From the Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595 and the § Department of Biology, University of Oslo, 0317 Oslo, Norway
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ABSTRACT |
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Protein seryl/threonyl phosphatase inhibitors
such as calyculin A block inside-out and outside-in platelet signaling.
Our studies demonstrate that the addition of calyculin A blocks
platelet adhesion and spreading on fibrinogen, responses that depend on integrin Effective hemostasis depends on three basic platelet responses
(1). First, adhesion, which includes attachment and spreading reactions, initiates events that lead to formation of a hemostatic plug. Second, secretion of factors aid in the activation of additional platelets and in the repair of the damaged vessel wall. Third, aggregation of recruited platelets to each other and to adhered platelets results in growth of the hemostatic plug. Both adhesive and
aggregatory responses are controlled by the coordinate actions of cell
surface glycoprotein receptors. Many of these receptors are dimers of
two different subunits (2). These include selective members of the
integrin family of cell adhesion molecules such as
The Suggesting that a phosphorylation event controls
Previous studies have shown that calyculin A, a serine/threonine
phosphatase inhibitor, inhibits platelet aggregation, which is
dependent on Materials--
Prostaglandin E1, apyrases,
aprotinin, leupeptin, benzamidine, protein A-Sepharose, human
fibrinogen, low molecular weight heparin (Mr
aprox. 3,000), phenylmethylsulfonyl fluoride, sodium vanadate, and
dithiothreitol were obtained from Sigma.
[32P]Orthophosphate was purchased from DuPont NEN. LC
Services supplied calyculin A. Rhodamine-labeled phalloidin was
purchased from Molecular Probes (Eugene, OR). Boehringer Mannheim
supplied sequencing grade AspN protease and trypsin. Hirudin (BKHV
recombinant) was obtained from Calbiochem. The C18 reverse phase
TSK-GEL ODS-120T column was purchased from Toso Haas (Montgomeryville,
PA). Anti- Platelet Preparations--
Human blood (10 parts) was drawn into
acid-citrate-dextrose (1 part). Platelet-rich plasma was obtained after
centrifugation at 210 × g for 10 min at 21 °C.
After the addition of prostaglandin E1 (0.4 µM) and apyrases (12.5 milliunits/ml), platelets were removed by centrifugation at 1100 × g for 15 min at
21 °C. Platelets were resuspended in a modified Tyrode's buffer, pH
6.4, containing 5 mM HEPES, 140 mM NaCl, 1 mM MgCl2, 2 mM KCl, 5.5 mM glucose, 12 mM NaHCO3, 0.2%
(w/v) bovine serum albumin, 0.2 µM prostaglandin E1, and apyrases (6 milliunits/ml), recentrifuged at
1100 × g, and suspended in a buffer containing 10 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM
MgCl2, 2 mM KCl, 5.5 mM glucose,
and 12 mM NaHCO3 at a concentration of 2 × 109/ml.
32P Labeling of Phosphoamino Acid Analysis--
Phosphoproteins extracted from
SDS gels were hydrolyzed in 6 N HCl for 1 h at
110 °C, and phosphoamino acids were separated by two-dimensional
electrophoresis on cellulose plates using 2.5% formic acid and 7.8%
acetic acid, pH 1.9, in the first dimension and 0.5% pyridine and 5%
acetic acid, pH 3.5, in the second dimension (24).
Identification of Phosphorylation Sites--
Immunoprecipitated
32P-labeled Static Platelet Adhesion Studies--
Washed platelets (5 × 108/ml) were treated with either buffer or 100 nM calyculin A for 5 min at 21 °C. After the addition of
buffer or A23187 (0.25 µM), the platelets were added to
fibrinogen-coated slides and allowed to settle for 20-30 min. In some
experiments, apyrase (0.5 units, Sigma) or ADP (10 µM,
Sigma) were included in the incubations before adding the cells to the
slides. Adhered platelets were processed for confocal microscopy as
described below.
