(Received for publication, July 7, 1995)
From the
We have reported that platelets exposed to thrombin or thrombin
receptor-directed ligand activate phospholipase C and rapidly
accumulate
phosphatidylinositol(3, 4, 5) -trisphosphate
(PtdIns(3,4,5)P) and phosphatidylinositol (3, 4) -bisphosphate (PtdIns(3,4)P
) as a
function of the activation of phosphoinositide (PI) 3-kinases in a
GTP-binding protein-dependent manner. In such platelets, serine- and
threonine-directed phosphorylation of pleckstrin also occurs and has
been attributed to protein kinase C activation. We now report that the
phosphorylation of pleckstrin is partially dependent upon PI 3-kinase.
Pleckstrin phosphorylation in response to thrombin receptor stimulation
is progressively susceptible to inhibition by wortmannin, a potent and
specific inhibitor of platelet PI 3-kinases. PI 3-kinase thus seems to
play a gradually increasing role in promoting pleckstrin
phosphorylation. The IC
for wortmannin in inhibiting
SFLLRN-stimulated 3-phosphorylated phosphoinositide accumulation is 10
nM, and that (i.e. 50% of maximum inhibition) for
inhibiting pleckstrin phosphorylation is 15 nM. Synthetic
PtdIns(3,4,5)P
, when added to saponin-permeabilized (but
not intact) platelets, causes wortmannin-insensitive phosphorylation of
pleckstrin. PtdIns(3,4,5)P
also overcomes the inhibition by
wortmannin of thrombin- or guanosine
5`-3-O-(thio)trisphosphate-stimulated pleckstrin
phosphorylation. In contrast, PtdIns(4,5)P
or inositol (1, 3, 4, 5) -tetrakisphosphate are
ineffective in these respects. The pattern of phosphorylation of
pleckstrin activated by PtdIns(3,4,5)P
is not
distinguishable from that of pleckstrin phosphorylated in intact
platelets exposed to protein kinase C-activating
-phorbol
myristate acetate, mimicking diacylglycerol. Activation of protein
kinase(s) by PtdIns(3,4,5)P
thus offers a route for
pleckstrin phosphorylation in vivo that is an alternative to
activation of phospholipase C
diacylglycerol
protein
kinase C.
It is being appreciated increasingly that the metabolism of
phosphoinositides catalyzed by phosphoinositide (PI) ()3-kinase has signal-transducing consequences that rival
those of phosphoinositidase C activation in a variety of cells.
PtdIns(3,4,5)P
and PtdIns(3,4)P
are both major
physiological products of activated PI 3-kinase. They have been
proposed to be modulators of protein kinase(s), thereby amplifying an
initial limited amount of signal(1) , analogously with
diacylglycerol and PKC. Indeed, several members of the PKC family have
been reported recently to be stimulated by PtdIns(3,4,5)P
,
including a mixture of rat brain PKC isozymes(2) ,
PKC
(3) , and PKC
,
, and
(by either
PtdIns(3,4,5)P
or PtdIns(3,4)P
)(4) .
Other protein kinases may also be stimulated. We have described the
activation of two species of PI 3-kinase by a variety of agonists,
including thrombin, thromboxane A
analogue, GTP
S, and
thrombin receptor-directed ligand, leading to the sequential
accumulation of PtdIns(3,4,5)P
and PtdIns(3,4)P
in intact or permeabilized
platelets(5, 6, 7) . Another response to
platelet activation, one of the earliest reported (8, 9) , is the phosphorylation of an apparent M
40,000-47,000 protein known as pleckstrin (platelet and leukocyte C kinase
substrate) (10) at serine and threonine. As the name
implies, pleckstrin is considered to be one of the major (and is
certainly one of the most readily identifiable) substrates for PKC in
the platelet. Recently, an inhibitory effect of nM concentrations of the mycotoxin wortmannin on pleckstrin
phosphorylation, which is not attributable to inhibition of PKC, has
been reported(11) . Since wortmannin has since proved to be a
potent and rather selective irreversible inhibitor of PI
3-kinases(12, 13, 14) , blocking both the
lipid kinase and intrinsic protein kinase activities of this enzyme (15) , we decided to determine whether the inhibitory effects
of wortmannin on pleckstrin phosphorylation were attributable to
inhibition of PtdIns(3,4,5)P
formation.
