From the Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
Received for publication, July 1, 2002, and in revised form, December 30, 2002
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ABSTRACT |
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To better understand the molecular mechanisms of
platelet granule secretion, we have evaluated the role of type II
phosphatidylinositol (PtdIns) 5-phosphate 4-kinase in agonist-induced
platelet Phosphoinositide phosphorylation has been shown to play a prominent
role in regulated granule secretion. Initial observations in chromaffin
cells demonstrated that the maintenance of polyphosphoinositides is
crucial for vesicle secretion (17). Subsequently, type I PIPK (18) and
phosphatidylinositol transfer protein (19, 20) were found to mediate
ATP-dependent events required for granule secretion.
Activation-dependent translocation of type I PIPK to membranes has emerged as a key event in the regulated synthesis of
PtdIns(4,5)P2. Regulated translocation of type I PIPK is
directed by ADP-ribosylation factors (21-23). Investigators have
hypothesized that focal sites of PtdIns(4,5)P2 form in
areas of PIPK activity following translocation to membranes. According
to this model, the nidus of PtdIns(4,5)P2 that is
subsequently formed on the cytoplasmic face of the cell membrane binds
components of the secretory machinery that contain
PtdIns(4,5)P2-binding domains. Support for this model is
derived from studies in chromaffin cells in which agonist-induced
secretion is blocked by inhibitory PtdIns(4,5)P2-binding proteins that localize to focal membrane sites (24). Thus,
translocation of type I PIPK to membranes may initiate its function in
granule secretion.
Type II PIPK can also undergo activation-dependent
translocation (25, 26) and contains an activation loop that directs localization of this kinase to specific subcellular locals (27, 28).
Although cellular functions for type II PIPK have been difficult to
demonstrate (29), type II PIPK has been shown to participate in
MgATP-dependent, Ca2+-triggered Chemicals and Reagents--
All buffer constituents, Triton
X-100, and CaCl2 were purchased from Sigma. Sepharose 2B
was obtained from Amersham Biosciences. Reduced SL-O was
purchased from Corgenix (Peterborough, United Kingdom).
Bisindolylmaleimide I, PMA, protein kinase C inhibitor peptide (19-31,
RFARKGALRQKNV), and 5,6-dichloro-1- Platelet Preparation--
Blood from healthy donors who had not
ingested aspirin in the 2 weeks prior to donation was collected by
venipuncture into 0.4% sodium citrate. Citrate-anticoagulated blood
was centrifuged at 200 × g for 20 min to prepare
platelet-rich plasma. Platelets were then purified from platelet-rich
plasma by gel-filtration using a Sepharose 2B column equilibrated in
PIPES/EGTA buffer (25 mM PIPES, 2 mM EGTA, 137 mM KCl, 4 mM NaCl, 0.1% glucose, pH 6.4).
Final gel-filtered platelet concentrations were 1-2 × 108 platelets/ml.
Permeabilization of Platelets--
Platelets were permeabilized
using reduced SL-O. The ability of each batch of SL-O to permeabilize
platelets was tested by analyzing for incorporation of FITC-dextran
sulfates by flow cytometry as described previously (4).
Analysis of P-selectin Surface Expression--
For analysis of
P-selectin surface expression from agonist-stimulated,
SL-O-permeabilized platelets, 20 µl of gel-filtered platelets
(1-2 × 108/ml) were permeabilized by exposure to the
indicated concentration of reduced SL-O. Samples were adjusted to pH
6.9 immediately following permeabilization and then incubated with
SFLLRN, PMA, or buffer. The timing of the addition of inhibitors or
recombinant proteins is indicated in the figure legends. Following a
20-min incubation after the addition of agonist, 10 µl of reaction
mixture was transferred to 5 µl of phycoerythrin-conjugated AC1.2
anti-P-selectin antibody. Phosphate-buffered saline (500 µl) was
added to the sample after a 20-min incubation and the platelets were
analyzed immediately by flow cytometry using the FL2 channel as
described below. For these experiments, the amount of P-selectin
surface expression above baseline observed in permeabilized platelets
exposed to the indicated agonist was set to 100% and all other
P-selectin expression is expressed as a percent of this control.
In experiments using BAPTA-AM-treated, SL-O-permeabilized platelets, 20 µl of gel-filtered platelets (1-2 × 108/ml) were
incubated for 30 min with the indicated concentration of BAPTA-AM.
Platelets were then permeabilized by exposure to the indicated
concentration of reduced SL-O. Samples were adjusted to pH 6.9 immediately following permeabilization. The timing of the addition of
agonists, inhibitors, and 10 µM Ca2+ is
indicated in the figure legends. The amount of CaCl2
required to give a free Ca2+ concentration of 10 µM in the presence of 2 mM EGTA at pH 6.9 was
calculated for each condition using a computer program based on the
algorithms described by Fabiato and Fabiato (32). Following an
additional 15-min incubation after the addition of Ca2+, 10 µl of reaction mixture was transferred into 5 µl of
phycoerythrin-conjugated AC1.2 anti-P-selectin antibody and processed
as described above. For these experiments, the amount of P-selectin
surface expression in the absence of agonist or inhibitor was set at
100% and all other P-selectin expression is expressed as a percent of
this control.