Perfusion Adhesion Studies--
Perfusions were conducted using
one of three parallel plate perfusion chambers as described previously
(26). Blood was drawn into acid-citrate-dextrose at a ratio of 1:10
(acid-citrate-dextrose:blood), hirudin (200 units/ml), or low molecular
weight heparin (6 units/ml). Under the flow conditions described below,
platelets adhered poorly in the presence of low molecular weight
heparin, an anticoagulant that does not chelate calcium
(n = 3),2
precluding its use in subsequent studies. This is analogous to its
effect on platelet adhering to fibronectin (32). The blood (4 ml) was
prewarmed at 37 °C for 10 min before the addition of dimethyl
sulfoxide (Sigma, 0.001% final) or varying concentrations of calyculin
A. After an additional 5-min incubation at 37 °C, the blood was
perfused through the chamber containing a fibrinogen-coated coverslip
positioned 70 mm from the inlet valve. Whole blood was used within
3 h of withdrawal. Flow was conducted using two different approaches. In one approach, blood was allowed to recirculate for 10 min at 10 ml/min through an eight-roller precalibrated peristaltic pump
(Cole Parmer). Under these conditions, wall shear rates of 650 and
100/s were achieved. Alternatively, whole blood passed through the
chambers one time at a constant flow rate for 1-2 min. Flow rates of 1 and 2 ml/min produced 65 and 520/s wall shear rates, respectively,
which corresponds to flow in veins and small arteries. When indicated,
platelet-rich plasma was prepared by centrifugation at 1100 × g for 3 min at 21 °C. Platelet counts in the
platelet-rich plasma were standardized to 350,000/µl using a modified
Tyrode's buffer. Calyculin A treatment and perfusion of platelet-rich
plasma through the chamber were conducted as described for whole blood.
For preparation of surfaces for flow studies, suspensions of 0.1 mg/ml
fibrinogen, prepared in phosphate-buffered saline, were sprayed onto
plastic coverslips using a retouching airbrush (model 100; Badger,
Franklin Park, IL) at a nitrogen pressure of 1 atm as described
previously (26). Coverslips (18 × 22 mm) were spray-coated with
~300 ul to a final surface density of ~5 µg/cm2. The
coverslips were kept at 21 °C for up to 12 h. Before perfusion with whole blood or platelet-rich plasma, coverslips were washed for 5 min with a modified Tyrode's buffer, pH 7.4. For static adhesions,
drops of the fibrinogen solution were pipetted onto glass slides and
dried at room temperature. The matrix was washed with the modified
Tyrode's buffer before the adhesion assay. After perfusion with blood,
the coverslips were immediately washed under flow conditions with the
modified Tyrode's buffer for 1 min. The coverslips were removed, fixed
in a 2.5% glutaraldehyde solution for 15 min, and washed twice with
phosphate-buffered saline. After permeabilization with 0.1% Triton
X-100 for 3 min, platelets were washed twice with TBS, blocked for 3 min with 0.1% bovine serum albumin, and stained with
rhodamine-conjugated phalloidin. Adhered platelets were imaged using a
Bio-Rad MRC-1000 confocal microscope. Platelets were viewed after
excitation with an argon laser beam using either an inverted- or
upright-staged microscope equipped with a 60× oil immersion or 40×
dry objective and an epifluorescent illumination attachment. The
percentage of area covered by platelets was determined using a
digitized palette and Sigmascan software.
To determine the effect of calyculin A on platelet adhesion in a
physiological setting, we used flow conditions. Citrated blood was
treated with either vehicle or 100 nM calyculin A and recirculated through a parallel plate perfusion chamber at a wall shear
rate of 650/s. The percent area covered by vehicle-treated platelets
was ~40% (n = 5) on fibrinogen-coated surfaces (Fig. 1A). Adhesion was blocked by
80 ± 5% (mean ± S.E.; n = 4) by
preincubating the blood with 100 nM calyculin A (Fig.
1B). Similarly, calyculin A added directly to platelet-rich
plasma inhibited platelet deposition to fibrinogen, establishing that
the drug was directly affecting platelets.
As seen in Fig. 1, substantial aggregation occurred when a
recirculating system was used. This likely reflects the activation of
platelets as a consequence of being exposed to the fibrinogen matrix
and to activating agents release from adhered cells. To minimize this
aggregatory response, whole blood was passed through the chambers one
time at wall shear rates of 65 and 520/s. Under these conditions
platelets adhered as singlets (Fig. 2,
A and C). Regardless of the flow rate, a decrease
in deposition by >90% (n = 4) was observed by
pretreating whole blood with calyculin A (Fig. 2, B and
D). The decrease in platelet adhesion in citrated blood
occurred in the presence of 100 nM calyculin A. As
discussed later, the response to calyculin A occurs at a very sharp
concentration threshold. These findings were confirmed using varying
preparations of calyculin A.
