From the
The major platelet integrin
Platelets are nonproliferative, terminally differentiated cells,
which offer an attractive model system to study the various biochemical
events leading to structural and functional alterations in activated
cells. When platelets are exposed to stimuli such as thrombin and
collagen, platelets become activated, undergo a dramatic shape change,
adhere to each other, and aggregate. In these processes, the major
platelet integrin
Protein-tyrosine phosphorylation has been
implicated in most biochemical events that involve transmembrane
signaling and cytoskeletal reorganization. Agonist-stimulated platelets
show rapid changes in tyrosine phosphorylation of multiple
proteins
(5, 6) , some of which are dependent on platelet
aggregation mediated through the binding of immobilized fibrinogen to
However,
little information exists about the physiological role or regulation of
PTPases. Like the PTK family, PTPases are grouped into two forms:
non-transmembrane cytoplasmic PTPases and receptor-type transmembrane
PTPases
(17) . Non-transmembrane PTPases consist of a single
catalytic domain and N- or C-terminal extensions, which are important
for enzymatic regulation and intracellular localization. For example,
PTP1B
(18) and T-cell PTPase
(19) are localized to the
endoplasmic reticulum or the particulate fraction via C-terminal
targeting sequences, whereas other non-transmembrane PTPases, PTP1C
(20) (also known as SH-PTP1C, HCP, SHP, or PTP6N; Refs. 21-24)
and SH-PTP2
(25) (also called PTP2C, SH-PTP3, Syp, or PTP1D;
Refs. 26-29), contain two Src homology 2 (SH2) domains. PTP1C is
expressed predominantly in hematopoietic cells
(22) , whereas
PTP1B and SH-PTP2 are expressed ubiquitously
(18, 25) .
Until now, two forms of non-transmembrane PTPases have been documented
in platelets
(30, 31) , whereas receptor-type
transmembrane PTPases, such as CD45, have not been found in
platelets
(32) .
We previously reported that
When we first studied
protein-tyrosine phosphorylation of subcellular fractions after
platelets were activated and lysed in the lysis buffer that had been
conventionally used in previous
studies
(14, 16, 34) , we experienced difficulty
in recovering the same tyrosine dephosphorylation patterns in
subcellular fractions as were observed in the whole cell lysate.
Tyrosine-phosphorylated proteins were recovered mostly in the
cytoskeleton, but there was little tyrosine dephosphorylation observed
on these proteins. This suggested to us that some PTKs were still
active in the platelet lysate for unknown reasons, while PTPases were
inactivated by orthovanadate included in the lysis buffer. Therefore,
we tried to modify the conventionally used lysis buffer by adding
various inhibitors in order to preserve tyrosine dephosphorylation
following platelet lysis. Although inhibitors of PTKs, such as
genistein or tyrphostin, were expected to be suitable for this purpose,
they were found to be insufficient to prevent protein-tyrosine
phosphorylation in the platelet lysate in our preliminary experiments.
The addition of cytochalasin D to the lysis buffer enabled us to
show the reproducible patterns of tyrosine dephosphorylation in the
cytoskeleton with similar kinetics to those observed in the whole
lysate. This suggests that actin polymerization is still persistent,
even after platelets are lysed, and that newly polymerized cytoskeleton
keeps associated PTKs active and maintains protein-tyrosine
phosphorylation. It is possible that ATP required for such
phosphorylation could be supplied from the platelet dense granules
which contain abundant ATP
(41) after platelets are lysed. Thus,
we demonstrated for the first time the presence of tyrosine
dephosphorylation in the cytoskeleton which was dependent on
Frangioni et al.(30) have recently shown that
The occurrence of dephosphorylation of
tyrosine-phosphorylated proteins in the cytoskeleton suggests that the
responsible PTPases must be associated with the cytoskeleton to act on
their substrates. Among the PTPases investigated in this study, we
found that PTP1B and PTP1C were associated with the cytoskeleton in
activated platelets, but SH-PTP2 was not. Interestingly, not only
intact PTP1B but also its cleaved 42-kDa form were present in the
cytoskeleton of aggregated platelets. It appears that PTP1B itself
could be responsible for more PTPase activity than its cleaved form for
dephosphorylation of tyrosine-phosphorylated proteins, since the
inhibition of PTP1B cleavage by calpain inhibitors did not affect
tyrosine dephosphorylation of those proteins. However, there is still
the possibility that the cleaved 42-kDa form associated with the
cytoskeleton could elicit PTPase activity on tyrosine-phosphorylated
proteins that were not detected in this study. Furthermore, it is
tempting to speculate that PTP1B cleavage by calpain may occur after
PTP1B relocated into the cytoskeleton, since the cleaved 42-kDa form
was found to be present only in the cytoskeleton but not in the other
fractions of aggregated platelets. The intact PTP1B localizes to the
endoplasmic reticulum membrane via its C-terminal sequence
(18) .
Lee and Chen
(43) suggested the involvement of the cytoskeleton
in motility of the endoplasmic reticulum in interphase cells. The
demonstration of PTP1B association with the cytoskeleton in the present
work may also suggest the dynamic association of the endoplasmic
reticulum with the cytoskeleton in platelets.
