(Received for publication, October 4, 1994; and in revised form, January 3, 1995)
From the
Stimulation of platelets by thrombin leads to an increased
association of activated phosphoinositide 3-kinase (PI 3-K) with a
membrane cytoskeletal fraction (CSK). Activation of PI 3-K is dependent
upon GTP-binding protein(s), since PI 3-K in permeabilized platelets is
stimulated by GTPS (guanosine 5`-3-O-(thio)triphosphate),
and stimulation of platelet cytosolic PI 3-K by GTP
S requires a
functional small G-protein, Rho. Recent reports indicate that cytosolic
PI 3-Ks can also be activated by the
subunits of
heterotrimeric G-proteins (G
). We now report that the
activated PI 3-K that is associated with CSK can be inhibited by a
recombinant protein containing the G
-binding pleckstrin
homology domain of
-adrenergic receptor kinase 1 (
ARK-PH).
Inhibition is blocked by G
. PI 3-K in nonactivated platelet
CSK is activated by GTP
S but unaffected by
ARK-PH or
G
. Western blots indicate that activated platelet CSK
contains a novel 110-kDa PI 3-K(
) that has been shown to be
stimulated by G
and to lack binding sites for the 85-kDa
subunit of conventional PI 3-K. PI 3-K in immunoprecipitates obtained
via p85 subunit-directed antibodies can be activated by GTP
S but
not by G
. PI 3-K that is stimulatable by G
remains
soluble, as does PI 3-K(
), and is unaffected by Rho. In contrast,
ADP-ribosylation of Rho present in p85 immunoprecipitates is
inhibitory. Further, activation of PI 3-K in permeabilized platelets
exposed to thrombin or GTP
S is inhibited by
ARK-PH and/or
Rho-specific ADP-ribosylating enzymes. We conclude that Rho and
G
each, respectively, contributes to the activation of
different PI 3-Ks (p85-containing heterodimer and PI 3-K (
)) in
thrombin-stimulated platelets.
GTP-binding proteins (G-proteins), ()both
heterotrimeric and small, are known to be involved in platelet
activation by receptor-directed agonists, controlling the inhibition of
adenylyl cyclase, the stimulation of phosphatidylinositide-specific
phospholipase C, and platelet
aggregation(1, 2, 3, 4, 5) .
The significance of phospholipase C and adenylyl cyclase in cell
signaling is by now well documented. More recently, attention has
focused on an enzyme which, in other cells, can regulate events such as
the mitogenic response to growth factors (6) and the
respiratory burst in activated neutrophils (7) ,
phosphoinositide 3-kinase (PI 3-K). PI 3-K has been proposed to play a
role in cytoskeletal/integrin reorganization, and activated PI 3-K
becomes associated with the
integrin-containing membrane cytoskeleton of thrombin-activated
platelets(8) . In platelets exposed to thrombin, PI 3-K rapidly
phosphorylates PtdIns(4,5)P
at the 3-OH position to form
PtdIns(3,4,5)P
(9) . We have reported that PI 3-K in
permeabilized platelets (10) and in platelet cytosolic
fractions (11) can be activated by GTP
S and that such
activation in cytosol is dependent upon the small G-protein Rho, since
ADP-ribosylation of endogenous Rho by C3 transferase is
inhibitory(11) . Rho has also been implicated in the control of
platelet aggregation(5) , a response to platelet agonists that
is dependent upon activated
(12) . PI 3-K may
participate in regulating
activation.
Studies with cytosolic fractions from myeloid
cells (13) and platelets (14) indicate that PI 3-K
activity also can be stimulated by a product of heterotrimeric
G-protein dissociation, G, although direct evidence for the
role of either pathway in regulating the activation of PI 3-K in
receptor-stimulated cells has not yet been provided. We have addressed
this issue, and the nature of the PI 3-Ks involved, in the present
work. In doing so, we have taken advantage of the observations that the
carboxyl-terminal portion of
ARK1 (which contains a PH domain)
binds G
(15, 16) and that EDIN or C3
transferase can ADP-ribosylate Rho selectively in permeabilized
platelets, whose PI 3-K activity is ordinarily responsive to thrombin
or GTP
S(10) .