Immunoblot Analysis--
Gel-filtered platelets (1-2 × 107/ml) were pelleted and solubilized in sample buffer
(62.5 mM Tris-HCl, 0.2% SDS, 0.5% Isolation of Platelet Cytoskeletal Fractions--
For samples
using SFLLRN-stimulated platelets, gel-filtered platelets (2-4 ml)
were incubated with 25 µM BAPTA-AM for 30 min and
subsequently incubated with SFLLRN or buffer for 10 min. Samples were
then exposed to 4 units/ml SL-O for 15 min. The permeabilized platelets
were pelleted and solubilized in Triton lysis buffer (2% Triton X-100,
100 mM Tris, 10 mM EDTA, 6 mM EGTA,
2 mM dithiothreitol, 0.5 µg/ml leupeptin, 1 µg/ml
pepstatin, and 2 µg/ml aprotinin). Cytoskeletons were isolated by
centrifugation at 10,000 × g at 4 °C for 20 min. As
a positive control for the precipitation of the cytoskeleton,
gel-filtered platelets were incubated with 1 unit/ml thrombin at
37 °C and stirred. Platelets were then pelleted and solubilized in
Triton lysis buffer as described above. Fractions were analyzed for
type II PIPK by immunoblotting.
Isolation of Triton X-100-insoluble Fractions--
For samples
using SFLLRN-stimulated platelets, gel-filtered platelets (2-4 ml)
were incubated with 25 µM BAPTA-AM for 30 min and
subsequently exposed to 100 µM SFLLRN or buffer for 10 min. Noncytoskeletal, Triton X-100-insoluble fractions were isolated by
centrifugation of the 10,000 × g supernatant (derived
from the cytoskeletal preparation) at 100,000 × g at
4 °C for 3 h. Under conditions in which the 10,000 × g pellet demonstrated no type II PIPK, noncytoskeletal,
Triton X-100-insoluble fractions were isolated by centrifugation of
Triton X-100 lysate at 100,000 × g at 4 °C for
3 h. Pellets were heated in SDS-PAGE sample buffer and analyzed by
immunoblotting for the presence of type II PIPK. For samples using
PMA-stimulated platelets, gel-filtered platelets were stimulated with
0.2 µM PMA and solubilized in Triton lysis buffer. The
Triton X-100-insoluble fraction was then isolated and analyzed as
described above.
Analysis of FITC-QRLFQVRKGRR Binding to Platelets--
For
analysis of the association of the FITC-labeled gelsolin
PtdIns(4,5)P2-binding domain and FITC-labeled control
peptides to BAPTA-AM-treated, SL-O-permeabilized platelets, 20 µl of
gel-filtered platelets (1-2 × 108/ml) were incubated
for 30 min with the indicated concentration of BAPTA-AM. The timing of
addition of inhibitors varied according to the inhibitor that was being
evaluated and is indicated in the figure legend. Platelets were then
incubated for 10 min with SFLLRN, PMA, or buffer and permeabilized by
exposure to the indicated concentration of SL-O in the presence of
FITC-labeled peptide (5 µM). Following an additional
20-min incubation, the sample was diluted in 500 µl of
phosphate-buffered saline and analyzed immediately by flow cytometry
using the FL1 channel as described below. For these experiments,
binding of the FITC-labeled gelsolin PtdIns(4,5)P2-binding
domain to nonpermeabilized platelets was determined to be background.
The amount of FITC-labeled peptide fluorescence greater than background
observed in permeabilized, BAPTA-AM-treated platelets exposed to the
indicated agonist was set to 100% and all other secretions are
expressed as a percent of this control.
Flow Cytometry--
Flow cytometry was performed on gel-filtered
platelet samples using a BD Biosciences FACSCalibur flow cytometer.
Fluorescent channels were set at logarithmic gain. Five to ten-thousand
particles were acquired for each sample. A 585/42 band pass filter was
used for FL1 fluorescence and a 530/30 band pass filter was used for FL-2 fluorescence. FITC was measured in the FL-1 channel. Phycoerythrin was measured in the FL-2 channel. Data were analyzed using CellQuest software on a MacIntosh PowerPC.
Type II PIPK Participates in SFLLRN-stimulated PKC Participates in Type II PIPK-mediated Type II PIPK Mediates an Agonist-dependent Step Prior
to Ca2+-triggered Secretion--
We have previously used a
MgATP-dependent, Ca2+-triggered platelet
secretory model to demonstrate that type II PIPK acts at an
ATP-dependent step prior to Ca2+-triggered
secretion (15). Based on these studies, we hypothesized that type II
PIPK acts prior to Ca2+-mediated triggering during
agonist-induced
Like SFLLRN, PMA supported Ca2+-triggered Agonist-induced Translocation of Type II PIPK--
Translocation
of PIPKs has been shown to be involved in the regulation of their
activity (21-26, 41-43). We next sought to determine whether agonist
stimulation resulted in translocation of type II PIPK in our platelet
secretory model. In these experiments, BAPTA-AM-treated platelets were
incubated in the presence or absence of SFLLRN. Samples were then
incubated in the presence or absence of SL-O. Platelets from these
samples were pelleted and platelet-associated type II PIPK assayed by
immunoblot analysis. Under these conditions, the majority of type II
PIPK diffused of SL-O-permeabilized, BAPTA-AM-treated platelets
that were not exposed to SFLLRN (Fig.