Li et al.(31) have recently reported that PTP1C is associated with the
cytoskeleton in an aggregation-dependent manner. In contrast to their
report, we found that PTP1C became associated with the cytoskeleton in
activated platelets regardless of the occurrence of aggregation, since
PTP1C was found to relocate into the cytoskeleton in activated
thrombasthenic platelets. However, in view of the aggregation-induced
enhancement of PTP1C association with the cytoskeleton, the apparent
discrepancy between our findings and those of Li et al. could
be due to the difference in sensitivity for detection of associated
PTP1C. Therefore, it is reasonable to conclude that the relocation of
PTP1C into the cytoskeleton is regulated by two steps: first by the
stimulation of thrombin and second by the
In addition to the relocation of PTPases, one
interesting observation was that PTP1C became phosphorylated on
tyrosine as platelet aggregation proceeded. It has been reported that
PTP1C becomes tyrosine-phosphorylated in the BAC1.2F5 macrophage cell
line in response to colony-stimulating factor
1
(45, 46) , in the megakaryoblastic leukemia cell line
Mo7e in response to stem cell factor
(46) , and in a T-cell
hybridoma line or normal murine thymocytes in response to CD4 or CD8
cross-linking
(40) . Similarly to these previous reports, we
failed to define the biochemical consequences of PTP1C tyrosine
phosphorylation in vivo which may regulate its enzymatic
activity, possibly because of autodephosphorylation. However, it should
be noted that PTP1C was associated with the cytoskeleton but was not
tyrosine-phosphorylated in thrombin-activated platelets without
aggregation; PTP1C tyrosine phosphorylation was dependent on
Another important new aspect of PTP1C is that
A model summarizing our findings and the possible mechanisms of
We are grateful to Dr. M. Tamai for providing the EST,
to Dr. E. H. W. Bohme for providing the MDL, and to Shoko Okamoto for
secretarial assistance.
(glycoprotein IIb-IIIa) has been implicated in the regulation of
tyrosine phosphorylation and dephosphorylation in activated platelets.
To investigate the mechanisms of the
-dependent tyrosine
dephosphorylation, normal platelets or thrombasthenic platelets lacking
were stimulated with thrombin and
fractionated into Triton X-100-soluble or -insoluble subcellular
matrices. We then examined the kinetics of the tyrosine-phosphorylated
proteins and distribution of protein-tyrosine phosphatases in these
fractions and whole cell lysates. First,
-dependent tyrosine
dephosphorylation was recovered mainly in the cytoskeleton with similar
kinetics to the whole cell lysate. Second, protein-tyrosine phosphatase
(PTP) 1B and its cleaved 42-kDa form were associated with the
cytoskeleton in an aggregation-dependent manner, whereas association of
PTP1C with the cytoskeleton was regulated differentially both by
thrombin stimulation and by
-mediated aggregation. Several
calpain inhibitors did not affect either tyrosine phosphorylation and
dephosphorylation or relocation of PTP1B, but they did inhibit cleavage
of PTP1B. Cytochalasin D blocked relocation of both PTP1B and PTP1C but
not PTP1B cleavage. SH-PTP2 was distributed in the other fractions than
the cytoskeleton and showed no relocation on thrombin stimulation.
Finally, the cytoskeleton-associated PTP1C became
tyrosine-phosphorylated in an
-mediated aggregation-dependent
manner. Thus, integrin
was
involved differentially in the regulation of PTP1B and PTP1C.
(also called
glycoprotein IIb-IIIa), which is a heterodimeric adhesion
receptor
(1) , plays a critical role. Integrin
can recognize
Arg-Gly-Asp-containing adhesive ligands including fibrinogen,
fibronectin, von Willebrand factor, and
vitronectin
(2, 3) . Among these adhesive molecules, the
binding of fibrinogen to
is the
major mechanism for platelet aggregation. Besides functioning as an
adhesive receptor, integrin
is
involved in signal transduction from the extracellular matrix to the
cytoplasm
(4) .
under stirring
condition
(7, 8) . The net phosphorylation of tyrosine
residues on substrate proteins is regulated by activities of both
protein-tyrosine kinases (PTKs)
(
)
and
protein-tyrosine phosphatases (PTPases). Platelets contain numerous
cytosolic PTKs including five members of the Src family
(pp60
, pp60
,
pp60
, pp61
, and
pp54/58
)
(9, 10, 11) ,
pp125
(12) , and
pp72
(13) .
pp60
, which is the most abundant
PTK in platelets
(9) , shows an increase in tyrosine kinase
activity with thrombin-induced activation
(14) and translocates
to the Triton X-100-insoluble, cytoskeleton-rich fraction dependent on
platelet aggregation
(15, 16) . pp125
,
which is located in focal adhesion, is phosphorylated on tyrosine and
activated in an aggregation-dependent manner
(12) .
is involved in the
tyrosine-specific dephosphorylation of certain proteins, suggesting for
the first time the engagement of
in the activation of some PTPases present in
platelets
(33) . Here we report that cytoskeletal reorganization
and protein-tyrosine phosphorylation are also involved in the
-dependent regulation of PTPases in
platelets.