The dramatic effect of phosphatase inhibitors on the adhesive
properties of platelets observed in citrated blood might possibly be
exaggerated by the effects of citrate itself in the experimental system. It is known that divalent cations modulate the binding of
Adhesion represents the attachment and spreading of platelets on
matrices. This latter response confers tight adhesion, allowing platelets to withstand high shear forces associated with arterial and
venous flow. To test whether the effect of calyculin A on platelet
adhesion seen under flow conditions correlates with decreased platelet
spreading, we monitored the effect of calyculin A on platelet spreading
to fibrinogen. On adherence, >90% nontreated platelets spread (Fig.
3A). In contrast, calyculin
A-treated platelets adhered, but only as singlets (Fig. 3B).
Thus, under conditions in which type 1 and 2A phosphatases are
inhibited, platelets attach but do not spread. Their ability to attach
was further demonstrated by Western analysis of adhered "control"
and "calyculin A-treated" platelets using Identifying Phosphorylation Sites in
Phosphoamino acid analysis of
Direct sequence analyses of other peptide peaks obtained from the same
AspN digests gave recoveries of ~5-8 pmol (data not shown). Because
these peptides represented extracellular regions of Conclusion--
The present study tests the hypothesis that
decreased type 1 and 2A phosphatase activity affects platelet responses
by altering the structure of IIb
3 signaling. We
hypothesized that this reflects a change in
IIb
3 structure caused by a specific state
of phosphorylation. We show that addition of calyculin A leads to
increased phosphorylation of the
3 subunit, and
phosphoamino acid analysis reveals that only threonine residues become
phosphorylated; sequence analysis by Edman degradation established that
threonine 753 became stoichiometrically phosphorylated during
inhibition of platelet phosphatases by calyculin A. This region of
3 is linked to outside-in signaling such as platelet
spreading responses. The effect of calyculin A on platelet adhesion and
spreading and on the phosphorylation of T-753 in
3 is
reversed by the calcium ionophore A23187, demonstrating that these
effects of calyculin A are not generally toxic ones. We propose that
phosphorylation of
3 on threonine 753, a region of
3 linked to outside-in signaling, may be a mechanism by
which integrin
IIb
3 function is regulated.
INTRODUCTION
Top
Abstract
Introduction
References
2
1 (collagen-binding),
6
1(laminin),
5
1 (fibronectin), and
IIb
3 (fibrinogen and von Willebrand
factor) (for review, see Ref. 1).
IIb
3 complex (glycoprotein IIb-IIIa)
is unique among integrin molecules expressed on platelets; it functions
as a binary switch. On resting platelets,
IIb
3 exists in an inactive conformation in which the binding pocket for soluble forms of fibrinogen and von
Willebrand factor is cryptic. Thus, before activation, the interaction
of
IIb
3 is limited to surface-bound
fibrinogen, which supports adhesion under low shear forces (2-5). As a
consequence of platelet activation,
IIb
3
converts to an active conformation (via a process referred to as
inside-out signaling) and becomes capable of binding other
surface-bound molecules and soluble forms of fibrinogen and von
Willebrand factor (6). This latter activity facilitates platelet
aggregation. The molecular events that switch
IIb
3 into a high affinity state are not
known. Structural correlations have identified potential cytoplasmic
motifs in
3 that regulate its activation. First, a
naturally occurring mutation in the cytoplasmic domain of
IIb
3 negatively modulates its activity:
serine 752 to proline mutation in the cytoplasmic portion of
3, a variant associated with Glanzmann's
thrombasthenia, inhibits
IIb
3 signaling (7, 8). Second, site-directed mutagenesis of Asp723 in
3 effectively disrupts potential salt bridges between
the
and
subunits and results in a constitutively active
integrin molecule (9). Thus, it appears that changes in charge in the cytoplasmic portion of
3 may have important consequences
with respect to integrin structure and function. Phosphorylation of
3 on a threonine residue, which would change the charge
in
3, has been implicated in exposing binding sites on
IIb
3 (10, 11), although the role that
this phosphorylation plays in integrin function is unknown (12).