We present evidence that wortmannin inhibits the accumulation
of 3-PPI and phosphorylation of pleckstrin (p47) in response to
thrombin, thrombin receptor-directed peptide, or GTPS, and that
the inhibition of pleckstrin phosphorylation can be overcome in
permeabilized platelets by diC8- or diC16-PtdIns(3,4,5)P
,
but not by PtdIns(4,5)P
or Ins(1,3,4,5)P
. These
findings implicate a PtdIns(3,4,5)P
-activated protein
kinase, rather than the intrinsic protein kinase activity of PI
3-kinase. Phosphorylation of pleckstrin can be initiated by
PtdIns(3,4,5)P
in a time and concentration-dependent
manner, and does not lead to accumulation of
P-PtdOH or
further accumulation of
P-3-PPI. The pattern of
phosphorylation of pleckstrin achieved in platelets in response to
PtdIns(3,4,5)P
is not distinguishable from that resulting
from exposure to PMA, although this does not constitute proof that PKC
is being activated by PtdIns(3,4,5)P
.
Fig. 1indicates the dose-dependence for wortmannin's
inhibitory effects on both 3-PPI accumulation (Panel A) and
p47 phosphorylation (Panel B). These are seen to be quite
similar, with IC values of 10-15 nM, where
maximum inhibition of p47 (pleckstrin) phosphorylation is 50-60%.
Figure 1:
Dose-dependent inhibition by wortmannin
of SFLLRN-activated 3-PPI accumulation and pleckstrin phosphorylation.
Labeled platelets were exposed to 0-100 nM wortmannin
for 5 min at 37 °C prior to the addition of SFLLRN (10
µM) for 120 s. Lipids and proteins were extracted,
resolved, and quantitated as described. Data are the mean ± S.D.
of two experiments performed in duplicate, where 100% = the
maximum stimulated response. Panel A, 3-PPI accumulation:
PtdIns(3,4,5)P (
) and PtdIns(3,4)P
(
). Panel B, effects on pleckstrin
phosphorylation.
As shown in Fig. 2, phosphorylation of pleckstrin occurs
rapidly in platelets exposed to SFLLRN, and net phosphorylation is
inhibited only slightly by 100 nM wortmannin within the first
15 s. In contrast, net accumulation of PtdIns(3,4,5)P peaks
by 30 s, followed by sustained accumulation of
PtdIns(3,4)P
, and the levels of both are inhibited
completely by 100 nM wortmannin in this period (not shown;
IC
10 nM)). Accumulation of
P-PtdOH (not shown), indicative of phosphoinositidase C
and DG kinase activation in platelets(21) , is unaffected by
wortmannin. With time, however, following accumulation of the 3-PPIs,
the inhibitory effects of wortmannin on
P-p47 levels
become more pronounced, reaching a maximum of about 50% by 60 s, and
decreasing by 120 s. The delayed nature of wortmannin-inhibitable
phosphorylation of p47 is emphasized by the broken line in this figure.
Similar effects are observed if platelets are exposed to wortmannin for
5 min and then washed prior to stimulation. Thus, the delayed effects
of wortmannin on p47 phosphorylation are not due to a time-dependent
direct inhibition by wortmannin of pleckstrin-directed protein kinase.
Figure 2:
Accumulation of P-labeled
3-PPI and pleckstrin in platelets exposed to SFLLRN. Labeled platelets
were exposed to 100 nM wortmannin or Me
SO for 5
min prior to the addition of SFLLRN (10 µM) or buffer for
varied periods. In some studies, platelets were exposed to wortmannin
prior to washing and labeling. Lipids and proteins were extracted,
resolved, and quantitated as described. Data are the average ±
range of duplicates (some ranges are included within symbols)
and are representative of two experiments. 3-PPI are presented as a
percent of the activity (basal) in the absence of SFLLRN:
PtdIns(3,4,5)P
- wortmannin (
) and
PtdIns(3,4)P
- wortmannin (
). For p47
(pleckstrin): - wortmannin (
); + wortmannin (
);
(- wortmannin)-(+ wortmannin) (
) in
disintegrations/min. Wortmannin inhibited completely 3-PPI accumulation
at all times, but did not affect basal
P-pleckstrin, which
did not change in the absence of SFLLRN.
