's mutant peptide failed to bind the 200-kDa
protein in renatured blots. Furthermore, an unrelated diphosphorylated
peptide based on the sequence of the
chain of the T cell receptor
complex containing an immune receptor tyrosine-based activation motif (ITAM) domain, was also negative in this assay.
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The Tyrosine Residues within the 3 Cytoplasmic
Domain Are Important for
3-dependent Fibrin
Clot Retraction--
The results reported above indicate that the
phosphorylation of the
3 cytoplasmic tyrosine residues
might influence integrin-cytoskeletal interactions. This finding
predicts that mutating the tyrosine residues of
3 should
affect cellular functions dependent upon integrin-cytoskeletal
interactions. One such function is the
3-dependent retraction of fibrin clots,
where the integrin is believed to function as a transmembrane linkage
between extracellular adhesion proteins and the cytoskeleton. Due to
the difficulty of genetically manipulating platelets, we used a CHO
cell system that has proven useful in the study of integrin function
(19, 39) to directly address this issue. It has previously been shown
that CHO cells transfected with wild-type
3 will express
the
3 on the cell surface in conjunction with endogenous
v chains (19). In contrast to nontransfected CHO cells,
the
3-transfected CHO cells gain the ability to retract
fibrin clots (Ref. 19, and data not shown). We generated stable CHO
cell lines expressing either wild-type
3 or
3 bearing the conservative Y747F and Y759F mutations. As illustrated in Fig. 5A, FACS
analysis with the
v
3-specific antibody LM609, confirmed that these transfectants expressed similar levels of
v
3 or
v
3(Y747F, Y759F) at the cell surface.
When the two CHO cell lines were used in the fibrin clot retraction
assay, it was found that clot retraction was reduced markedly in the Y747F, Y759F transfectants, as demonstrated by a 50 ± 11.9%
increase in clot weight compared with the clot weights obtained with
the wild-type
3-expressing CHO cells (Fig.
5B).
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DISCUSSION |
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A well established function of integrin cytoplasmic domains is as
a bridge between extracellular matrix proteins and the
cytoskeletal/contractile machinery within a cell. The data presented in
this study indicate that the two tyrosine residues within the
3 cytoplasmic domain may be important in mediating some
of these interactions and suggest a way in which a modification of
these residues, namely by phosphorylation, may also be involved in
these bridging processes. We have found that the tyrosine
phosphorylation of the
3 subunit of
IIb
3 occurs as a general consequence of
platelet aggregation. That this phosphorylation may in turn affect the
interaction of the
3 integrin with the platelet
cytoskeleton is indicated by the following data. First, phosphorylated
3 is located preferentially within the
detergent-insoluble cytoskeletal fraction of aggregated platelets; and
second, the contractile protein myosin can bind directly, in a
phosphotyrosine-dependent manner, to peptide corresponding to the cytoplasmic domain of
3. Furthermore, functional
data obtained by analysis of CHO cells bearing a
3 in
which both cytoplasmic tyrosine residues were mutated to phenylalanines
also indicates the importance of these
3 tyrosines in
the
3-dependent retraction of fibrin clots.
We have previously demonstrated the interaction of
tyrosine-phosphorylated
3 with the signaling proteins
SHC and Grb2 (1) and hypothesized that tyrosine phosphorylation of
3 allowed for the recruitment of signaling complexes to
the membrane. Our new studies indicate that in addition to the role of
3 tyrosine phosphorylation in binding signaling
proteins, the phosphorylation may also influence the interaction of
3 with the myosin-based contractile apparatus and in
doing so play an important role in integrin-dependent
functions involving cytoskeletal reorganization.
The present data demonstrate that platelet aggregation is both
necessary and sufficient to induce tyrosine phosphorylation of
3. First, conditions that only induced the active
conformation of
IIb
3 and did not allow
for aggregation to occur, such as ADP stimulation in the absence of
fibrinogen or in the presence of an inhibitory RGD peptide, did not
induce
3 tyrosine phosphorylation. Second, platelet
aggregation induced by LIBS6 independent of platelet stimulation also
resulted in
3 tyrosine phosphorylation. Third,
3 tyrosine phosphorylation did not occur under
conditions that induced ligand occupancy of
IIb
3 but not platelet aggregation, such
as the addition of ADP and fibrinogen in the absence of stirring. Last,
a reversal of aggregation-induced
3 tyrosine
phosphorylation was observed upon reversal of platelet aggregation. In
all instances, platelet aggregation, with the subsequent
platelet-platelet interactions, was absolutely required for
3 tyrosine phosphorylation.
Early studies established that a significant portion of
IIb and
3 could be isolated with
cytoskeletal structures in thrombin-aggregated, but not activated,
platelets (21), suggesting that platelet aggregation induces the
association of
IIb
3 with the platelet cytoskeleton. It was proposed that this integrin became Triton X-100
detergent-insoluble because of the macromolecular associations between
the platelet membrane surfaces and actin filaments. Morphological studies have also demonstrated that fibrinogen binding to
IIb
3 induced its interaction with the
cytoskeleton since the membrane-bound integrin appeared to be
co-aligned with cytoskeletal structures of the platelet (40). Further,
Fox and co-workers demonstrated an aggregation-dependent
redistribution of
IIb
3 from the membrane skeleton to the Triton X-100-insoluble fraction of platelets (22). Several tyrosine kinases and other tyrosine-phosphorylated proteins also redistributed to the cytoskeleton upon platelet aggregation (22).
