(Received for publication, August 30, 1995)
From the
Platelet stimulation by thrombin or the thrombin receptor
activating peptide (TRAP) results in the activation of phosphoinositide
3-kinase and the production of the novel polyphosphoinositides
phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P) and
phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P
).
We have shown previously that these lipids activate calcium-independent
protein kinase C (PKC) isoforms in vitro (Toker, A., Meyer,
M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns,
D. J., Ballas, L. M. and Cantley, L. C.(1994) J. Biol. Chem. 269, 32358-32367). Activation of platelet PKC in response to
TRAP is detected by the phosphorylation of the major PKC substrate in
platelets, the p47 phosphoprotein, also known as pleckstrin. Here we
provide evidence for two phases of pleckstrin phosphorylation in
response to TRAP. A rapid phase of pleckstrin phosphorylation (<1
min) precedes the peak of PtdIns-3,4-P
production and is
unaffected by concentrations of wortmannin (10-100 nM)
that block production of this lipid. However prolonged phosphorylation
of pleckstrin (>2 min) is inhibited by wortmannin concentrations
that block PtdIns-3,4-P
production. Phorbol ester-mediated
pleckstrin phosphorylation was not affected by wortmannin and
wortmannin had no effect on purified platelet PKC activity.
Phosphorylation of pleckstrin could be induced using permeabilized
platelets supplied with exogenous
-
P[ATP]
and synthetic dipalmitoyl PtdIns-3,4,5-P
and dipalmitoyl
PtdIns-3,4-P
micelles, but not with dipalmitoyl
phosphatidylinositol 3-phosphate or phosphatidylinositol
4,5-bisphosphate. These results suggest two modes of stimulating
pleckstrin phosphorylation: a rapid activation of PKC (via
diacylglycerol and calcium) followed by a slower activation of
calcium-independent PKCs via PtdIns-3,4-P
.
The activation of phosphoinositide 3-kinase (PI 3-K) ()in agonist-stimulated cells results in the rapid formation
of the two novel polyphosphoinositides PtdIns-3,4-P
and
PtdIns-3,4,5-P
. A critical requirement for PI 3-K
activation in a variety of cellular functions has been established.
These include growth factor dependent mitogenesis, chemotaxis, receptor
down-regulation, insulin-induced glucose transport, and actin-filament
rearrangements leading to membrane ruffling (for a review, see (1) ). Despite these correlations, the direct targets for these
polyphosphoinositides remain undescribed. PtdIns-3,4-P
and
PtdIns-3,4,5-P
have been proposed to act as second
messengers as they are not hydrolyzed by any known phospholipase type C
enzymes(2, 3) . The other product of PI 3-K activity,
PtdIns-3-P, does not increase in agonist-stimulated cells and may be
important in intracellular protein sorting mechanisms(4) . Two
enzymes, pp70S6 kinase (5) and the serine-threonine protein
kinase Akt(6, 7) have been shown to be downstream of
activated PI 3-K, but there is no evidence that these are the immediate
targets of PtdIns-3,4-P
and PtdIns-3,4,5-P
.
Recent data from our laboratory points to the calcium-independent
isoforms of PKC (
,
, and
) as direct targets of these
lipids(8) , although in vivo evidence for this is
still lacking. The diacylglycerol-insensitive isoform PKC
may
also be a target for these phosphoinositides (9) .
Thrombin
activation of platelets results in the rapid activation of PI
3-K(10, 11) . The major product of PI 3-K in activated
platelets is PtdIns-3,4-P, which peaks at 2-3 min
following stimulation. A small peak of PtdIns-3,4,5-P
at 30
s to 1 min has also been reported(11) . Although the exact
function of these novel phosphoinositides in platelet activation is
undescribed, we recently established a critical requirement for PI 3-K
activation in integrin-mediated platelet aggregation, leading to
activation of the integrin GPIIb-IIIa(12) . Furthermore, PI 3-K
activation occurs both upstream and downstream of the integrin,
suggesting that this enzyme contributes both to
``inside-out'' and ``outside-in'' signaling in
platelets. Using the potent PI 3-K inhibitor wortmannin, we also showed
that there is no critical requirement for PI 3-K in mediating actin
assembly in response to thrombin-receptor activation(12) ,
suggesting that the D4 phosphoinositides PtdIns-4-P and
PtdIns-4,5-P
are the phospholipid mediators of this event
in human platelets(13) .