4A). In contrast, >50% of
platelet type II PIPK remained associated with permeabilized platelets
incubated with SFLLRN prior to permeabilization. These results suggest
that type II PIPK translocates from the cytosol upon stimulation with
platelet agonists. We next sought to determine whether translocation of
type II PIPK was distal to activation of PKC following stimulation with
SFLLRN. Platelets were incubated in the presence or absence of the PKC
inhibitor peptide and subsequently exposed to SFLLRN, permeabilized,
and pelleted. SFLLRN-induced translocation of type II PIPK was
inhibited by incubation of platelets with the PKC inhibitor peptide
(Fig. 4B). Type II PIPK also remained platelet-associated in
SL-O-permeabilized platelets incubated in the presence of PMA and both
SFLLRN- and PMA-induced translocation was inhibited by
bisindolylmaleimide I (data not shown). These results demonstrate that
agonist-induced translocation of type II PIPK is mediated by PKC.
Type II PIPK has previously been shown to translocate to the platelet
actin cytoskeleton in an aggregation-dependent manner (25).
However, under the conditions of our assay, platelet aggregation did
not occur. To determine whether type II PIPK translocated to the actin
cytoskeleton of the BAPTA-AM-treated platelets, we solubilized the
BAPTA-AM-treated, SFLLRN-exposed platelets in Triton X-100 and isolated
the insoluble actin cytoskeleton by centrifugation at 10,000 × g. No type II PIPK was associated with the platelet actin
cytoskeleton of the BAPTA-AM-treated, SFLLRN-exposed platelets (Fig.
5A). Consistent with the
results of Hinchliffe et al. (25), however, type II PIPK did
translocate to the platelet actin cytoskeleton when untreated platelets
were exposed to thrombin and stirred to induce aggregation (Fig.
5A). These results demonstrate that type II PIPK does not
translocate to the actin cytoskeleton in BAPTA-AM-treated,
SFLLRN-exposed platelets. However, when BAPTA-AM-treated platelets were
incubated in the presence or absence of SFLLRN, pelleted, and
solubilized in 2% Triton X-100, type II PIPK was detected in Triton
X-100-insoluble material isolated by centrifugation at 100,000 × g. Type II PIPK was detected only in pellets from samples
exposed to SFLLRN (Fig. 5B). In contrast, no type II PIPK was detected in lyophilized supernatants of SFLLRN-exposed samples following centrifugation at 100,000 × g (data not
shown). Type II PIPK was also pelleted at 100,000 × g
from the Triton X-100-insoluble material from platelets treated with
PMA (Fig. 5C). These data demonstrate that type II PIPK
translocates to a Triton X-100-insoluble compartment in an
activation-dependent manner.
Agonist-induced, Type II PIPK-dependent Recruitment of
a PtdIns(4,5)P2-binding Domain--
Studies in other
secretory systems have suggested that translocation of PIPKs results in
the recruitment of PtdIns(4,5)P2-binding proteins to
membranes (24, 42). We, therefore, sought to assess the hypothesis that
type II PIPK mediates the recruitment of
PtdIns(4,5)P2-binding proteins in platelets in an
agonist-dependent manner. For these experiments,
BAPTA-AM-treated platelets were incubated in the presence or absence of
SFLLRN and subsequently permeabilized in the presence of a FITC-labeled
PtdIns(4,5)P2-binding peptide, FITC-QRLFQVKGRR, derived
from segment 2 of gelsolin (amino acids 160-169) (30, 44, 45). The
rhodamine B-conjugated form of this peptide, termed PBP10, binds
PtdIns(4,5)P2 preferentially to PtdIns(4)P or PtdIns and
fails to interact significantly with phosphatidylserine or
phosphatidylcholine (30). In these experiments, binding of the
FITC-labeled PtdIns(4,5)P2-binding domain of gelsolin to
platelets was analyzed by flow cytometry. Incubation of
BAPTA-AM-treated platelets with SFLLRN increased the association of
FITC-QRLFQVKGRR with platelets following permeabilization with SL-O
(Fig. 6A). Neomycin binds
PtdIns(4,5)P2 strongly (46) and has been used to study the
role of PtdIns(4,5)P2 in secretory processes (47). In these
experiments, neomycin abolished SFLLRN-induced FITC-QRLFQVKGRR binding
to BAPTA-AM-treated, permeabilized platelets, consistent with the
supposition that the FITC-labeled PtdIns(4,5)P2-binding domain of gelsolin associates with platelet PtdIns(4,5)P2
(Fig. 6A). Exposure to bisindolylmaleimide I also inhibited
SFLLRN-induced binding of the PtdIns(4,5)P2-binding peptide
to BAPTA-AM-treated platelets (Fig. 6A). In contrast,
neither neomycin nor bisindolylmaleimide I affected the levels of
FITC-QRLFQVKGRR associated with platelets that were not exposed to
SFLLRN. To assess the specificity of SFLLRN-induced binding of the
FITC-labeled gelsolin PtdIns(4,5)P2-binding domain, we
tested the ability of SFLLRN to enhance platelet association of a