The present data points to a possible mechanism for the redistribution
of
IIb
3 to the cytoskeleton in aggregated
platelets. Examination of the phospho-
3 distribution
between Triton X-100 soluble and insoluble fractions of aggregated
platelets demonstrated that phosphorylated
3
preferentially redistributes to the cytoskeletal fraction. The
observations that tyrosine phosphorylation of the
3
cytoplasmic domain is a common consequence of aggregation by a wide
range of platelet agonists and is a potential player in driving the
redistribution of
3 to the cytoskeleton prompted us to
examine biochemically whether
3 phosphorylation plays a part in mediating novel interactions of the receptor with the platelet
cytoskeleton.
The binding of integrin cytoplasmic domains to cytoskeletal proteins is
not unprecedented, and although little in vivo data exist,
due to the technical difficulties of working with poorly soluble
cytoskeletal proteins, a variety of in vitro strategies have
been used to discover and characterize such interactions. Interactions
between talin and 1 integrin cytoplasmic domain were
first studied using equilibrium gel filtration of purified proteins
(3). In solid phase binding assays, talin was found to bind directly
with
IIb
3 integrin cytoplasmic tail
sequences and to purified
IIb
3 (4).
-Actinin has been shown to bind directly to the cytoplasmic domain
of
1 as well as to purified
IIb
3 (6). In another study, the
cytoskeletal protein skelemin interacted with the
3
cytoplasmic domain in a yeast two-hybrid screen and with peptides
corresponding to the membrane proximal regions of
1 and
3 (41). Actin binding protein has also been demonstrated
to bind directly to the cytoplasmic domain of
2 integrin
using peptide affinity chromatography (42) and to the dimerized
1 cytoplasmic domains using a novel experimental
strategy (5). However, the mechanisms that regulate these
integrin-cytoskeletal interactions are unknown.
Given that tyrosine phosphorylation of 3 is a general
consequence of platelet aggregation and appears to direct the
redistribution of
IIb
3 to the
cytoskeleton, we postulate that
3 tyrosine
phosphorylation could be a general mechanism for regulating
integrin-cytoskeletal interactions. Fittingly, members of the Src
family of tyrosine kinases are known to selectively redistribute with a
subpopulation of
IIb
3 to the actin
cytoskeleton in aggregated platelets (22, 23). This redistribution is
reduced by treatment of platelets with tyrosine kinase inhibitors,
suggesting that tyrosine kinases, either directly or through the
phosphorylation of other proteins, may regulate the cytoskeletal
attachment of
IIb
3 (43). Further,
IIb
3-mediated clot retraction is
inhibited by tyrosine kinase inhibitors (43). Although there is
circumstantial evidence that certain integrin-cytoskeletal interactions
are phosphotyrosine-dependent, experimental data directly
supporting this hypothesis are lacking.
In the present work, we used in vitro ligand binding
methodology to observe a novel interaction between myosin and a
3 integrin cytoplasmic domain peptide that was regulated
by tyrosine phosphorylation. Phosphorylated and nonphosphorylated
integrin cytoplasmic domain peptides were synthesized and used to
identify a direct and tyrosine phosphorylation-dependent
interaction between the
3 cytoplasmic domain peptide and
platelet myosin heavy chain. Since the doubly phosphorylated
3 peptide bound specifically to myosin, this contractile protein may possess tandem phosphotyrosine binding regions analagous to
the tandem SH2 domains of the tyrosine kinases ZAP-70 or Syk, which
bind ITAM domains in immune receptor complexes (44, 45). However, to
our knowledge, classic phosphotyrosine binding motifs in platelet
myosin heavy chain have not yet been identified. We further observed
that the tail domain of myosin was responsible for its interaction with
3. Interestingly, the tail region of myosin serves as an
anchor so that it can translocate actin and has been hypothesized to
bind certain myosin isoforms to cell or organelle membranes (46).
Together these data suggest that the tyrosine phosphorylated
3 binding domain of myosin exists on the tail region of
myosin heavy chain and that this domain contains previously
unrecognized phosphotyrosine binding motifs. These binding motifs may
allow for the interaction of phosphorylated
3 with the
cytoskeletons of aggregated platelets, providing alignment for certain
postaggregation contractile events, such as clot retraction, to
occur.
Although our in vitro data strongly suggest that tyrosine
phosphorylation of the 3 cytoplasmic domain can allow
for
IIb
3 interaction with myosin, this
conclusion was not possible to confirm in vivo because of
the aforementioned problems associated with working with many
cytoskeletal proteins. Indeed, myosin is insoluble at physiological
salt concentrations; only highly stringent co-immunoprecipitation conditions could be employed using detergent lysates that are well
known to disrupt protein-protein interactions. In this case, the
problem is compounded by the fact that the major portion of tyrosine
phosphorylated
IIb
3 does itself
translocate to the insoluble cytoskeleton in aggregated platelets.