The involvement of PtdIns(3,4,5)P in this event is made
clear in Fig. 3and Fig. 4. When added to permeabilized
platelets, PtdIns(3,4,5)P
causes phosphorylation of p47 in
a time- and dose-dependent manner (Fig. 3), achieving close to
maximum effects after a 5-min exposure to 4 µm
diC8-PtdIns(3,4,5)P
.Approximately twice that concentration
of diC16-PtdIns(3,4,5)P
is required to attain the same
results, attributable to the different solubility characteristics of
these isoforms (not shown). Both isoforms of PtdIns(3,4,5)P
overcome the inhibitory effects of 100 nM wortmannin on
p47 phosphorylation induced by GTP
S (Fig. 4) or thrombin,
implicating PtdIns(3,4,5)P
in regulating pleckstrin
phosphorylation in response to a variety of agonists that are dependent
upon GTP-binding proteins. Exogenous diC8- or
diC16-PtdIns(3,4,5)P
is not acting via contaminant diC8-DG
or diC16-DG, or via phosphoinositidase C action leading to diC8-DG or
diC16-DG, since 1) intact platelets are not affected, 2) no
P-labeled PtdOH is formed in response to diC8- or
diC16-PtdIns(3,4,5)P
, whereas diC8-DG is known to activate
PKC in intact or permeabilized platelets, and become converted to PtdOH (
)(22) and 3) PtdIns(3,4,5)P
is not a
substrate for any known phosphoinositidase C(23, 24) .
In comparison, one can estimate that platelets exposed to thrombin for
15 s accumulate 8-10 pmol of PtdIns(3,4,5)P
/10
cells(17) , or 0.8-1 µM (10
µl/10
), local concentrations at the plasma membrane, of
course, being higher.
Figure 3:
Phosphorylation of pleckstrin in platelets
incubated with PtdIns(3,4,5)P. Permeabilized platelets were
exposed to varied concentrations of diC8-PtdIns(3,4,5)P
for
5 min (A) or to 2 µM diC8-PtdIns(3,4,5)P
for various periods (B) after an initial 60-s labeling
period with [
-
P]ATP + saponin, and
incubations were terminated with Laemmli SDS-reducing buffer and
boiling, followed by SDS-polyacrylamide gel electrophoresis and
quantitation of
P in p47 protein, as above. Similar
results were obtained using diC16-PtdIns(3,4,5)P
, which was
about half as efficient. No accumulation of labeled PtdOH or 3-PPI was
observed in response to exogenous PtdIns(3,4,5)P
. No
increase in
P-pleckstrin occurred when intact platelets,
labeled as in Fig. 2, were exposed to
PtdIns(3,4,5)P
. Results are the averages ± ranges of
duplicates, included within symbols.
Figure 4:
The effects of wortmannin on GTPS- or
PtdIns(3,4,5)P
-induced
P-pleckstrin
accumulation in permeabilized platelets. Platelets were exposed to
wortmannin (100 nM) or Me
SO for 5 min at 37 °C
before washing and permeabilization as above. Wortmannin did not affect
basal levels of
P-pleckstrin. Subsequent incubations were
± diC8-PtdIns(3,4,5)P
(2 µM) or
PtdIns(4,5)P
(4 µM) for 6 min, or with these
agents for 6 min + GTP
S (10 µM) for the final 1
min. Similar effects were observed when thrombin (5 units/ml) was
substituted for GTP
S. Results (A) are the average
disintegrations/min ± range of duplicate determinations, counted
from the gels at 47 kDa whose autoradiograph is shown in (B),
corresponding to samples 1-8. Results are representative of three
experiments.