control peptide, FITC-QALFQVAKGAA, in which the basic amino acids of
the PtdIns(4,5)P2-binding domain are replaced with alanine. The relative fluorescence of unstimulated permeabilized platelets incubated in the presence of 5 µM of the mutant
FITC-binding protein was 49 ± 14% of that of permeabilized
platelets incubated in the presence of 5 µM native
FITC-binding protein. Binding of FITC-labeled alanine mutant to
permeabilized BAPTA-AM-treated, SFLLRN-exposed platelets was 114 ± 17% of that of binding to unexposed permeabilized BAPTA-AM-treated
platelets. Thus, the mutant form of the peptide failed to interact with
permeabilized, BAPTA-AM-treated platelets in an
agonist-dependent manner. Neither neomycin nor
bisindolylmaleimide I had a significant effect on the binding of the
FITC-labeled alanine mutant peptide to BAPTA-AM-treated platelets in
either the presence of absence of SFLLRN (data not shown). Like SFLLRN, PMA also induced binding of FITC-QRLFQVKGRR to BAPTA-AM-treated, permeabilized platelets (Fig. 6B). Neomycin and
bisindolylmaleimide I inhibited PMA-induced binding, but failed to
effect binding of FITC-QRLFQVKGRR to unstimulated platelets. Similarly,
binding of FITC-labeled mutant peptide to BAPTA-AM-treated, PMA-exposed permeabilized platelets was 110 ± 18% of that of binding to
unexposed, BAPTA-AM-treated, permeabilized platelets. These data
demonstrate that exposure of BAPTA-AM-treated platelets to agonists
induces the generation of intracellular sites capable of binding the
FITC-labeled gelsolin PtdIns(4,5)P2-binding domain and that
PKC participates in the events that lead to increased binding of this
peptide.
We next determined whether type II PIPK mediates agonist-induced
increases in binding of the gelsolin PtdIns(4,5)P2-binding domain. In these experiments, anti-type II PIPK antibodies were infused
into BAPTA-AM-treated, SL-O-permeabilized platelets either prior to or
following exposure to SFLLRN. Association of FITC-QRLFQVKGRR with
platelets was subsequently analyzed by flow cytometry. Addition of
anti-type II PIPK antibodies to permeabilized platelets prior to
addition of SFLLRN inhibited the association of FITC-QRLFQVKGRR with
platelets (Fig. 7B). In
contrast, addition of anti-type II PIPK antibodies to permeabilized
platelets following incubation with SFLLRN failed to influence the
association of FITC-QRLFQVKGRR with platelets. Similarly, incubation
with nonimmune antibody had no effect on platelet association of
FITC-QRLFQVKGRR. Anti-type II PIPK antibodies also inhibited the
PMA-induced platelet association of FITC-QRLFQVKGRR when added prior
to, but not after, exposure to PMA (Fig. 7B). The anti-type
II PIPK antibodies had no effect on binding of the FITC-labeled
gelsolin PtdIns(4,5)P2-binding domain to nonpermeabilized
platelets or platelets that had not been exposed to agonist. In
addition, these antibodies did not affect binding of the alanine mutant
form of the peptide to agonist-exposed, permeabilized platelets. These
data demonstrate that agonist-induced recruitment of the gelsolin
PtdIns(4,5)P2-binding domain is mediated through type II
PIPK.
PtdIns(4,5)P2 is required for regulated granule
secretion in numerous secretory systems. In most cells,
PtdIns(4,5)P2 required for granule secretion is synthesized
via a type I PIPK-dependent pathway (18, 48). A role for
type II PIPK, however, has been proposed for platelet A role for PKC in Ca2+-triggered granule secretion has been
demonstrated in several secretory systems. Phorbol esters enhance Ca2+-triggered granule secretion from adrenal chromaffin
cells (52) and PC12 cells (53) as well as synaptosomal vesicle release induced by hypertonic sucrose (54). Purified PKC augments
Ca2+-triggered secretion in PC12 cells (55) and in
platelets (13). In our permeabilized platelet model, Regulated translocation of PIPKs to membrane compartments has
previously been observed in other cell models. Recruitment of type I
PIPK Our studies using the agonist-responsive -granule secretion. SFLLRN-stimulated
-granule secretion
from SL-O-permeabilized platelets was inhibited by either antibodies
directed at type II PtdIns 5-phosphate 4-kinase or by a kinase-impaired
point mutant of type II
PtdIns 5-phosphate 4-kinase. In contrast,
recombinant type II
PtdIns 5-phosphate 4-kinase augmented
SFLLRN-stimulated
-granule secretion from SL-O-permeabilized
platelets. SFLLRN-stimulated
-granule secretion was inhibited by a
protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated
-granule secretion was
inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the
kinase-impaired point mutant of type II
PtdIns 5-phosphate 4-kinase
and augmented by recombinant type II
PtdIns 5-phosphate 4-kinase.