PKC is also rapidly activated in
thrombin-stimulated platelets. PKC comprises a large family of
closely-related serine/threonine protein kinases, classed according to
their co-factor requirements (reviewed in (14) ). Conventional
(,
I,
II, and
) family members are dependent on
calcium, phospholipid, and diacylglycerol for activation, whereas
non-conventional isoforms (
,
,
(L),
,
and µ) are insensitive to calcium. Atypical PKC
and
/
are insensitive to diacylglycerol and calcium. An often
used measure of PKC activation in agonist-stimulated cells is the
phosphorylation of defined substrate proteins. In platelets and other
cells of hematopoietic origin, the major PKC substrate is the p47
phosphoprotein, pleckstrin (platelet and leukocyte C kinase substrate),
which is rapidly phosphorylated in response to a variety of agonists,
including thrombin, thrombin receptor activating peptide (TRAP), as
well as phorbol ester(15, 16) . The physiological
function of pleckstrin is undefined, although recent studies reveal
that interaction with polyphosphoinositides such as PtdIns-4,5-P
may be mediated by the pleckstrin homology (PH) domain at the N
terminus of this protein(17) . PH domains may also be involved
in protein-protein interactions; in particular, various PKC isoforms
have been reported to interact with the PH domains of the Akt protein
kinase (18) and the tyrosine kinase Btk(19) .
In
this report we use the phosphorylation of pleckstrin to study the
contribution of PI 3-K lipid products in activating platelet PKC. We
present data demonstrating that PI 3-K is essential for a late phase of
TRAP-stimulated pleckstrin phosphorylation and, more specifically, that
the phosphoinositides PtdIns-3,4-P and PtdIns-3,4,5-P
mediate this effect.
For experiments involving
platelet phospholipid labeling, platelets isolated by two cycles of
centrifugation were incubated for 1 h at 37 °C with 2 mCi/ml
[P]orthophosphoric acid, gel filtered as
described above, and allowed to rest for 1 h before use. Phospholipids
were extracted as described previously(12, 21) .
Lipids were quantitated using a Radiomatic A500 on-line radioactivity
counter (Packard Instrument Co., Downers Grove, IL).
Phosphoinositides were stored at -70 °C in methanol:chloroform:1 N HCl (2:1:0.1). For permeabilization studies, phosphoinositides were prepared by drying under a steam of nitrogen, followed by resuspension in 20 mM Hepes, 1 mM EGTA, pH 7.5, and sonicated in an ice-cold cup horn bath sonicator at 50% output for 10 min (Branson Ultrasonics, Danbury, CT). Sonicated phosphoinositides were used immediately for permeabilization studies.
Figure 1:
Wortmannin inhibits the TRAP-stimulated
phosphorylation of pleckstrin. P-Labeled gel-purified
platelets were preincubated with the indicated doses of wortmannin (WM) for 15 min, then stimulated with 25 µM TRAP
in the presence of 500 µg/ml fibrinogen under stirring conditions.
The reaction was terminated by addition of SDS-polyacrylamide gel
electrophoresis sample buffer, and heating to 100 °C for 5 min.
Platelet phosphoproteins were analyzed on a 13% SDS-polyacrylamide gel
followed by autoradiography. In panel A, platelets were
stimulated with TRAP for 30-s intervals up to 6 min prior to lysis. In panel B, platelets were preincubated with 100 nM
wortmannin, then stimulated with TRAP. The arrow points to the
position of pleckstrin, with a molecular mass of 47 kDa, determined
according to appropriate standards.
A dose response of wortmannin
inhibition in platelets stimulated for 3 min reveals that doses as low
as 10 nM are capable of inhibiting pleckstrin phosphorylation
(17%) induced by TRAP (Fig. 2, A and B), and
100 nM wortmannin was maximally effective (61.3% inhibition).
This inhibition did not increase with wortmannin concentrations above
100 nM and up to 1 µM, indicating that other
wortmannin-insensitive pathways contribute to the phosphorylation of
pleckstrin. The dose-response inhibition of pleckstrin phosphorylation
by wortmannin closely correlates with that found for TRAP-stimulated
PtdIns-3,4-P and PtdIns-3,4,5-P
production(12) . In contrast, concentrations as high as 1
µM did not inhibit the initial burst of pleckstrin
phosphorylation at 30 s following TRAP stimulation (data not shown).