IIb
3 activation is in accordance with
protein phosphorylation events regulating numerous platelet responses,
including adhesion and aggregation (13). Phosphorylation of proteins on
serine, threonine, and tyrosine residues is controlled by the competing
activities of protein kinases and phosphatases. Thus, increased
phosphorylations can represent increased kinase activity or decreased
phosphatase activity. Platelets contain numerous protein kinases (13)
and protein seryl/threonyl phosphatase types 1, 2A, and 2B (14, 15).
Type 1 and 2A protein phosphatases are active in resting and stimulated
platelets (14) whereas type 2B, a
Ca2+-dependent, calmodulin-stimulated enzyme,
is only active after platelet activation (15). To understand the
physiological significance of these enzymes in platelets,
membrane-permeable inhibitors have been used. Type 1 and 2A
phosphatases have been implicated in controlling platelet responses
including aggregations (16-23). Very little is known, however, about
how these enzymes modulate molecular events linked to the aggregatory response.
IIb
3 function. The present
study tests whether calyculin A affects other
IIb
3 functions and tests the hypothesis that altered
IIb
3 function is linked to
altered structure of this integrin. The data show that, in addition to
an effect on aggregation, calyculin A blocks platelet adhesion and
spreading on fibrinogen, reactions that depend on
IIb
3 outside-in signaling. Biochemical
analysis links calyculin A effects to
3 phosphorylation. Sequence analysis demonstrates that threonine 753, which is in the
intracellular segment of
3, becomes stoichiometrically
phosphorylated. These data suggest that structural modification of
3, through phosphorylation, may regulate outside-in, and
perhaps inside-out, signaling through the
IIb
3 integrin.
EXPERIMENTAL PROCEDURES
3 polyclonal antiserum (E8) was a gift from
Drs. David Phillips and Debbie Law (COR Therapeutics, San Francisco, CA).
3--
Platelet
suspensions (4 ml) were incubated with
[32P]orthophosphate (0.2 mCi/ml) for 3 h at 37 °C
before treatment with calyculin A (100 nM) for 5 min.
Platelets were directly solubilized using Nonidet P-40 lysis buffer
containing 1% Nonidet P-40, 137 mM NaCl, 20 mM
Tris-HCl, pH 8.0, 2 mM EDTA, 1 mM sodium
vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 0.15 units/ml aprotinin, and 10 mM
-nitrophenyl phosphate. Detergent-insoluble material
was removed by centrifugation at 10,000 × g for 5 min, and the resulting soluble fraction was incubated with anti-
3 (10 µg/mg of lysate) for 16 h at 4 °C followed by a 1-h
incubation with protein A-Sepharose at 4 °C. The immunoprecipitates
were washed three times with Nonidet P-40 lysis buffer and solubilized with SDS sample buffer. Proteins were separated by 10%
SDS-PAGE,1 stained using
Coomassie Blue, destained, and either dried for autoradiography or
maintained hydrated for efficient extraction of
3 for
identification of phosphorylation sites.
3 was reduced and alkylated by
the method of Talmadge et al. (29) before SDS-PAGE. The
Coomassie Blue-stained
3 was cut from the gel using a
scalpel and digested with AspN in situ and extracted (25).
The extracts were concentrated under vacuum, resuspended in 0.1%
trifluoroacetic acid in 6 M guanidine-HCl, and fractionated
on a 120T C18 reverse phase column (reverse phase HPLC), collecting
fractions by peak detection (30). Isolated peptides were sequenced by
automated Edman degradation on a model 477/120A pulse liquid sequencer
(Applied Biosystems) using standard chemistry.