Materials
Anti-phosphotyrosine monoclonal
antibodies 4G10 and PY20 were purchased from Upstate Biotechnology,
Inc. (UBI, Lake Placid, NY) and ICN Biomedicals, Inc. (Costa Mesa, CA),
respectively. Affinity-purified rabbit polyclonal antibodies against
PTP1B, PTP1C, and SH-PTP2 were from UBI. Monoclonal antibody 327 is
specific for pp60 and was obtained
from Oncogene Science, Inc. (Uniondale, NY). Prostaglandin E
was kindly provided by Ono Pharmaceutical Co. (Osaka, Japan).
Cytochalasin D and aprotinin were obtained from Sigma. Leupeptin was
from Peptide Institute, Inc. (Minoh, Japan). Calpeptin was from LC
Laboratories (Woburn, MA). EST and MDL were generous gifts of Dr. M.
Tamai of Taisho Pharmaceutical Co. (Saitama, Japan) and Dr. E. H. W.
Bohme of Marion Merrell Dow Research Institute (Cincinnati, OH),
respectively. All other reagents were obtained as described
previously
(33) .
Preparation and Activation of Platelets
After
informed consent was obtained, venous blood was collected from healthy
adult donors or from patients with Glanzmann's thrombasthenia.
Anti-coagulation of blood and preparation of washed platelets were
performed as described previously
(33) . Washed platelets
(0.5-1.0 10
cells/ml) were activated by 1
unit/ml thrombin with or without stirring at 1,000 rpm in an
aggregometer at 37 °C for appropriate intervals. In some
experiments, washed platelets were preincubated for 10 min at 37 °C
with the following inhibitors prior to activation with thrombin: 1
mM RGDS, 20 µM cytochalasin D, 500
µM EST, 250 µM calpeptin, or 250
µM MDL.
Fractionation of Platelets
Subcellular
fractionation of platelets was carried out using a modification of the
method described by Fox et al.(34) . After washed
platelets (5 10
cells/ml) were activated with
thrombin, reactions were stopped by lysing the cells with 0.5 volume of
3
lysis buffer containing 3% Triton X-100, 15 mM EGTA,
15 mM EDTA, 30 mM benzamidine, 3 mM
phenylmethylsulfonyl fluoride (PMSF), 3 mM
Na
VO
, 60 µg/ml leupeptin, 60 µg/ml
aprotinin, 30 µM cytochalasin D, and 50 mM
Tris-HCl, pH 7.4. In some experiments, cytochalasin D was omitted from
the lysis buffer. This and all subsequent steps were performed at 4
°C. The cytoskeleton was sedimented immediately by centrifugation
of the lysate at 10,000
g for 5 min. The membrane
skeleton was isolated from the 10,000
g supernatant by
centrifugation at 100,000
g for 3 h in a 1.5-ml
microcentrifuge tube (Eppendorf, Hamburg, Germany) with a Beckman
TL-100 using a TLA-100.3 rotor (Beckman Instruments, Inc., Palo Alto,
CA). The cytoskeleton and the membrane skeleton then were washed three
times with 1
lysis buffer containing 1% Triton X-100, 5
mM EGTA, 5 mM EDTA, 10 mM benzamidine, 1
mM PMSF, 1 mM Na
VO
, 20
µg/ml leupeptin, 20 µg/ml aprotinin, and 50 mM
Tris-HCl, pH 7.4, and solubilized in 1
SDS sample buffer (2%
SDS, 5% glycerol, 5%
-mercaptoethanol, 62.5 mM Tris-HCl,
pH 6.8). The Triton X-100-soluble fraction from the 100,000
g supernatant was diluted with 0.5 volume of 3
concentrated SDS sample buffer. When the samples of the whole lysate of
platelets were needed, thrombin-activated platelets were solubilized
directly by addition of 0.5 volume of 3
concentrated SDS sample
buffer. Following solubilization in SDS sample buffer, the samples were
promptly boiled for 5 min.
Immunoblotting
The samples (proteins from 1
10
platelets/lane) were subjected to 10%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
(35) .
Separated proteins were transferred electrophoretically to a
nitrocellulose membrane (Bio-Rad) with a semidry blotter. The membranes
were blocked for 1 h at room temperature or overnight at 4 °C with
3% nonfat dried milk in TPBS (0.1% Tween 20, 137 mM NaCl, 2.7
mM KCl, 8 mM Na
HPO
, 1.5
mM KH
PO
) and washed three times in
TPBS. The membranes then were incubated for 2 h with primary antibodies
against proteins of interest in TPBS containing 1% bovine serum
albumin. The primary antibodies used were as follows: a mixture of
anti-phosphotyrosine monoclonal antibodies 4G10 and PY20 (1 µg/ml
each); anti-pp60
monoclonal
antibody 327 (1 µg/ml); polyclonal antibodies against PTP1B (0.5
µg/ml), PTP1C (1 µg/ml), or SH-PTP2 (1 µg/ml). The
membranes were washed four times in TPBS and incubated for 1 h either
with horseradish peroxidase-conjugated goat anti-mouse (1 µg/ml) or
anti-rabbit (0.5 µg/ml) IgG in TPBS containing 1% bovine serum
albumin. Immunoreactivity was determined using the ECL
chemiluminescence reaction (Amersham International plc, Little
Chalfont, United Kingdom). In some experiments, the membranes once
probed were stripped of bound antibodies by incubation in buffer
containing 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM
Tris-HCl, pH 6.7, for 30 min at 60 °C and then reprobed with other
antibodies as described above.