As demonstrated in Fig. 1, enzymes known to
ADP-ribosylate Rho (either EDIN or C3 transferase) inhibit the
activation of PI 3-K in permeabilized platelets in a dose- and
NAD-dependent manner. This inhibitory effect is both
selective, i.e. not observed with respect to phospholipase C
activation (gauged indirectly by PtdOH, Fig. 2, A and B) or PtdIns(4,5)P
metabolism (not shown), and
transient (Fig. 2, C and D). The transience is
not attributable to a reversal of ADP-ribosylation, since levels of
[
P]ADP-ribosylated Rho remain essentially
constant during the period of incubation with agonist (not shown). It
is possible that a portion of Rho is protected from ADP-ribosylation by
binding to RhoGDI (21) and is later freed of RhoGDI after
exposure to platelet agonists but further protected by interaction with
effector(s). It also seemed possible, however, that inhibition might be
overcome by increasing accumulations of G
(13, 14) as a function of the stimulated dissociation
of heterotrimeric G-protein(s).
Figure 1:
Effect of varied ADP-ribosylation of
Rho on GTPS-stimulated PI 3-K in permeabilized platelets.
Permeabilized platelets were incubated with
[
P]ATP and the indicated concentrations of
EDIN (
) or C3 transferase (
), in the presence or absence
(*) of NAD
, 5 min prior to incubation with buffer or
GTP
S for 2 min. Labeled lipids were extracted and quantitated as
described. Basal quantities of labeled lipids were unaffected by
ADP-ribosylation conditions. Results are presented as the average
percentage of inhibition of stimulated accumulations of
PtdIns(3,4)P
± the range for a representative
experiment performed in duplicate. Error bars are included within symbols. Similar effects were observed for
PtdIns(3,4,5)P
. No significant GTP
S-stimulated changes
were observed in PtdIns3P during this period, as noted(10) .
Changes in PtdIns(4,5)P
and PtdOH were unaffected by
ADP-ribosylation. 3PPI, 3-phosphorylated
phosphoinositide.
Figure 2:
Effects of EDIN on GTPS- or
thrombin-stimulated accumulations of PtdIns(3,4,5)P
,
PtdIns(3,4)P
, and PtdOH in permeabilized platelets with
time. Platelets were incubated, as in Fig. 1, with (opensymbols) or without (closedsymbols) 22
µg/ml EDIN prior to addition of buffer, GTP
S (A and C), or thrombin (B and D) for 2 min. Lipids
were resolved and quantitated as described. No significant changes were
observed in the absence of GTP
S or thrombin. Data are shown as the
averages ± ranges (included within symbols) for a
representative experiment performed in duplicate. C and D, PtdIns(3,4,5)P
(
,
) and
PtdIns(3,4)P
(
,
) are shown. A and B, PtdOH (
,
) is shown. Similar results were
observed when C3 transferase and GTP
S/SFLLRN were employed in
place of EDIN and thrombin.
That G becomes available
to activate PI 3-K in thrombin-stimulated platelets is indicated by the
data in Fig. 3. The CSK fraction from platelets exposed to
thrombin for 45 s exhibits an elevated specific activity of PI 3-K in
comparison with the Triton-soluble fraction (supernatant) from
similarly incubated cells (Fig. 3A), and this elevated
PI 3-K activity gradually decreases as increasing concentrations of
G
-binding
ARK-PH are added to CSK. Equivalent amounts of
glutathione S-transferase are without effect (not shown). The
inhibitory effects of
ARK-PH are maximal at 5 µM and
bring the specific activity of CSK PI 3-K down to that of supernatant,
which is unaffected by
ARK-PH. That PI 3-K in supernatant is
unaffected by
ARK-PH argues against the PH domain of
ARK
acting as an inhibitor by binding to PtdIns(4,5)P
substrate(22) . Furthermore, G
additionally
activates the PI 3-K activity of CSK from stimulated platelets and
competes with
ARK-PH, consistent with a specific effect of
ARK-PH on G
. The effect of thrombin in promoting an
increased specific activity of PI 3-K in CSK is rapid, achieving near
maximal levels within 15 s of thrombin addition (Fig. 3B). Most of this increase is inhibited by 5
µM
ARK-PH, causing the specific activity to approach
that of PI 3-K in unstimulated platelet CSK.