RESULTS AND DISCUSSION
3 Phosphorylation Correlates with Decreased
Outside-in Signaling--
Previous studies have shown that calyculin
A, an inhibitor of protein seryl/threonyl phosphatase types 1 and 2A,
markedly reduces platelet aggregation induced by collagen (18) and low doses of thrombin (17). These data support the idea that calyculin A
blocks inside-out signaling linked to
IIb
3. In the presence of high thrombin
doses, calyculin A does not inhibit aggregation but does block
cytoskeletal assembly that is usually coupled to an aggregatory
response (17). This indicates that calyculin A blocks outside-in
signaling by activated
IIb
3. To further test this hypothesis, we examined the effect of calyculin A on platelet
adhesion to immobilized fibrinogen, a process that depends on
outside-in
IIb
3 signaling. Platelet
adhesion was measured in both flow and static assays.
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Fig. 1.
Effect of calyculin A on platelet adhesion to
fibrinogen under flow. Citrated blood was treated with dimethyl
sulfoxide (0.001%) or 100 nM calyculin A for 5 min before
perfusion over fibrinogen-coated coverslips. Perfusion at 650/s was
conducted in a recirculating setting for 10 min, and adhered cells were
viewed with a 60× immersion objective. The data are representative of
five experiments.
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Fig. 2.
Effect of calyculin A on platelet adhesion:
single pass conditions and various anticoagulants. Blood drawn
into acid-citrate-dextrose (ACD; A-D) or hirudin
(E and F) were treated without (A, C,
and E) or with (B, D, and F) 100 nM calyculin (Cal A). Perfusions were conducted
for 2 min at 65/s (A and B) or for 1 min at 520/s
(C-F), and adhered cells were viewed with a 40× objective.
The data are representative of three to five experiments.
IIb
3 to fibrinogen, and it is possible
that citrate, as a divalent cation chelator, is modifying platelet
interactions that are exaggerating the effect of calyculin A (31). To
determine whether the effect of phosphatase inhibition was dependent on citrate, we repeated the adhesion studies using the anticoagulant hirudin, which is not an ion chelator. Platelets from hirudin-treated blood adhered as singlets in the absence of calyculin A (Fig. 2E; n = 2), and, as with the citrated blood,
adhesion was reduced by 90% in the presence of calyculin A (Fig.
2F; n = 2). Therefore, the effect of
phosphatase inhibition by calyculin A on platelet adhesion is not
dependent on the anticoagulant, strongly suggesting that
phosphorylation plays a direct role in regulating platelet adhesion
under physiological conditions.
3
antibodies; similar levels of protein were found, indicating that equal
numbers of platelets adhered (data not shown). Increased spreading of
calyculin A-treated platelets can be restored by applying calcium
ionophore (A23187) to the platelet suspension before they settle onto
fibrinogen. The addition of 0.25 µM A23187 to a
suspension of calyculin A-treated platelets resulted in increased
platelet spreading (Fig. 3C). The reversal of calyculin A
inhibition by A23187 demonstrates that inhibition of phosphatases has a
specific effect on platelet functions. The apparent effect of A23187
did not result from the release of ADP; the addition of apyrases did
not reverse the ionophore effect, and the addition of 10 µM ADP did not overcome the effect of calyculin A. The
effect of the calcium ionophore appears to be
calmodulin-dependent; trifluoperazine prevents the effect
of A23187 (Fig. 3D).
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Fig. 3.
Light micrographs of platelet adhesion to
fibrinogen under static conditions: effect of A23187 on reversing the
effect of calyculin A. Washed platelets were treated with dimethyl
sulfoxide (A) or 100 nM calyculin A (Cal
A; B-D) for 5 min before treatments with A23187 alone
(C), or A23187 plus 10 µM trifluoperazine
(D). Platelets were allowed to settle onto fibrinogen-coated
slides for 20 min. Nonadhered cells were removed by aspiration, and the
adhered cells were fixed, permeabilized, stained with
phalloidin-labeled rhodamine, and visualized using confocal microscopy
with a 60× immersion lens. These images are representative micrographs
of four to six separate experiments. The phosphorylation states of
3 from cells treated with buffer (A),
calyculin A alone (B), or calyculin A and A23187
(C) are shown adjacent to each panel title. Phosphorylation
was determined by immunoprecipitation as described under
"Experimental Procedures," and the bands corresponding to
3 are shown.