Immunoprecipitation
Washed platelets (1
10
cells/ml) were stimulated with thrombin for appropriate
periods and lysed for 1 h in 0.5 volume of 3
radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 15
mM EGTA, 3% Triton X-100, 3% sodium deoxycholate, 0.3% SDS, 3
mM PMSF, 3 mM Na
VO
, 60
µg/ml leupeptin, 60 µg/ml aprotinin, and 50 mM
Tris-HCl, pH 7.4). This and all subsequent steps were carried out at 4
°C. The lysates were clarified by centrifugation at 16,000
g for 20 min, precleared with 50 µl of protein A-Sepharose
CL-4B (50% slurry) (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden)
by sedimentation at 16,000
g for 3 min, and then
incubated for 2 h with anti-PTP1B antibody (2 µg/ml), anti-PTP1C
antibody (4 µg/ml), anti-SH-PTP2 antibody (10 µg/ml), or rabbit
anti-mouse IgG (10 µg/ml) as a control. Immune complexes were
incubated for 1 h with 50 µl of protein A-Sepharose CL-4B (50%
slurry). Immunoprecipitates were sedimented by brief centrifugation and
washed four times in 1
RIPA buffer (150 mM NaCl, 5
mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1
mM PMSF, 1 mM Na
VO
, 20
µg/ml leupeptin, 20 µg/ml aprotinin, and 50 mM
Tris-HCl, pH 7.4). For immunoprecipitation of proteins from the
subcellular fractions, the cytoskeleton and the membrane skeleton of
thrombin-activated platelets were solubilized in RIPA buffer following
fractionation of cells. Immunoprecipitated PTP1C was obtained from each
solubilized fraction in RIPA buffer. Immunoprecipitated proteins (from
4
10
platelets/lane) were eluted from protein
A-Sepharose beads in 1
SDS sample buffer, boiled for 5 min,
resolved on SDS-PAGE, and analyzed by immunoblotting as described
above.
RESULTS
Protein-tyrosine Phosphorylation and Dephosphorylation
in the Subcellular Fractions of Thrombin-activated Platelets
To
investigate the involvement of integrin in protein-tyrosine phosphorylation and dephosphorylation, we
first examined tyrosine-phosphorylated proteins by immunoblot assay in
whole lysates of platelets from normal donors and from patients with
Glanzmann's thrombasthenia. As shown in Fig. 1(A and B), thrombasthenic platelets lacking
showed different patterns from
normal platelets in tyrosine dephosphorylation as well as in tyrosine
phosphorylation on stimulation with thrombin. In normal platelets,
tyrosine-phosphorylated protein bands with molecular masses of 130,
120, and 115 kDa peaked at 10 s, 30 s, and 2 min, respectively, then
quickly declined, and disappeared. However, in thrombasthenic
platelets, the 130- and 120-kDa protein bands persisted and were not
dephosphorylated until 5 min after stimulation, whereas the 115-kDa
protein band did not appear. Two clusters of protein bands with
molecular masses of 68-78 and 53-60 kDa also showed
tyrosine-specific dephosphorylation in normal platelets but did not in
thrombasthenic platelets. Immunoblot analysis with
anti-pp60
antibody revealed that
the most prominent band in the 53-60-kDa bundle was
pp60
(data not shown). The 102-kDa
tyrosine-phosphorylated protein band appeared at 2 min and persisted
until 5 min following thrombin stimulation in normal platelets, but did
not appear in thrombasthenic platelets. The 95-kDa band peaked at 30 s
and was persistent until 5 min either in normal or thrombasthenic
platelets. These results indicated that
was involved in the tyrosine phosphorylation of the 115- and
102-kDa protein bands and the tyrosine dephosphorylation of the 130-,
120-, 68-78-, and 53-60-kDa protein bands. Thrombasthenic
platelets also differed from normal platelets in kinetic patterns of
other doublet bands of 36/37 and 33/34 kDa in tyrosine phosphorylation
and dephosphorylation. Following stimulation with thrombin, these
doublets peaked at 10 s, quickly declined, and disappeared in normal
platelets, whereas these bands in thrombasthenic platelets peaked at 30
s and declined slowly.
Figure 1:
Time course of thrombin-induced
protein-tyrosine phosphorylation in whole lysates and subcellular
fractions of normal and thrombasthenic platelets. Washed platelets from
normal donors (A, C, E, and G) or
integrin -deficient subjects with
Glanzmann's thrombasthenia (B, D, F,
and H) were stimulated with 1 unit/ml thrombin for the
indicated times under stirring conditions. The platelets were lysed
directly in SDS sample buffer to obtain whole cell lysates (A and B) or in 1% Triton X-100-containing lysis buffer with
cytochalasin D, and fractionated into the cytoskeleton (1% Triton
X-100-insoluble, 10,000
g pellets) (C and
D), the membrane skeleton (1% Triton X-100-insoluble, 100,000
g pellets) (E and F), and the 1%
Triton X-100-soluble fraction (G and H) as described
under ``Experimental Procedures.'' The same experiment as
shown in panelC was also performed using the lysis
buffer without cytochalasin D (C` in parentheses).