Several proteins are labeled with P in permeabilized platelets at ``rest'' or in
response to exogenous PtdIns(3,4,5)P
, however, p47 is the
protein most conspicuously phosphorylated in response to physiological
platelet agonists or
-phorbol esters. As seen in Fig. 5A, which shows results with pleckstrin-directed
immunoprecipitations,
P-labeled pleckstrin accumulates in
platelets exposed to PtdIns(3,4,5)P
, and other labeled
proteins do not co-immunoprecipitate appreciably. Upon CNBr digestion,
the pattern of phosphorylation, which is alkali-labile, is very similar
to that achieved in response to PKC-activating PMA (Fig. 5B). Phosphorylation by PKC of pleckstrin appears
to occur primarily on three residues: Ser
,
Thr
, and Ser
,
and it appears
that this same preference is maintained for
PtdIns(3,4,5)P
-activated protein kinase. It is thus
possible that PKC(s) is(are) activated by PtdIns(3,4,5)P
in
platelets, consistent with the observation that PKC species, notably
PKC
, are activated by PtdIns(3,4,5)P
or
PtdIns(3,4)P
. In fact, both species of phosphoinositide may
be present when PtdIns(3,4,5)P
is added to permeabilized
platelets, since 5-phosphatase activity capable of acting on this
substrate is present in platelets. (
)As is true for
SFLLRN-stimulated intact platelets (Fig. 2), however, p47
phosphorylation is transient, whereas PtdIns(3,4)P
is
increasing (Fig. 3), therefore only PtdIns(3,4,5)P
may be effective here. Of further interest, the cytoskeleton of
activated platelets, with which PI 3-kinases become
associated(7) , also contains increased amounts of PKC
, as
detected by Western blotting,
constituting a potential
locus for the pleckstrin phosphorylation observed.
Figure 5:
Immunoprecipitation of P-pleckstrin and CNBr digestion. Pleckstrin from
permeabilized platelets preincubated with 100 nM wortmannin
and subsequently labeled with [
-
P]ATP
± diC8-PtdIns(3,4,5)P
was extracted and
immunoprecipitated as described. An autoradiograph of the
immunoprecipitated
P-labeled material is shown (A), where the first lane is the immunoprecipitate from
buffer-incubated platelets, and the subsequent three lanes are from
triplicate incubations with diC8-PtdIns(3,4,5)P
. The
autoradiograph of CNBr-digested and undigested immunoprecipitate,
electrophoresed on a tricine polyacrylamide gel, is shown (B, right) in comparison with a digest of immunoprecipitated
P-pleckstrin from labeled platelets exposed to PMA (B, left). The numbers shown indicate molecular mass
(kDa).
At present, the
function of pleckstrin (or phosphorylated pleckstrin) in activated
platelets is unknown. It may play a role in the reorganization of
integrin , whose active
conformation binds fibrinogen and is involved in platelet aggregation.
We have observed (
)that addition of PtdIns(3,4,5)P
to permeabilized platelets, in addition to promoting pleckstrin
phosphorylation, potentiates the increase in active
formed in response to SFLLRN.
Recent studies in which pleckstrin has been expressed in COS-1 or
HEK-293 cells indicate that it can inhibit the activation of
phosphoinositidase C
and phosphoinositidase C
initiated by
several receptors, including that for thrombin, and the findings are
consistent with an interaction between pleckstrin and
PtdIns(4,5)P
(20) . Given its two pleckstrin
homology domains, pleckstrin may also play a part in regulating PI
3-kinase (
) activity by binding to G
subunits(7) , a function currently under investigation in our
laboratory. Phosphorylation of pleckstrin in response to
PtdIns(3,4,5)P
/PtdIns(3,4)P
offers an alternate
route to that provided by the activation of phosphoinositidase C,
formation of DG, and activation of PKC. This may serve a redundant
function, allowing, e.g. a phosphoinositidase C deficiency to
have less severe consequences for protein phosphorylation under certain
circumstances, or it may affect the duration of the PKC response,
permitting a more sustained activation after DG is metabolized, or it
may be relevant to the localization of PKC activation. In any case, our
data constitute unique evidence that PtdIns(3,4,5)P
plays a
second messenger role in activating protein kinase(s) in an
agonist-responsive cell, the human platelet.