Figure 3:
Effects of ARK-PH and G
on
PI 3-K activity in platelet CSK and supernatant. CSK and supernatant
derived from platelets exposed to thrombin for 45 s (A) were
incubated with varied concentrations of
ARK-PH in the absence
(
) or presence (
) of 1 µM G
and
assayed for PI 3-K activity. Supernatant was also assayed with varied
concentrations of
ARK-PH (
). Results are presented as the
mean ± S.D. for three incubations in duplicate. CSK fractions
were also obtained from platelets incubated for 0-3 min with
thrombin (B) and assayed for PI 3-K activity in the presence
(
) or absence (
) of
ARK-PH (5
µM).
ARK-PH also
inhibits the accumulation of labeled PtdIns(3,4,5)P
and
PtdIns(3,4)P
but not PtdOH (or metabolism of
PtdIns(4,5)P
; not shown) in permeabilized platelets
stimulated with GTP
S (Fig. 4), implying an impairment of PI
3-K but not of phospholipase C activities. The combination of EDIN and
ARK-PH is more inhibitory than is either agent alone; however,
neither totally blocks the activation of PI 3-K after 5 min, leaving it
likely that an additional factor, relatively insensitive to EDIN and
ARK-PH, contributes to the stimulated activity observed.
Figure 4:
Effects of varied concentrations of
ARK-PH with or without EDIN on GTP
S-stimulated accumulations
of PtdIns(3,4,5)P
, PtdIns(3,4)P
, and PtdOH in
permeabilized platelets. Platelets were incubated as in Fig. 1with up to 10 µM
ARK-PH in the presence
(*) or absence of 22 µg/ml EDIN, prior to addition of buffer or
GTP
S (10 µM). Incubations were terminated after 5
min, and lipids were extracted and resolved as described. Results shown
are the averages ± ranges (in some cases ranges are included
within symbols) of two experiments performed in duplicate and
are presented as the -fold increase over GTP
S-free controls. Basal
values with or without
ARK-PH or EDIN were not significantly
different. No effects of glutathione S-transferase in
ARK-PH buffer were observed.
, PtdIns(3,4,5)P
;
, PtdIns(3,4)P
;
, PtdOH. Note that values for
PtdOH with EDIN are not shown, since they were not different from
values without EDIN (see also Fig. 2).
Interestingly, the lower PI 3-K activity associated with the small
amount of CSK present in unstimulated platelets (8) is
unaffected by ARK-PH (Fig. 3B). Not only is
ARK-PH without inhibitory effect (which might have been attributed
to a lack of G
), but, as illustrated in Fig. 5,
G
is not stimulatory. Yet, PI 3-K in this fraction (which
contains Rho) can be stimulated by GTP
S. The stimulation is
impaired by ADP-ribosylation of Rho and restored by exogenous Rho,
implying that Rho/GTP
S-stimulatable PI 3-K is different from
G
-stimulatable PI 3-K. Indeed, the Rho-containing
p85-directed immunoprecipitates derived from resting platelet cytosol
(using polyclonal antibody to the 85-kDa subunit of PI 3-K (Fig. 6) or a combination of monoclonals to p85 isoforms (Fig. 7)) show similar characteristics: no activation of PI 3-K
by G
but activation by GTP
S ( Fig. 6and Fig. 7), in a manner inhibited by ADP-ribosylation (Fig. 7). The antibody has no effect on total PI 3-K activity
with or without G
(Fig. 6). As gauged by Western
blotting, the efficiency of p85 immunoprecipitation achievable with the
polyclonal antibody ranges from 38 to 42% and is equally efficacious
for
,
, and
isoforms, whereas the combination of
monoclonal antibodies is more efficient (up to 96%).