Proteins from the same number of platelets were subjected to 10%
SDS-PAGE and probed by immunoblotting with anti-phosphotyrosine
antibodies. Molecular masses of major tyrosine-phosphorylated protein
bands and molecular mass markers are indicated in kDa on the left and right of the panels,
respectively.
Because previous reports have suggested that
many tyrosine-phosphorylated proteins are components of Triton
X-100-insoluble, cytoskeleton-rich fractions of activated platelets, we
explored whether the Triton X-100-soluble or -insoluble subcellular
fraction was the site of the tyrosine dephosphorylation observed in the
whole lysate of thrombin-activated platelets. This investigation led us
to the finding that the conventionally used lysis buffer
(34) should be modified by including cytochalasin D to observe
the protein-tyrosine dephosphorylation patterns in the subcellular
fractions with similar kinetics to those in the whole cell lysate. When
we used the lysis buffer without cytochalasin D, protein-tyrosine
phosphorylation observed in the cytoskeleton of normal platelets was
persistent and quite different from that in the whole cell lysate
(Fig. 1C`, parentheses). On the
contrary, using the modified lysis buffer containing cytochalasin D as
described under ``Experimental Procedures,'' nearly identical
patterns of tyrosine phosphorylation and dephosphorylation of proteins
observed in the whole cell lysate were noted in the cytoskeletons of
normal and thrombasthenic platelets (Fig. 1, C-H),
except for several protein bands. One exception to these observations
was pp60.
pp60
was redistributed from the
Triton X-100-soluble fraction and the Triton X-100-insoluble membrane
skeleton into the cytoskeleton in aggregated normal platelets but not
in thrombasthenic platelets, as has been reported by several
laboratories
(14, 15, 16) . Other exceptions were
the 36/37- and 33/34-kDa tyrosine-phosphorylated protein bands. We
could not obtain reproducible results for the cytoskeletal localization
of these protein bands. However, we detected only the 36/37-kDa bands
in the cytoskeleton of normal platelets. When normal platelets were
activated with thrombin in the presence of RGDS, an inhibitor of
-fibrinogen binding, or without
stirring to prevent platelet aggregation, we obtained the same patterns
of tyrosine phosphorylation and dephosphorylation as observed in
thrombasthenic platelets (data not shown).
Cytochalasin D but Not Calpain Inhibitors Affects the
Patterns of Tyrosine Phosphorylation
Next, we examined whether
an inhibitor of actin polymerization, cytochalasin D, influenced the
state of tyrosine-phosphorylated proteins in the whole cell lysate and
in the cytoskeleton. As shown in Fig. 2, pretreatment of normal
platelets with 20 µM cytochalasin D did not inhibit
thrombin-induced aggregation (data not shown), but markedly reduced the
thrombin-induced tyrosine-phosphorylated protein bands both in the
whole cell lysate and in the cytoskeleton. This suggested that tyrosine
phosphorylation of the cytoskeletal components was largely dependent on
actin polymerization. Furthermore, cytochalasin D blocked the
relocation of pp60 to the
cytoskeleton in aggregated normal platelets (Fig. 2, C,
E, and G), confirming the findings by Oda et
al.(16) .
Figure 2:
Effects of an actin polymerization
inhibitor, cytochalasin D, and a calpain inhibitor, EST, on
thrombin-induced tyrosine phosphorylation in whole lysates and
subcellular fractions of normal platelets. Washed platelets from normal
donors were pretreated with 20 µM cytochalasin D
(A, C, E, and G) or 500
µM EST (B, D, F, and
H) for 10 min and stimulated with 1 unit/ml thrombin for the
indicated times under stirring conditions. The platelets were lysed
directly in SDS sample buffer to obtain whole cell lysates (A and B) or in 1% Triton X-100-containing lysis buffer with
cytochalasin D, and fractionated into the cytoskeleton (C and D), the membrane skeleton (E and
F), and the 1% Triton X-100-soluble fraction (G and
H) as described under ``Experimental Procedures.''
Proteins from the same number of platelets were subjected to 10%
SDS-PAGE and probed by immunoblotting with anti-phosphotyrosine
antibodies. Molecular masses of major tyrosine-phosphorylated protein
bands and molecular mass markers are indicated in kDa on the left and right of the panels,
respectively.
Recently, Frangioni et al.(30) have reported that, in activated platelets, a
calcium-dependent neutral protease, calpain, cleaves PTP 1B in an
aggregation-dependent fashion, and that the cleaved 42-kDa form of
PTP1B relocates from the membrane to the cytosol and shows 2-fold
higher enzymatic activity than the intact form. Hence we studied the
effects of calpain inhibitors EST (36), MDL
(37) , and calpeptin
(38) on the -regulated
tyrosine dephosphorylation in platelets. However, as shown in
Fig. 2
, EST affected neither aggregation (data not shown) nor the
patterns of tyrosine phosphorylation and dephosphorylation in the whole
cell lysate and the subcellular fractions, particularly the
cytoskeleton. At the same time, we confirmed that the cleavage of PTP1B
occurred in thrombin-activated normal platelets as aggregation
proceeded but not in thrombin-activated thrombasthenic platelets which
showed no aggregation; EST completely inhibited this
aggregation-dependent cleavage of PTP1B (Fig. 3, a,
b, and d). The other calpain inhibitors, MDL and
calpeptin, did not affect either aggregation or tyrosine
phosphorylation and dephosphorylation. However, calpeptin inhibited the
cleavage of PTP1B less than EST or MDL (data not shown). By contrast,
cytochalasin D did not prevent cleavage of PTP1B
(Fig. 3c).