Also, robust tyrosine dephosphorylation mechanisms are present in
platelets (47), which make it difficult to preserve tyrosine
phosphorylation of
3 except under denaturing conditions
(e.g. by the addition of SDS-containing sample buffer) or in
rapid postlytic fractionations (e.g. cytoskeleton isolation)
hampering immunoprecipitation experiments. Therefore, our data do not
preclude other, possibly phosphotyrosine-independent, interactions
between the cytoplasmic domains of
IIb
3
and myosin. If other such interactions do indeed exist, it is
attractive to hypothesize that tyrosine phosphorylation of
3 cytoplasmic domain, possibly at only one of the
tyrosine residues, could induce a more stable and avid interaction
between previously-associated
IIb
3 and
myosin.
Thus, our data suggest that tyrosine phosphorylation of the
3 cytoplasmic tail may regulate a direct association
with myosin, providing anchorage of surface
3 integrins
to the contractile apparatus. A possible consequence of this
interaction is to allow for
IIb
3-mediated
clot retraction in platelets. We addressed the role of the
3 cytoplasmic tyrosines in clot retraction using CHO
cells transfected with
3. Previous studies using such a
CHO cell expression system have proven useful for analyzing the role of
both
IIb and
3 integrin cytoplasmic
domains in
IIb
3 signaling and adhesive
functions (17-19, 36). In particular, CHO cells transfected with
IIb
3 gain the ability to contract fibrin
clots, whereas both untransfected CHO cells and cells expressing the S752P Glanzmann's mutant
IIb
3 fail to do
so (36). Another expression system, in which CS-1 melanoma cells are
transfected with a cDNA encoding the integrin
3
subunit, has been used to characterize the roles of the
v
3 integrin cytoplasmic domains in
adhesion, spreading, and migration on vitronectin (20). Although clot
retraction was not addressed in this study, mutating either tyrosine
747 or 759 on
3 to phenylalanine had little or no effect on other
v
3 adhesive events (20). In the
present work, CHO cells bearing the Y747F and Y759F
3
cDNA displayed a pronounced defect in fibrin clot retraction: the
first demonstration of an effect of
3 tyrosine to
phenylalanine mutations on a biologically relevant event. Since
integrins are believed to support clot retraction by providing the
transmembrane linkage between extracellular adhesive proteins and the
contractile cytoskeleton (19), it is interesting to hypothesize that,
by mutating the tyrosine residues within the
3
cytoplasmic domain, we have disrupted the
phosphotyrosine-dependent integrin-myosin interaction and
that this could account for the defective clot retraction observed in
the mutant CHO cell transfectants.
Our working hypothesis of the role of 3 cytoplasmic
domain tyrosine phosphorylation in platelet function can be summarized as follows: the receptor is phosphorylated as a common consequence of
aggregation in response to a number of platelet agonists. Although direct associations of known tyrosine kinases with
IIb
3 have not yet been detected, members
of the Src family of tyrosine kinases are capable of phosphorylating
the receptor in vitro (1) and Src and Lyn can be
cross-linked to
3 in intact platelets treated with
chemical cross-linking agents (48). Once phosphorylated, the
3 integrin tails are capable of associating with
signaling proteins SHC and Grb2 to potentially initiate outside-in
signaling cascades (1). In addition to providing a scaffold for the
recruitment of signaling complexes to the membrane, the doubly
phosphorylated cytoplasmic domain of
3 can also bind to
cytoskeletal proteins. In particular, the present work demonstrates
direct binding of a doubly tyrosine-phosphorylated
3
integrin cytoplasmic domain peptide to myosin and further reveals that
replacement of these tyrosine residues with phenylalanines in
IIb
3-transfected CHO cells results in
defective
3 integrin-mediated retraction of fibrin
clots. In light of these data, we postulate that phosphorylation of
3 integrin cytoplasmic domain may be an important
mechanism for regulating a direct myosin-integrin interaction.
Inhibition of this interaction may interfere with the transmission of
the mechanical forces that regulate processes such as clot retraction and cell motility. Proving or disproving such hypotheses will be the
focus of future work.
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ACKNOWLEDGEMENT |
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The assistance of Liping Gao in tissue culture work is gratefully acknowledged.
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Note Added in Proof |
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While this manuscript was in review, a
manuscript was published which also reported effects of
3 tyrosine mutations on clot retraction (Blystone,
S. D., Williams, M. D., Slater, S. E., and Brown,
E. J. (1997) J. Biol. Chem. 272, 28757-28761).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL 48728 (to M. H. G).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.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: COR Therapeutics, Inc., 256 E. Grand Ave., South San Francisco, CA 94080. Tel.: 650-244-6884; Fax: 650-244-9270; E-mail: david_phillips{at}corr.com.
1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HBB, HEPES blot buffer; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; ITAM, immune receptor tyrosine-based activation motif.
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REFERENCES |
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