GTP
S-stimulated activity in the post-immunoprecipitation
supernatant decreases in proportion to decreases in p85, whereas
G
-stimulatable PI 3-K remains in the supernatant at levels
relatively unaffected by immunoprecipitation. Notably, platelet Rho
also sediments with p85-containing PI 3-K, in proportion to the
efficiency of immunoprecipitation. Under conditions in which
38-42% of p85 is immunoprecipitated, approximately the same
amount of Rho is sedimentable, and when more than 90% of p85 is
immunoprecipitated, most of the Rho is immunoprecipitated as well. One
might predict, therefore, that, in contrast to the case for the assayed
immunoprecipitate, ADP-ribosylation of the Rho-depleted
post-immunoprecipitation supernatant would have no effect on PI 3-K
activity. Indeed, GTP
S is ineffective in stimulating, and
ADP-ribosylation cannot inhibit this activity (Fig. 7). Even
when exogenous Rho is provided to the p85 and Rho-depleted supernatant,
however, there is no enhancement of PI 3-K activity (Fig. 8).
Thus, in contrast to the case for p85-containing PI 3-K,
G
-stimulatable PI 3-K is not modulated by Rho. The platelet
cytosol presumably contains relatively low but variable amounts of
heterotrimeric G-protein(s), since we have found GTP
S-stimulated
PI 3-K to be inhibited variably by
ARK-PH. This supposition is
confirmed by the variable detectability of G
in Western blots of
platelet cytosol (data not shown).
Figure 5:
Effects of G, ADP-ribosylation (ADP-R), Rho, and GTP
S on PI 3-K activity in CSK from
unstimulated platelets. Triton-insoluble (CSK) fractions from
unstimulated platelets were incubated under various conditions as
described under ``Experimental Procedures,'' and PI 3-K
activity was assayed. *, with Rho (5 µM), added after
ADP-ribosylation of endogenous Rho. Results are the means ± S.D.
of two experiments in duplicate. Openbars, without
GTP
S; hatchedbars, with
GTP
S.
Figure 6:
Effects of polyclonal antibody to p85 on
G- or GTP
S-stimulatable PI 3-K activity. Platelet
cytosol (Cytosol) was incubated with buffer (- p85
Ab for control and later ``mock'' immunoprecipitation)
or polyclonal rabbit antibody to the p85 subunit of PI 3-K (+ p85 Ab) at 4 °C. A portion of these incubation mixtures was
combined as well with protein A-Sepharose, followed by sedimentation.
The supernatant (Cytosol, Post-spin) was removed and saved,
and the pellet was resuspended in the same volume of original buffer as
the cytosol from which it came. Efficiency of immunoprecipitation was
monitored by Western blotting with polyclonal antibody to p85, and the
percentage of the original p85 present was calculated (values in parentheses). PI 3-K activities in cytosol, post-spin cytosol,
and resuspended pellets (from true and mock immunoprecipitations) were
assayed as described under ``Experimental Procedures'' in the
presence (hatchedbars) or absence (openbars) of GTP
S (5 µM) with or without
G
(1 µM). Data are results of an experiment
performed in duplicate and are representative of two
experiments.
Figure 7:
Effects of immunoprecipitation with
combined monoclonal antibodies to p85 on activation of PI 3-K by
GTPS or G
and inhibition by EDIN. Incubations proceeded
as in Fig. 6except that antibodies were added to all cytosols
and assayed, or immunoprecipitated and then assayed, i.e. no
mock immunoprecipitations were performed. Combined monoclonal
antibodies to the
,
, and
forms of p85 were added to
cytosol, and immunoprecipitation with anti-mouse IgG/protein
A-Sepharose was performed. Pellets were suspended to the same original
volume. Western blotting was with combined monoclonal antibodies to
p85. The percentage of original p85 is shown in parentheses.