Figure 3:
Aggregation-dependent cleavage of PTP1B in
thrombin-activated platelets is inhibited completely by EST but not by
cytochalasin D. Untreated normal (a) or thrombasthenic
platelets (b) were stimulated with 1 unit/ml thrombin for the
indicated times under stirring conditions. Normal platelets pretreated
with 20 µM cytochalasin D (c) or 500
µM EST (d) were stimulated with thrombin.
Platelets were lysed in SDS sample buffer. Proteins from the same
number of platelets were subjected to 10% SDS-PAGE and probed by
immunoblotting with anti-PTP1B antibody. The positions of PTP1B
(arrow) and the cleaved form of PTP1B (arrowhead) are
indicated on the right of the
blots.
Association of PTP1B and Its Cleaved 42-kDa Form with the
Cytoskeleton
In order to show the presence of PTPases in the
cytoskeleton, we studied subcellular distributions of PTPases, PTP1B,
PTP1C, and SH-PTP2 by immunoblot analysis. As shown in
Fig. 4A, PTP1B translocated from the detergent-soluble
fraction to the cytoskeleton in thrombin-activated normal platelets
under stirring conditions. Interestingly, not only PTP1B itself but
also its cleaved 42-kDa form were associated with the cytoskeleton in a
time-dependent manner as platelets aggregated (Fig. 4A,
a). In thrombasthenic platelets stimulated with thrombin,
PTP1B did not translocate to the cytoskeleton (Fig. 4B,
a). We confirmed that PTP1B did not translocate to the
cytoskeleton if normal platelets were activated with thrombin in the
presence of RGDS or under unstirring conditions to prevent aggregation
(data not shown). Pretreatment of normal platelets with cytochalasin D
prior to thrombin stimulation blocked the relocation of PTP1B to the
cytoskeleton, whereas a calpain inhibitor, EST, which prevented the
cleavage of PTP1B, did not interfere with the relocation of PTP1B
(Fig. 4B, b and c). These results
indicated that PTP1B association with the cytoskeleton was dependent on
an -mediated aggregation as well as
actin polymerization.
Figure 4:
PTP1B
translocates into the cytoskeleton in thrombin-activated platelets in
an aggregation- and actin polymerization-dependent manner. A,
normal platelets were stimulated with 1 unit/ml thrombin for the
indicated times under stirring conditions, lysed in 1% Triton
X-100-containing lysis buffer with cytochalasin D, and fractionated
into the cytoskeleton (CSK) (a), the membrane
skeleton (MSK) (b), and the detergent-soluble
fraction (SOL) (c) as described under
``Experimental Procedures.'' B, thrombasthenic
(a) and normal platelets, which were pretreated with 20
µM cytochalasin D (b) or 500 µM EST
(c) for 10 min, were stimulated with 1 unit/ml thrombin for
the indicated times under stirring conditions and lysed. Cytoskeletons
were subjected to 10% SDS-PAGE and probed by immunoblotting with
anti-PTP1B antibody. The positions of PTP1B (arrow) and the
cleaved form of PTP1B (arrowhead) are indicated on the
right of the blots.
Redistribution of PTP1C in the Subcellular
Fractions
As shown in Fig. 5, PTP1C was present in the
detergent-soluble fraction and the membrane skeleton of unstimulated
platelets. When normal platelets were activated with thrombin under
stirring conditions, PTP1C relocated to the cytoskeleton
(Fig. 5A). PTP1C was redistributed to the cytoskeleton
also in thrombin-activated thrombasthenic platelets
(Fig. 5B, a). However, the intensity of the
PTP1C band was weaker in the cytoskeleton of thrombasthenic platelets
than in aggregated normal platelets (Fig. 5A and
B, panels a). When normal platelets were activated
with thrombin in the presence of RGDS or without stirring, we obtained
the same results as in thrombasthenic platelets (data not shown).
Pretreatment of normal platelets with cytochalasin D prior to thrombin
stimulation attenuated the association of PTP1C with the cytoskeleton
and diminished the intensity of the PTP1C band in a time-dependent
manner as platelet aggregation proceeded (Fig. 5B,
b). These findings may be derived from detachment of the
associated PTP1C from the cytoskeleton. Another PTPase containing SH2
domains, SH-PTP2, was distributed in the membrane skeleton and in the
detergent-soluble fraction but not in the cytoskeleton of unstimulated
platelets. Stimulation with thrombin did not change the subcellular
distribution of SH-PTP2 (data not shown).