Immunoblot analysis demonstrated that type II PtdIns 5-phosphate
4-kinase remained associated with SL-O-permeabilized platelets when
incubated in the presence, but not the absence, of SFLLRN. This
SFLLRN-induced translocation of type II PtdIns 5-phosphate 4-kinase was
blocked by either the protein kinase C-specific inhibitor peptide or
bisindolylmaleimide I. In addition to stimulating
-granule
secretion, both SFLLRN and PMA enhanced the association of a
fluorescein isothiocyanate-labeled peptide derived from the
PtdIns (4,5)P2-binding domain of gelsolin to
permeabilized platelets. Agonist-induced recruitment of the PtdIns
(4,5)P2-binding domain was inhibited by neomycin,
bisindolylmaleimide I, and anti-type II PtdIns 5-phosphate 4-kinase
antibody. These results suggest a mechanism whereby protein kinase
C-mediated translocation of type II PtdIns 5-phosphate 4-kinase leads
to the recruitment of PtdIns (4,5)P2-binding proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Granules are the dominant platelet granule and contain many
components that have been implicated in thrombosis. The molecular mechanisms that direct membrane fusion events required for regulated secretion of
-granules from platelets have been studied intensely in
recent years (for reviews see Refs. 1 and 2). The
SNARE1 protein isoforms
SNAP-23, syntaxin 2 and 4, and VAMP-3 are found in platelets and have
been demonstrated to mediate
-granule secretion (3-7). Yet, while
purified SNARE proteins are capable of fusing lipid membranes in
vitro (8, 9), regulated secretion necessitates that the SNARE
protein apparatus responds to activation-dependent signals.
Several molecules, including Munc-18c,
N-ethylmaleimide-sensitive fusion protein, Rab4,
PKC
, calpain, myristoylated alanine-rich C kinase substrate,
and PtdIns(4,5)P2, have recently been shown to participate
in platelet granule secretion and are proposed to influence distal
events in the secretory pathway (5, 10-16). The organization,
localization, and sequence of interactions of these components,
however, remain unknown. In particular, the mechanism by which
ligand-receptor interactions at the platelet surface direct membrane
fusion has not been detailed in platelets at the molecular level.
-granule
secretion from platelets. In these studies, type II PIPK was shown to
act at a priming step prior to Ca2+-triggered secretion
(15). Whether type II PIPK participates in agonist-induced
-granule
secretion and how type II PIPK activity is regulated in platelets,
however, has not previously been assessed. We therefore sought to study
the role of type II PIPK in agonist-mediated
-granule secretion.
Using an agonist-stimulated SL-O-permeabilized platelet model, we
demonstrate that type II PIPK participates in agonist-induced
-granule secretion. We also demonstrate that stimulation of
platelets by SFLLRN results in PKC-dependent translocation of type II PIPK. Type II PIPK, in turn, mediates recruitment of a
FITC-labeled PtdIns(4,5)P2-binding protein derived from gelsolin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranosyl benzimidazole were purchased from Calbiochem (San Diego, CA). Phycoerythrin-conjugated AC1.2 anti-P-selectin antibody was purchased from BD Biosciences (San Jose, CA). FITC-QRLFQVKGRR (30),
FITC-QALFQVAKGAA, irrelevant control peptide (VFLSREEANSVLREE), and
SFLLRN were synthesized using solid phase Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied
Biosystems model 430A peptide synthesizer. Goat polyclonal
affinity-purified antipeptide antibodies to type II PIPK in
phosphate-buffered saline were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). The antibody was raised against a peptide consisting
of the 19 amino-terminal amino acids of type II PIPK. This antibody
recognized a single band of 53 kDa in platelet lysates and recognized
recombinant type II PIPK glutathione S-transferase fusion
proteins in immunoblot analysis (15). Recombinant type II
PIPK was
expressed as a glutathione S-transferase fusion protein in
bacteria using a cDNA (31) kindly provided by Dr. Moses V. Chao
(New York University School of Medicine, New York, NY). Recombinant type II
PIPK D216A mutant was expressed as a glutathione
S-transferase fusion protein in bacteria using a cDNA
kindly provided by Dr. Lewis C. Cantley (Beth Israel Deaconess Medical
Center, Boston, MA). Recombinant proteins were subsequently purified to
homogeneity in Tris buffer (50 mM Tris-Cl, 5 mM
reduced glutathione, pH 7.5) using standard techniques. The type II
PIPK D216A mutant demonstrated 5% of the enzymatic activity of the
native type II
PIPK.2
Purification of recombinant proteins was confirmed by SDS-PAGE. All
solutions were prepared using water purified by reverse-phase osmosis
on a Millipore Milli-Q purification water system.