PI 3-K was assayed following pretreatment for 5 min with
-NAD
(200 µM) with or without EDIN
(20 µg/ml). Assays were in the presence (hatchedbars) or absence (openbars) of
GTP
S (5 µM) with or without G
(1
µM) and are the results of an experiment in duplicate. ADP-R, ADP-ribosylation; I. P.,
immunoprecipitate.
Figure 8:
Effects of exogenous Rho on
G-stimulated PI 3-K activity in cytosol depleted of p85 and
Rho. Platelet cytosol was incubated with a mixture of monoclonal
antibodies to p85
,
, and
isoforms as in Fig. 7and then centrifuged with anti-mouse IgG and protein
A-Sepharose. PI 3-K activity in the resulting supernatant fractions was
assayed in the presence or absence of recombinant Rho-glutathione S-transferase (1 µM) with or without G
(1 µM) and with or without GTP
S (5 µM). Hatchedbars, with GTP
S. The antibody mixture
was >90% efficient in immunoprecipitating both p85 and endogenous
Rho. Results are the average ± the range of an experiment
performed in duplicate.
Using Western blotting, we have
detected a new form of PI 3-K in platelet cytosol and in activated CSK (Fig. 9). This PI 3-K() has been cloned and expressed
recently and has been detected in U937 and K562 cells.
A
cDNA sequence encoding this novel kinase is also present in a leukemic
cell line derived from a platelet progenitor cell, the megakaryoblast. (
)PI 3-K(
) is not present in CSK of quiescent platelets
but appears in the CSK after platelet activation (Fig. 9), as
does G
-stimulatable (Fig. 9,) and
ARK-PH-inhibitable (Fig. 9, *) PI 3-K activity, and G
(not shown). Further, PI 3-K(
) is not immunoprecipitated by our
p85-directed antibodies, even when >90% of p85 is immunoprecipitated (Fig. 9).
Figure 9:
Presence of PI 3-K() in cytoskeleton
and cytosol of human platelets. Platelet cytosol before (lane1) and after (lane2)
immunoprecipitation with a mixture of monoclonal antibodies to p85
isoforms (as in Fig. 8, >90% of p85 immunoprecipitated) was
monitored by Western blotting with an affinity-purified antibody to PI
3-K(
), after resolution on SDS-polyacrylamide gel electrophoresis
and transfer to nitrocellulose. Similarly, Triton-insoluble
cytoskeletal fractions were prepared from quiescent (lane3) and thrombin-activated (45 s) platelets (lane4), as described in Fig. 3. The same amount of
protein from resting and activated platelets was applied per lane for
SDS-polyacrylamide gel electrophoresis and Western blotting. The arrow indicates PI 3-K(
). For cytoskeletal fractions (lanes3 and 4) assayed for PI 3-K activity
(see also Fig. 3and Fig. 5), inhibition by
ARK-PH
is shown (*), and stimulation by G
(**) is
shown.
Our studies indicate that thrombin-induced stimulation of
platelet PI 3-K activity is dependent upon both functional Rho and
G. This conclusion is based upon the inhibitory effects of
ADP-ribosylating C3 transferase/EDIN and G
-binding
ARK-PH, respectively. Further, the Rho-responsive versus G
-responsive forms of PI 3-K differ. Our findings
suggest that G
-stimulatable PI 3-K is a distinct PI 3-K,
probably not a conventional p85-containing PI 3-K. Rho-responsive PI
3-K activity precipitates with antibodies directed to the 85-kDa
subunits of the most thoroughly characterized (to date) form of PI 3-K, i.e. the heterodimer. Consistent with this, the
immunoprecipitates also contain the 110-kDa catalytic subunit of PI 3-K
known as p110(
), detected by Western blotting (not shown), as well
as Rho and CDC42Hs, (
)another member of the Rho family of
small G-proteins(23) . These immunoprecipitates, however, do
not contain a newly identified PI 3-K, called PI 3-K(
).