IIb
3--
Because the platelet responses
affected by calyculin A are mediated by
IIb
3, we hypothesized that
hyperphosphorylation of
IIb
3 could be the
structural change that modifies
IIb
3
function. Thus, studies were undertaken to determine the
phosphorylation state of
IIb
3 in
calyculin A-treated platelets. The cytoplasmic portion of
3 (residues 716-762) contains 8 potential serine and threonine phosphorylation sites: 7 threonines and 1 serine (27). To
determine its phosphorylation state,
3 was
immunoprecipitated from platelets that had been labeled with
[32P]orthophosphate and treated with calyculin A. Immunoprecipitated
3 was analyzed by SDS-PAGE followed
by autoradiography. As seen in Fig. 3, little
3 was
phosphorylated in the absence of calyculin A (Fig. 3A),
whereas a loss of phosphatase activity by calyculin A treatment led to
increased phosphorylation of
3 (Fig. 3B). This correlated with a decrease in spreading. The addition of a calcium
ionophore, which partially restores spreading, caused a corresponding
dephosphorylation in
3 (Fig. 3C). The major
protein band that becomes phosphorylated by calyculin A treatment is
3, as identified by Western analysis (Fig.
4A). Maximal phosphorylation occurred at 100 nM calyculin A (Fig.
5; n = 3), a
concentration that inhibits adherence to fibrinogen (as shown in Figs.
1, 2, and 5) and collagen-induced aggregations (18). The same
concentration of ionophore that reversed the effect of calyculin A on
platelet spreading decreased the phosphorylation of
3
(shown in Fig. 3), which is likely attributable to the activation of
the calmodulin-dependent phosphatase, protein phosphatase
2B. Together, the data indicate that an inverse relationship exists
between
3 phosphorylation and platelet adhesion
reactions.
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Fig. 4.
Phosphorylation of 3 in
calyculin A-treated platelets. A,
[32P]-labeled platelets were incubated with either
vehicle (lane 1) or 100 nM calyculin A
(lane 2) for 5 min. Immunoprecipitated
3 was
subjected to SDS-PAGE and visualized by autoradiography.
3 was identified by Western analysis as the
phosphorylated band migrating at ~110 kDa. The entire gel is
shown in A. This autoradiogram is representative of six
different experiments. B, phosphorylated
3
was subjected to phosphoamino acid analysis as described under
"Experimental Procedures." PT, phosphothreonine;
PS, phosphoserine; PY, phosphotyrosine.
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Fig. 5.
Dose responsiveness of 3
phosphorylation and platelet adhesion to calyculin A. Washed
32P-labeled platelets and whole blood were treated with
varying concentrations of calyculin A before phosphorylation studies or
adhesion assays, respectively. In both cases, calyculin A treatment
lasted 5 min. Bands corresponding to immunoprecipitated
3 are shown and are representative of three different
experiments. For platelet adhesions, perfusions were conducted for 2-8
min using both recirculating and single pass conditions and chambers
that mimic arterial flow. Platelet adhesion values are means ± S.E. of three independent experiments.
3 showed the presence of
only phosphothreonine (Fig. 4B). The sites of
phosphorylation of
3 were determined directly by a
two-dimensional peptide isolation and sequencing approach.
3 was immunoprecipitated from 32P-labeled
platelets exposed to 100 nM calyculin A, reduced,
alkylated, and separated from coprecipitating polypeptides by SDS-PAGE.
The Coomassie Blue-stained
3 band was excised and
digested in situ with AspN. Proteolytic fragments were
separated by reverse phase HPLC. Peptide peaks (and gaps between peaks)
were collected, and their content of 32P was determined by
Cerenkov counting. Two prominent 32P-labeled peptides were
recovered (Fig. 6A,
N-1 and N-2). Approximately 40% of the
32P was recovered in N-1, and 60% was recovered in N-2.
These radiolabeled peptide fractions were digested with trypsin and
refractionated by reverse phase HPLC. All the radioactivity from each
digest was recovered in single peaks, indicating the presence of only one phosphopeptide from each digest. Both of these phosphopeptides were
sequenced, and the resulting analysis revealed that both peptides were
residues 749-760 of
3 (Fig. 6A). The
sequences of these peptides differed in that the peptide generated from N-1 was missing a detectable signal at threonine 753, whereas the
peptide generated from N-2 was missing the threonine residues at
positions 751 and 753. Because a phosphorylated residue is not released
for detection in the Applied Biosystems protein sequencers, the absence
of a signal at these positions is consistent with these threonine
residues being the sites of phosphorylation. Using quantitative
regression to extrapolate the initial yield of each peptide (Fig.