-mercaptoethanol, 14% glycerol, 0.01% bromphenol blue) at 95 °C for 5 min. Platelet proteins were then separated by SDS-PAGE on 14% gels. Immunoblotting was performed using anti-type II PIPK antibodies directed against the
amino-terminal of human type II PIPK (Santa Cruz, CA) and visualized
using enhanced chemiluminescence.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Granule
Secretion--
We have previously demonstrated that type II PIPK
participates in
-granule secretion in a MgATP-dependent,
Ca2+-triggered secretory system (15). In the present study,
we sought to determine the role of type II PIPK in agonist-mediated
granule secretion. In these experiments, platelets were permeabilized in the presence of either antibodies or recombinant proteins (33, 34)
and subsequently stimulated with the protease-activated receptor-1
activating peptide, SFLLRN. P-selectin surface expression was monitored
as an indicator of
-granule secretion (35). P-selectin surface
expression from platelets permeabilized prior to exposure to SFLLRN was
69 ± 12% that of P-selectin expression from intact platelets exposed
to SFLLRN. To assess the role of type II PIPK in SFLLRN-induced
-granule secretion, we used an antibody directed at the amino
terminus of type II PIPK that inhibits PIP2 synthesis in
permeabilized platelets (15). This antibody inhibited SFLLRN-induced P-selectin surface expression from SL-O-permeabilized platelets (Fig.
1A). Nonimmune antibody had no
effect in this assay. A kinase-impaired point mutant of type II
PIPK
also inhibited SFLLRN-induced
-granule secretion from permeabilized
platelets (Fig. 1B). In contrast, recombinant type II
PIPK augmented SFLLRN-induced
-granule secretion. Neither
recombinant type II
PIPK kinase-impaired point mutant nor
recombinant type II
PIPK affected baseline secretion of
-granules from SL-O-permeabilized platelets (data not shown). Furthermore, neither antibodies nor recombinant proteins affected SFLLRN-induced granule secretion from intact platelets. These data demonstrate that
type II PIPK mediates SFLLRN-induced
-granule secretion from
SL-O-permeabilized platelets.
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Fig. 1.
Type II PIPK mediates SFLLRN-induced
P-selectin surface expression from SL-O-permeabilized platelets.
A, gel-filtered platelets were permeabilized with 6 units/ml
SL-O in the presence of buffer (Buffer), 40 µg/ml
nonimmune IgG (Non-immune IgG), or 40 µg/ml anti-type II
PIPK IgG (Anti-type II PIPK IgG). Platelets were then
incubated in the presence or absence of SFLLRN. Following a 20-min
incubation, platelets were assayed for P-selectin surface expression.
For these experiments, the amount of P-selectin surface expression
above baseline observed in permeabilized platelets exposed to SFLLRN
was set to 100% and all other P-selectin expression is expressed as a
percent of this control. Error bars represent the S.D. of
five independent experiments. p value of nonimmune IgG
versus anti-type II PIPK IgG is 0.017. B,
gel-filtered platelets were permeabilized with 4 units/ml SL-O in the
presence of Tris buffer (Tris Buffer), 15 µg/ml
recombinant type II PIPK (rType II
PIPK), or 15 µg/ml kinase-impaired type II
PIPK point mutant
(Kinase-impaired rType II
PIPK). Platelets were then
exposed to either buffer or SFLLRN following addition of SL-O.
Following a 20-min incubation, platelets were assayed for P-selectin
surface expression. Error bars represent the S.D. of six to
nine independent experiments. p values for both Tris buffer
versus rType II
PIPK and Tris buffer versus
kinase-impaired rType II
PIPK are less than 0.0001.
-Granule Secretion
from Permeabilized Platelets--
We next sought to identify
downstream signaling elements required for agonist-induced, type II
PIPK-mediated
-granule secretion. In platelets, engagement of SFLLRN
with protease-activated receptor-1 results in the downstream activation
of PKC (36). In addition, PKC is thought to be involved in cellular
processes that render granules competent to secrete (37). We therefore
determined the effect of a PKC inhibitor peptide (38) and
bisindolylmaleimide I (39) on SFLLRN-induced
-granule secretion from
permeabilized platelets. Under these conditions, both the inhibitor
peptide and bisindolylmaleimide I inhibited SFLLRN-stimulated
-granule secretion (Fig.
2A). In contrast, an
irrelevant peptide had no effect. These results raise the possibility
that PKC is required for agonist-stimulated, type II PIPK-mediated
-granule secretion. Given the possibility that PKC participates in
agonist-responsive
-granule secretion, we next sought to determine
whether or not the PKC agonist, PMA, is capable of supporting
-granule secretion from permeabilized platelets. In these
experiments, platelets were permeabilized with SL-O and subsequently
exposed to PMA. Like SFLLRN, PMA was able to stimulate
-granule
secretion from permeabilized platelets. PMA is a potent PKC agonist.