Figure 5:
Redistribution of PTP1C between the
subcellular fractions in thrombin-activated platelets. A,
normal platelets were stimulated with 1 unit/ml thrombin for the
indicated times under stirring conditions, lysed in 1% Triton
X-100-containing lysis buffer with cytochalasin D, and fractionated
into the cytoskeleton (CSK) (a), the membrane
skeleton (MSK) (b), and the detergent-soluble
fraction (SOL) (c) as described under
``Experimental Procedures.'' B, thrombasthenic
(a) or normal platelets pretreated with 20 µM
cytochalasin D (b) were stimulated with 1 unit/ml thrombin for
the indicated times under stirring conditions and lysed. Cytoskeletons
were subjected to 10% SDS-PAGE and probed by immunoblotting with
anti-PTP1C antibody. Arrows indicate the position of
PTP1C.
Phosphorylation of PTP1C on Tyrosine Residues in an
Aggregation-dependent Manner
Tyrosine phosphorylation of
proteins, especially proteins containing SH2 domains, is an important
mechanism to regulate activities or interactions with other
molecules
(39) . We examined tyrosine phosphorylation of
immunoprecipitated PTPases from activated platelets with thrombin by
immunoblotting. We first found that stimulation of platelets with
thrombin under stirring conditions caused tyrosine phosphorylation of
PTP1C in a time-dependent fashion (Fig. 6A, a).
Furthermore, the tyrosine phosphorylation of PTP1C occurred only when
thrombin-activated platelets were stirred simultaneously to induce
aggregation (Fig. 6B, a). Blockage of
aggregation of thrombin-activated platelets by not stirring or by
addition of RGDS prevented tyrosine phosphorylation of PTP1C. In
addition, pretreatment of platelets with cytochalasin D did not inhibit
platelet aggregation but did abolish tyrosine phosphorylation of PTP1C
(Fig. 6B, a). Immunoblot analysis with
anti-PTP1C antibody showed that the amount of immunoprecipitated PTP1C
was nearly unchanged in these experiments (Fig. 6, A and
B, panelsb). We could not detect tyrosine
phosphorylation of PTP1B and SH-PTP2 in unstimulated platelets or in
thrombin-stimulated ones (data not shown). These data indicated that
tyrosine phosphorylation of PTP1C was dependent on
-mediated aggregation as well as
actin polymerization. Furthermore, tyrosine-phosphorylated PTP1C was
seen in the cytoskeleton but not in the membrane skeleton or the
detergent-soluble fraction (Fig. 6C, lanes
1-3). However, the intensity of tyrosine-phosphorylated
PTP1C band in the cytoskeleton was much weaker than that in the whole
cell lysate, whereas a considerable amount of PTP1C was
immunoprecipitated from each fraction (Fig. 6C,
lanes 4 and 5).
Figure 6:
Thrombin induces tyrosine phosphorylation
of PTP1C associated with the cytoskeleton, which is dependent on
aggregation and actin polymerization. A, normal platelets were
stimulated with 1 unit/ml thrombin for the indicated times under
stirring conditions, lysed in RIPA buffer, and immunoprecipitated with
anti-PTP1C antibody (-PTP1C) or rabbit anti-mouse IgG
(RAM) as a control. B, normal platelets were
untreated (lanes 1-3) or pretreated with 1 mM
RGDS (lane4) or 20 µM cytochalasin D
(lane5) for 10 min prior to stimulation.
Unstimulated platelets (lane1) or those stimulated
with 1 unit/ml thrombin for 2 min under stirring (lanes 2,
4, and 5) or unstirring conditions (lane3), were lysed inRIPA buffer, and
immunoprecipitated with anti-PTP1C antibody. C, normal
platelets were stimulated with 1 unit/ml thrombin for 2 min under
stirring conditions, lysed in 1% Triton X-100-containing lysis buffer
with cytochalasin D, and fractionated into the cytoskeleton
(CSK) (lanes 1 and 4), the membrane skeleton
(MSK) (lanes2 and 5), and the
detergent-soluble fraction (SOL) (lanes3 and 6). The proteins from each fraction were
immunoprecipitated with anti-PTP1C antibody. The immunoprecipitates
were subjected to 10% SDS-PAGE, transferred to nitrocellulose membrane,
and probed by immunoblotting with anti-phosphotyrosine antibodies
(A, a; B, a; and C,
lanes 1-3). The same membranes were reprobed with
anti-PTP1C antibody (A, b; B, b;
and C, lanes 4-6). Arrows mark the
position of PTP1C.
We also studied whether
tyrosine-phosphorylated PTP1C increased its enzymatic activity compared
to unphosphorylated PTP1C by an immune complex PTPase assay using
para-nitrophenyl phosphate; we did not detect any significant
difference in PTPase activity (data not shown). However, this negative
result agrees with the suggestions by Lorenz et al.(40) that autodephosphorylation of PTP1C takes place in
vitro and would make it difficult to prove any difference in PTP1C
activity due to tyrosine phosphorylation. In fact,
autodephosphorylation may explain why tyrosine-phosphorylated PTP1C was
largely decreased as described above during preparation of the
cytoskeleton fraction.