Figure 2: Dose-response inhibition of pleckstrin phosphorylation in TRAP-stimulated platelets. A, platelets were preincubated with increasing doses of wortmannin up to 1000 nM, then stimulated with TRAP for 180 s. Pleckstrin phosphorylation was detected as described in the legend to Fig. 1. B, the 47-kDa band in panel A was quantified by densitometry and is plotted against increasing concentrations of wortmannin. Inhibition of pleckstrin phosphorylation is plotted as a percentage of control (no wortmannin). The results are representative of six different experiments.
Figure 3:
TRAP stimulates production of D3
phosphoinositides in isolated human platelets. A, P-labeled gel-purified platelets were stimulated with 25
µM TRAP in the presence of 500 µg/ml fibrinogen under
stirring conditions, and in the presence or absence of 100 nM wortmannin. The reaction was stopped at the appropriate time
points by the addition of the lipid extraction mixture. Lipids were
extracted and analyzed as described under ``Experimental
Procedures.'' B, the 47-kDa bands in Fig. 1(A and B) were quantified on a Molecular Imager and plotted
against the time course of PtdIns-3-P, PtdIns-3,4-P
, and
PtdIns-3,4,5-P
production.
The sustained production of
PtdIns-3,4-P therefore correlates temporally with sustained
pleckstrin phosphorylation in response to TRAP. This correlation is
further examined in Fig. 3B. Between 2 and 4 min,
activation of the thrombin receptor with TRAP under aggregating
conditions results in the sustained accumulation of PtdIns-3,4-P
and the sustained phosphorylation of p47. Both of these events
are inhibited in platelets pretreated with 100 nM wortmannin.
The decline in PtdIns-3,4-P
production at 4-6 min
also correlates with a decline of p47 phosphorylation. The initial
burst of pleckstrin phosphorylation also correlates with the appearance
of PtdIns-3,4,5-P
, but at this early time point (30 s), p47
phosphorylation is not affected by wortmannin pretreatment. Activation
of PKC from DAG synthesis has been reported at 30-60 s (30) , and this may account for pleckstrin phosphorylation at
these early times, although it is likely that PtdIns-3,4,5-P
may also contribute.
Figure 4:
Phorbol ester-stimulated pleckstrin
phosphorylation is not inhibited by wortmannin. A, platelets
were preincubated with the indicated doses of wortmannin for 15 min, or
staurosporine for 5 min. After the addition of 500 µg/ml
fibrinogen, platelets were activated with 100 nM PMA for the
times indicated. The reaction was stopped, and platelet phosphoproteins
were detected as described in the legend to Fig. 2. The arrow points to the position of pleckstrin. B, PKC
purified from resting platelets was preincubated with the indicated
doses of wortmannin for 5 min, then assayed for the ability to transfer P from [
-
P]ATP into histone
III-S or peptide
as described under ``Experimental
Procedures.'' section. The results are plotted as percentage of
stimulation above basal, in the absence of
phosphatidylserine/DAG.
Second, PKC was partially
purified from resting platelets by column chromatography and assayed in
the presence of increasing concentrations of wortmannin. Using
concentrations as high as 10 µM, there was no effect of
wortmannin on the ability of platelet PKC to phosphorylate either
histone III-S or the peptide substrate (Fig. 4B).
Histone III-S was used to assay for conventional calcium-dependent PKCs
(
,
, and
). However, histone III-S is a poor substrate
for non-conventional and atypical PKCs (
,
,
,
,
, and
) and therefore peptide
, based on the PKC
pseudosubstrate sequence was used(23) . These results are in
agreement with previous observations where wortmannin failed to
significantly affect PKC
activity(22, 31, 32) .
Figure 5:
Synthetic PtdIns-3,4-P and
PtdIns-3,4,5-P
induce pleckstrin phosphorylation in
permeabilized platelets. Isolated human platelets were permeabilized
with saponin as described under ``Experimental Procedures,''
in the presence of exogenously-added
[
-
P]ATP. Under non-aggregating conditions,
25 µM TRAP in the presence or absence of 100 nM wortmannin (WM) or 100 nM PMA were added to
detect pleckstrin phosphorylation. A, synthetic short-chain
DiC
PtdIns-3,4,5-P
(C
PIP
, 5 µM) was
presented to the permeabilized platelets as a free monomer in solution,
in untreated or wortmannin (WM, 100 nM) pretreated
platelets. Similarly, long-chain
DiC
PtdIns-3,4,5-P
(C
PIP
, 5 µM)
was sonicated in the absence of carrier phospholipids and presented as
micelles in untreated or wortmannin pretreated platelets (NP,
non-permeabilized platelets). B, the specificity of
phosphoinositide-mediated pleckstrin phosphorylation was assayed using
synthetic DiC
PtdIns-3-P (PI3P),
DiC
PtdIns-3,4-P
(PI3,4P2),
DiC
PtdIns-3,4,5-P
(PIP3), or
PtdIns-4,5-P
(PI4,5-P2) micelles (5
µM) in the absence of carrier phospholipids, as described
above. The arrow points to the position of pleckstrin. The
results are representative of four separate
experiments.