However, it also targets other proteins that participate in granule
secretion such as Munc-13 family proteins and Rac-GTPase-activating
proteins (40). We therefore sought to confirm that the effects of PMA observed in the agonist-responsive platelet secretion model are secondary to activation of PKC. PMA-induced
-granule secretion was
inhibited by both the PKC inhibitor peptide and bisindolylmaleimide I
(Fig. 2B). We next determined whether PKC-responsive
-granule secretion from permeabilized platelets was dependent on
type II PIPK. Anti-type II PIPK antibodies, but not nonimmune
antibodies, infused into SL-O-permeabilized platelets inhibited
PMA-induced P-selectin surface expression (Fig. 2C).
Similarly, permeabilization of platelets in the presence of the
kinase-impaired mutant of type II
PIPK also inhibited PMA-induced
P-selectin surface expression (Fig. 2D). In contrast,
permeabilization of platelets in the presence of native recombinant
type II
PIPK augmented PMA-induced P-selectin surface expression.
Neither antibodies nor recombinant proteins affected either baseline
P-selectin expression levels or PMA-induced P-selectin expression from
intact platelets (data not shown). These data demonstrate that type II
PIPK participates in PMA-stimulated
-granule secretion from
SL-O-permeabilized platelets.
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Fig. 2.
PKC-mediates P-selectin surface expression
from SL-O-permeabilized platelets in a type II
PIPK-dependent manner. A, gel-filtered
platelets were permeabilized with 6 units/ml SL-O in the presence of
buffer (Buffer), 5 µM irrelevant peptide
(Control peptide), 5 µM PKC inhibitor peptide
(PKC inhibitor peptide), or 1 µM
bisindolylmaleimide I (BIM I). Platelets were then exposed
to SFLLRN following addition of SL-O. Following a 20-min incubation,
platelets were assayed for P-selectin surface expression. Error
bars represent the S.D. of six to nine independent experiments.
p values for control peptide versus PKC inhibitor
peptide and for buffer versus BIM I are both less than
0.0001. B, gel-filtered platelets were incubated in the
presence of 6 units/ml SL-O in the presence of buffer
(Buffer), irrelevant peptide (Control peptide) 5 µM PKC inhibitor peptide (PKC inhibitor
peptide), or 1 µM bisindolylmaleimide I (BIM
I). Platelets were then exposed to 0.2 µM PMA
following addition of SL-O and assayed for P-selectin surface
expression following a 20-min incubation. Error bars
represent the S.D. of five to six independent experiments. p
value for control peptide versus PKC inhibitor peptide is
0.016 and p value for buffer versus BIM I is less
than 0.0001. C, gel-filtered platelets were incubated in the
presence of 6 units/ml SL-O in the presence of buffer
(Buffer), 40 µg/ml nonimmune IgG (Non-immune
IgG), or 40 µg/ml anti-type II PIPK IgG (Anti-type II PIPK
IgG). Platelets were then exposed to 0.2 µM PMA
following addition of SL-O and assayed for P-selectin surface
expression following a 20-min incubation. Error bars
represent the S.D. of five independent experiments. p value
of nonimmune IgG versus anti-type II PIPK IgG is less than
0.0001. D, gel-filtered platelets were permeabilized with 3 units/ml SL-O in the presence of Tris buffer (Tris buffer),
15 µg/ml recombinant type II PIPK (rType II
PIPK),
or 15 µg/ml kinase-impaired type II
PIPK point mutant
(Kinase-impaired rType II
PIPK). Platelets were then
exposed to either buffer or 0.2 µM PMA following addition
of SL-O. Following a 20-min incubation, platelets were assayed for
P-selectin surface expression. Error bars represent the S.D.
of six to nine independent experiments. p value for Tris
buffer versus rType II
PIPK is 0.001 and p
value for Tris buffer versus kinase-impaired rType II
PIPK is less than 0.0001.
-granule secretion. To test this hypothesis, intact
platelets were incubated with the cell-permeant Ca2+
chelator BAPTA-AM to suppress rapid, Ca2+-mediated
triggering of granule secretion from intact platelets. Platelets were
subsequently exposed to SFLLRN. Following this exposure, platelets were
permeabilized in the presence of Ca2+ to elicit
-granule
secretion. This strategy allows for the temporal separation of
agonist-dependent events required for granule secretion from Ca2+-triggered secretory events. In these experiments,
intact BAPTA-AM-treated platelets failed to express significant amounts
of P-selectin on their surface in response to SFLLRN (Fig.
3A). In contrast, BAPTA-treated platelets incubated in the presence or absence of SFLLRN,
permeabilized, and then exposed to 10 µM free
Ca2+ following permeabilization expressed more P-selectin
on their surface following exposure to SFLLRN (Fig. 3A).
Analysis of flow cytometry demonstrated that nearly all of the
BAPTA-treated platelets exposed to SFLLRN showed some increase in
P-selectin expression (data not shown). This observation negates the
possibility that the increase in P-selectin expression is secondary to
a minority of fully activated platelets. The amount of P-selectin
surface expression observed under these conditions was only 15-30% of that observed in intact platelets stimulated with SFLLRN. To assess whether type II PIPK acts prior to or during Ca2+-mediated
triggering of
-granule secretion, antibodies directed at the amino
terminus of type II PIPK were infused into BAPTA-AM-treated, SL-O-permeabilized platelets either prior to or following exposure to
SFLLRN. Platelets were subsequently incubated with Ca2+ to
elicit
-granule secretion. Addition of anti-type II PIPK antibodies
to permeabilized platelets prior to addition of SFLLRN resulted in
inhibition of SFLLRN-mediated augmentation of
Ca2+-triggered P-selectin surface expression (Fig.