In these respects, the cytoskeleton of ``resting''
platelets and p85-directed immunoprecipitates resemble each other. PI
3-K(
) is a 110-kDa protein that shares the kinase domain of the
other mammalian p110 subunits (
and
) known to associate with
p85
and
subunits but lacks homology in the N-terminal
region responsible for binding to p85, which explains why recombinant
p110(
), in contrast to recombinant p110(
), does not bind p85.
Significantly, recombinant PI 3-K(
) is activated directly in
vitro by G
.
Platelet PI 3-K(
) remains
in the supernatant following immunoprecipitation of p85 PI 3-K, as does
G
-stimulatable PI 3-K activity, and both PI 3-K(
) and
G
-stimulatable PI 3-K activity appear in the cytoskeletal
fraction of thrombin-activated platelets. It therefore seems likely
that the G
-activated PI 3-K present in our platelet
subfractions is PI 3-K(
). These observations are consistent with
the finding of chromatographically separable PI 3-Ks in the cytosol of
myeloid cells and the lack of p85 isoforms
,
, or
in
the G
-activable PI 3-K from the cytosolic subfractions of
such cells(13) . Our results, however, differ from those of
Thomason et al.(14) who, using a monoclonal antibody
to p85 different from those that we employed, found that
G
-stimulatable PI 3-K co-precipitated with p85. Conceivably,
an epitope is shared between p85 and PI 3-K(
) or another component
to which PI 3-K(
) binds. Another possibility is that the platelets
from which the cytosolic fraction was derived were activated
inadvertently and that, in fact, a cytoskeletal complex containing both
p85/PI 3-K and PI 3-K(
) was isolated with the immunoprecipitates.
The observation that two forms of PI 3-K, regulated by G
and Rho, respectively, are activated in thrombin-stimulated platelets
is noteworthy. By definition, both routes of activation are controlled
by GTP, the former via the receptor and GTP-dependent dissociation of
G
from G
and the latter via the binding of GTP to the
small G-proteins of the Rho family (the connection between the receptor
and Rho being as yet unknown). Why two such mechanisms for regulating
PI 3-K are available in a cell exposed to one initial agonist is an
intriguing question. Further, although
ARK-PH was used as a
G
-sequestering tool in our experiments, platelets contain
ARK1 and
ARK2. (
)It is thus possible that platelet
ARK or other G
-binding entities in addition to G
play a physiological role in regulating platelet PI 3-K.
That
activated cytoskeletal PI 3-K is inhibited almost completely by
ARK-PH does not necessarily preclude Rho-dependent PI 3-K being
associated with activated CSK. Increased amounts of both Rho and PI 3-K
(gauged by the 85-kDa subunit of PI 3-K) have been found in the CSK of
activated platelets(8, 11) . It may be, however, that
Rho-dependent activation cannot be maintained easily during CSK
isolation, whereas G
is isolated with PI 3-K in CSK and is
activating as such. Some questions in this regard are whether PI
3-K(
) associates with CSK directly or via G
and how
p85/Rho PI 3-K and PI 3-K(
) each binds to CSK. An additional
complication is posed by the observation that Rho does not appear to
bind to PI 3-K directly (23) although it is present in
p85-directed immunoprecipitates from platelet cytosol, yet CDC42Hs,
also present in platelet cytosol,
has been shown in
reconstitution experiments to bind and activate p85-containing PI
3-K(23) . It thus seems likely that the presence of Rho in PI
3-K immunoprecipitates from platelet cytosol is dependent upon
additional factor(s) and that Rho may regulate the interaction between
endogenous platelet CDC42Hs and p85-containing PI 3-K. These are
subjects currently under investigation in our laboratory.