Figure 6: Phorbol ester can overcome wortmannin inhibition of irreversible platelet aggregation. Gel-filtered platelets were preincubated with 100 nM wortmannin (WM) for 15 min or with 100 nM staurosporine for 15 min prior to stimulation where indicated. After the addition of 500 µg/ml fibrinogen, aggregation was started by the addition of 25 µM TRAP (A) or 100 nM PMA (B) under constant stirring. At the indicated time point, 100 nM PMA was added to the aggregation cuvette (A). The tracings are representative of three experiments.
The results presented in this paper show that activation of
the thrombin receptor in isolated platelets results in the sustained
phosphorylation of the PKC substrate p47 pleckstrin. This event closely
correlates with the sustained accumulation of the PI 3-K lipid product,
PtdIns-3,4-P (Fig. 3). Pretreatment of platelets
with the potent PI 3-K inhibitor wortmannin leads to a loss of the
sustained phosphorylation of pleckstrin and inhibition of the sustained
synthesis of PtdIns-3,4-P
. An initial burst of pleckstrin
phosphorylation is also observed at 30 s to 1 min, but this is not
affected by wortmannin concentrations as high as 1 µM,
suggesting that PKC activation by diacylglycerol and/or other PI
3-K-insensitive pathways are responsible for mediating this early
event. Both DAG and PtdIns-3,4-P
may contribute at later
times as wortmannin only inhibits 61% of TRAP-stimulated pleckstrin
phosphorylation.
Wortmannin inhibition of pleckstrin phosphorylation has previously been reported, but has provided conflicting results. Yatomi et al.(22) have reported inhibition of platelet pleckstrin phosphorylation in response to suboptimal doses of thrombin and phorbol ester stimulation in wortmannin-treated platelets. Hashimoto et al.(33) , however, failed to reproduce the wortmannin inhibition of pleckstrin phosphorylation in response to phorbol ester. In this report, we have found no evidence for a direct inhibition of wortmannin on PKC. Phorbol ester-induced pleckstrin phosphorylation was not inhibited in platelets preincubated with wortmannin, although a complete inhibition was observed with the protein kinase inhibitor staurosporine (Fig. 4). Similarly, purified platelet PKC was not inhibited with wortmannin concentrations as high as 10 µM in in vitro assays. These results are in agreement with results from other laboratories (31, 32, 33) .
The specificity of
wortmannin as a PI 3-K inhibitor is of particular relevance to these
studies, as a recent report described a hormone-stimulated PI 4-kinase
activity that is sensitive to 100 nM wortmannin(34) .
We have shown that treatment of platelets with 100 nM wortmannin does not affect the synthesis of PtdIns-4-P and
PtdIns-4,5-P induced by TRAP(12) . Synthesis of the
D3 phosphoinositides, however, was completely abolished by 100 nM wortmannin. Wortmannin-sensitive PI 4-K activity is therefore
either absent in platelets or not stimulated by activation of the
thrombin receptor.
Based on the observations that sustained
PtdIns-3,4-P production correlates with pleckstrin
phosphorylation and our previous results showing activation of
calcium-independent PKCs
,
, and
(8) , we
devised a permeabilization scheme to introduce phosphoinositides into
platelets and measure pleckstrin phosphorylation. In this assay, both
TRAP and PMA were able to stimulate pleckstrin phosphorylation in
platelets provided with exogenous [
-
P]ATP.
Both PtdIns-3,4-P
and PtdIns-3,4,5-P
were also
able to stimulate pleckstrin phosphorylation above control levels (Fig. 5). Neither PtdIns-3-P nor PtdIns-4,5-P
was
able to stimulate pleckstrin phosphorylation, suggesting that this
event is limited to those phosphoinositides which accumulate in
TRAP-stimulated platelets. These data provide additional evidence that
the lipid products of PI 3-K activate one or more PKC family members,
which in turn phosphorylate pleckstrin. This model is further supported
by the finding that wortmannin inhibition of irreversible platelet
aggregation can be rescued by phorbol ester treatment (Fig. 6),
once again implicating PKC as a downstream target of PI 3-K.