6B), the singly (Thr753) phosphorylated peptide,
N-1, was ~ 3 pmol, and the doubly (Thr751 and
Thr753) phosphorylated peptide, N-2, was ~2 pmol. The
amount of peptide 32P sequenced was initially 160 cpm for
N-1 peptide and 230 cpm for N-2 peptide, and, assuming equal
efficiencies for initial coupling during sequencing of these samples,
the specific activity for N-1 is 53 cpm/pmol, and that for N-2 is 115 cpm/pmol. These results are consistent with one phosphorylation site in
peptide N-1 and two in peptide N-2; this independently confirms the
phosphothreonine assignment derived from direct Edman sequencing.
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Fig. 6.
Sequence analysis of 3
phosphorylation sites. A, 32P-labeled
3 was excised from gels, subjected to digestion with
AspN protease, and chromatographed by reverse phase HPLC. Peptides were
eluted with an acetonitrile gradient, and phosphopeptides were detected
by Cerenkov counting. Peptides N-1 and N-2 were further digested with
trypsin and subjected to sequence analysis. The amino acid sequences of
the tryptic fragments generated from N-1 and N-2 are presented in the
box. Similar results were obtained in two separate
experiments. B, quantitative regression analysis of amino
acids released from the tryptic peptides of N-1 and N-2 during each
cycle of the Edman degradation. The recovery of threonines in cycle 5 from N-1 and cycles 3 and 5 for N-2 were below delectability (<0.1
pmol).
3
not related to phosphorylation, the recovery of phosphorylated peptides
(~5 pmol) is consistent with a nearly stoichiometric phosphorylation
(63-100%) of platelet
3. Thus, inhibiting protein seryl/threonyl phosphatase activity in platelets results in the phosphorylation of almost all
3 molecules at
Thr753 and the phosphorylation at Thr751 in
~50% of
3 molecules.
IIb
3.
Indeed, the addition of calyculin A decreases platelet adhesion and
spreading on fibrinogen. A major finding of these studies is that
phosphatase inhibition leads to increased phosphorylation of
3 on threonine residues, linking this phosphorylation event to decreased outside-in signaling. These data suggest that protein phosphatases are critical for regulating
IIb
3 activity. The phosphorylation of
3 occurs on threonine residues adjacent to serine 752. Given that a point mutation of this serine to proline adversely affects
IIb
3 function and is linked to reduced
aggregation (7), outside-in signaling (8), and ability of
3 to recruit signaling proteins (33), it is tempting to
speculate that phosphorylation of threonines 753 and 751 disrupts
IIb
3 signaling in an analogous fashion.
Finally, being that threonine 753 in
3 is conserved in
the other
-integrin subunits (28), we hypothesize that
phosphorylation of this conserved residue may be a common mechanism to
negatively modulate integrin adhesiveness to ligands. In support of
this, preliminary studies indicate that calyculin A blocks platelet adhesion to collagen and laminin, which bind via integrins that contain
1 subunits.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. David Phillips and Debbie Law
for kindly providing anti-3 antiserum and Dr. Phillips
for critically evaluating the manuscript, Drs. Sansar Sharma and Marisa
Cotrina for expertise with confocal microscopy, Anne Marie Snow for
preparation of the figures, and Steve Mills, Kelly Pun, and Paul Thur
for discussions throughout these studies.
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FOOTNOTES |
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* This work was supported by Grant HL4489301 from the NHLBI, National Institutes of Health, and Grant NS29542 from the NINDS, National Institutes of Health (to V. A. F.).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.
Recipient of a Genentech established investigatorship from the
American Heart Association. To whom correspondence should be addressed:
Dept. of Cell Biology and Anatomy, Basic Science Bldg., New York
Medical College, Valhalla, NY 10595. Tel.: 914-594-4097; Fax:
914-594-4653; E-mail: ken_lerea{at}nymc.edu.
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.
2 R. I. Kirk and K. M. Lerea, unpublished observation.
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REFERENCES |
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