DISCUSSION
Until recently, the physiological roles or modes of
regulation of PTPases in platelets have received much less attention
than PTKs, although platelets have been suggested to possess several
PTPases
(30, 31) . Our previous work has shown for the
first time that integrin is
involved in protein-tyrosine dephosphorylation in activated
platelets
(33) . It has been suggested that many of the proteins
phosphorylated on tyrosine in activated platelets are components of the
cytoskeleton
(34) . If so, one would speculate that the
-regulated protein-tyrosine
dephosphorylation would be observed on the tyrosine-phosphorylated
proteins of the cytoskeleton in activated platelets. However, this
notion has not been successfully proven as yet, although there are a
few reports showing protein-tyrosine phosphorylation but not
dephosphorylation of cytoskeletons in activated
platelets
(14, 16, 34) .
-mediated aggregation, as was
observed in the whole lysate.
is engaged in inducing PTP1B cleavage by a
Ca
-dependent neutral protease calpain at a site
upstream from its C-terminal targeting sequence, resulting in
subcellular relocation of the PTP1B catalytic domain from the membrane
to the cytosol and a 2-fold increase in its enzymatic activity. They
proposed that the PTP1B cleavage, the subcellular relocation into the
cytosol, and the enzymatic activation account for the blunted increase
in protein-tyrosine phosphorylation seen with aggregation. However,
their proposed mechanism can not explain the regulation of the
aggregation-dependent dephosphorylation of tyrosine-phosphorylated
proteins observed in this study for the following reasons. First,
complete blockage of PTP1B cleavage by several calpain inhibitors
including EST did not affect the patterns of tyrosine phosphorylation
and dephosphorylation in any subcellular fractions as well as in the
whole cell lysate. Second, the tyrosine-phosphorylated proteins which
were observed to be dephosphorylated in the whole cell lysate were not
components of the cytosol but rather the cytoskeleton. In contrast to
our study, the central part of their findings was obtained from
experiments using washed platelets stimulated with 1 µM
A23187 under unstirring conditions such that platelets did not
aggregate. Interestingly, A23187 induces protein-tyrosine
phosphorylation and its subsequent dephosphorylation of platelets under
unstirring conditions, as originally reported by Takayama et
al.(42) . This indicates that A23187-induced
dephosphorylation of tyrosine-phosphorylated proteins is mediated by
different mechanisms from the aggregation-dependent ones that are
required for the physiologic agonists, such as thrombin-induced
tyrosine dephosphorylation.
-mediated aggregation. Such
two-step regulated association with the cytoskeleton is also the case
with pp60
(see Footnote 2) and
pp72
(44) . Cytochalasin D did not
completely inhibit the relocation of PTP1C into the cytoskeleton but
did that of PTP1B, indicating that actin polymerization is prerequisite
to the PTP1B relocation but not necessarily to the PTP1C relocation.
Since we have recently found that thrombin induces rapid
phosphorylation of PTP1C on serine residues,
(
)
such phosphorylation may be necessary for the PTP1C
relocation.
-mediated aggregation as well as
actin polymerization. Furthermore, we showed that tyrosine
phosphorylation of PTP1C was detected only in the cytoskeleton and not
in other fractions. Therefore, the functional role of PTP1C tyrosine
phosphorylation might not be related to its relocation into the
cytoskeleton but rather to the regulation of its enzymatic activity.
-dependent tyrosine phosphorylation
and dephosphorylation may be coupled, in part, through PTP1C
phosphorylated on tyrosine. Since Ferrell and Martin
(7) first
reported that
was involved in the
regulation of tyrosine phosphorylation of a specific set of proteins,
pp125
has been identified as one of these
proteins
(12) . However, it seems unlikely that PTP1C is a
substrate of pp125
that is activated by tyrosine
phosphorylation, since we observed that tyrosine phosphorylation of
PTP1C occurs before pp125
.
PTP1C appears to
be tyrosine-phosphorylated by unidentified PTKs whose activity is
regulated by
-mediated aggregation.
-dependent tyrosine
dephosphorylation is presented in Fig. 7. Further studies are
necessary to identify the tyrosine-phosphorylated proteins whose
dephosphorylations are regulated by
-mediated mechanisms and their
linkage with PTPases as well as PTKs. These efforts will lead to more
complete understandings of the functional roles of PTPases.
Figure 7:
A model for the aggregation-dependent
tyrosine dephosphorylation in the cytoskeleton of thrombin-activated
platelets. A, prior to aggregation, stimulation of platelets
with thrombin leads to activation of some PTKs, substrates of which
reside or relocate mainly in the cytoskeleton, translocation of PTP1C
to the cytoskeleton, and activation of
. Then fibrinogen is bound to
. B, under stirring
conditions, activated platelets aggregate through
-fibrinogen binding. Aggregation
induces cytoskeletal reorganization, calpain-catalyzed cleavage of
PTP1B, further relocation of PTP1C to the cytoskeleton, and activation
of some PTKs that phosphorylate proteins including PTP1C. In the
cytoskeleton, two forms of PTP1B and tyrosine-phosphorylated PTP1C
possibly dephosphorylate their substrates, which were phosphorylated on
tyrosine residues prior to aggregation. ThR, thrombin
receptor; PTKs, protein-tyrosine kinases; Tyr,
tyrosine; PY, phosphotyrosine
residues.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.