The
results presented here argue that activation of PKC family members by
TRAP-stimulated PI turnover, by PtdIns-3,4-P synthesis, or
by phorbol ester addition can result in similar cell responses. Protein
kinase C activation has previously been presumed to result from the
agonist-mediated activation of phospholipase activity leading to DAG
generation and calcium release from intracellular stores. However, we
have observed the lipid products of PI 3-K, PtdIns-3,4-P
and PtdIns-3,4,5-P
to be capable of activating novel,
calcium-independent PKCs in vitro(8) . The atypical
PKC family member, PKC
has also been shown to be activated by
PtdIns-3,4,5-P
(9) . Both calcium-dependent (
,
I, and
II) and calcium-independent (
,
,
,
and
) PKC family members have been detected in
platelets(30, 35, 36, 37, 38) .
PKC
may also be present in platelets, although only minor amounts
were detected by Western immunoblotting(38) . A number of
groups have investigated the mechanism of PKC activation in stimulated
platelets and have provided conflicting results. Baldassare et al.(30) showed thrombin activation of human platelets to
result in a rapid biphasic increase in DAG mass and correlated this
mass increase with translocation of PKC
,
, and
to the
membrane fraction. However, a subsequent report showed bryostatin
stimulation of platelets failed to affect a translocation of PKC
,
,
, or
(36) . Bryostatin is a macrocyclic
lactone that binds to and activates PKC and induces the phosphorylation
of PKC substrate proteins, including pleckstrin. A recent study
indicated that DAG levels in resting, unstimulated platelets can
fluctuate to levels comparable to that seen with thrombin stimulation
but that these fluctuations do not correlate with the phosphorylation
of pleckstrin (39) . This finding indicates that signaling
pathways other than those that lead to DAG production also may promote
PKC phosphorylation of pleckstrin. Consistent with this idea, platelets
subject to pathological stress and activated in response to von
Willebrand factor (vWF) have been shown to induce pleckstrin
phosphorylation, without stimulating PtdIns-4,5-P
hydrolysis and DAG generation(40) . vWF stimulation of
platelets has also been shown to lead to PI 3-K activation and
translocation to the cytoskeletal fraction(41) , suggesting
that pleckstrin phosphorylation in response to vWF may be mediated by
PI 3-K.
Little is known concerning the function of pleckstrin in
agonist-stimulated cells. Although the phosphorylation of pleckstrin
correlates with platelet aggregation, there is no direct evidence that
it is involved in the activation of the platelet integrin GPIIb-IIIa.
The observation that PH domains found in pleckstrin and other signaling
proteins bind specifically to phosphoinositides such as
PtdIns-4,5-P indicates that pleckstrin may under some
conditions be recruited to the plasma membrane(17) . It is
conceivable that phosphorylation of pleckstrin may affect this
interaction or promote its activity. The N-terminal PH domain of
pleckstrin was recently shown to inhibit both phospholipase C
- and
C
-mediated phosphoinositide hydrolysis(42) . PH domains
have also been shown to mediate protein-protein interactions, and
several PKC isoforms have now been shown to interact with the PH
domains of pleckstrin and of the Btk and Akt protein
kinases(18, 19) . PH domains may also tether proteins
to membranes by interacting with the
subunits of
heterotrimeric G-proteins. The PH domain of the
-adrenergic
receptor kinase (
ARK) has been shown to interact with both
PtdIns-4,5-P
and G
, and both of these ligands
appear to be necessary for the full catalytic activity of this
kinase(43, 44) .
The data presented here show that
activation of PI 3-K in response to TRAP correlates with a slow phase
of PKC activation leading to the phosphorylation of pleckstrin.
Addition of PtdIns-3,4-P and PtdIns-3,4,5-P
to
permeabilized platelets stimulates phosphorylation of pleckstrin. More
over, D3 phosphoinositides and DAG may act synergistically to modulate
GPIIb-IIIa. The initial wave of pleckstrin phosphorylation may result
from a DAG burst activating PKC, but DAG mass is then rapidly lost by
60 s(30) . Sustained PKC activation and pleckstrin
phosphorylation may require PI 3-K activation and synthesis of D3
phosphoinositides, particularly PtdIns-3,4-P
, whose product
correlates with sustained platelet aggregation. These processes could
sustain the activation of PKC.