3B). Antibodies inhibited SFLLRN-mediated augmentation of
P-selectin surface expression nearly to levels observed in samples not
exposed to SFLLRN. In contrast, anti-type II PIPK antibodies failed to
inhibit SFLLRN-responsive P-selectin surface expression in samples that
were exposed to SFLLRN prior to permeabilization in the presence of
antibodies (Fig. 3B). Thus, anti-type II PIPK antibodies
inhibited an SFLLRN-dependent step in
-granule secretion
prior to Ca2+-induced triggering of secretion. Nonimmune
antibodies had no effect on SFLLRN-responsive,
Ca2+-triggered P-selectin surface expression. These data
demonstrate that type II PIPK is required for an SFLLRN-stimulated
priming event that is necessary for
-granule membrane fusion in
response to Ca2+.
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Fig. 3.
Type II PIPK mediates an
agonist-dependent step prior to Ca2+-triggered
P-selectin surface expression in BAPTA-AM-treated, SL-O-permeabilized
platelets. A, gel-filtered platelets were incubated
with 25 µM BAPTA-AM for 30 min and subsequently exposed
to either buffer or SFLLRN for 10 min. Platelets were incubated in the
presence or absence of 1 unit/ml SL-O. Permeabilized platelets were
then incubated in either buffer alone or buffer containing 10 µM Ca2+. Following a 15-min incubation,
platelets were assayed for P-selectin surface expression. Error
bars represent the S.D. of four independent experiments.
B, BAPTA-AM-treated platelets (black bars) were
permeabilized with 6 units/ml SL-O in the presence of buffer
(Buffer), 40 µg/ml nonimmune IgG (Non-immune
IgG), or 40 µg/ml anti-type II PIPK IgG (anti-type II PIPK
IgG) and subsequently exposed to 100 µM SFLLRN and
10 µM Ca2+. A second group of
BAPTA-AM-treated platelets (white bars) was exposed to 100 µM SFLLRN 10 min prior to permeabilization in the
presence of buffer (Buffer), 40 µg/ml nonimmune IgG
(Non-immune IgG), or 40 µg/ml anti-type II PIPK IgG
(anti-type II PIPK IgG) and subsequently exposed to 10 µM Ca2+. Following a 15-min incubation,
platelets were assayed for P-selectin surface expression by flow
cytometry. Data are expressed as percent of P-selectin expression
compared with BAPTA-AM-treated samples exposed to Ca2+
immediately after permeabilization. Error bars represent the
S.D. of six independent experiments. p value of nonimmune
IgG versus anti-type II PIPK IgG is 0.004 in samples in
which IgG was added prior to SFLLRN exposure. C,
gel-filtered platelets were incubated with 50 µM BAPTA-AM
for 30 min and subsequently exposed to either buffer or 0.2 µM PMA for 10 min. Platelets were incubated in the
presence or absence of 1 unit/ml SL-O. Permeabilized platelets were
then incubated in either buffer alone or buffer containing 10 µM Ca2+. Following a 15-min incubation,
platelets were assayed for P-selectin surface expression. Error
bars represent the S.D. of four independent experiments.
D, gel-filtered platelets were incubated with 50 µM BAPTA-AM for 30 min. One group of platelets
(black bars) was permeabilized with 6 units/ml SL-O in the
presence of buffer (Buffer), 40 µg/ml nonimmune IgG
(Nonimmune IgG), or 40 µg/ml anti-type II PIPK IgG
(anti-type II PIPK IgG) and subsequently exposed to 0.2 µM PMA and 10 µM Ca2+. A second
group of platelets (white bars) was exposed to 0.2 µM PMA 10 min prior to permeabilization in the presence
of buffer (Buffer), 40 µg/ml nonimmune IgG
(Nonimmune IgG), or 40 µg/ml anti-type II PIPK IgG
(anti-type II PIPK IgG) and subsequently exposed to 10 µM Ca2+. Following a 15-min incubation,
platelets were assayed for P-selectin surface expression by flow
cytometry. Error bars represent the S.D. of seven
independent experiments. p value of nonimmune IgG
versus anti-type II PIPK IgG is less than 0.0001 in samples
in which IgG was added prior to PMA exposure.
-granule
release in BAPTA-AM-treated, SL-O-permeabilized platelets (Fig.
3C). In these studies, BAPTA-AM-treated platelets were
exposed to 0.2 µM PMA or buffer and then permeabilized
with SL-O in the presence of Ca2+. The degree of P-selectin
surface expression in BAPTA-AM-treated platelets exposed to PMA and
permeabilized in the presence of Ca2+ was 40-60% of that
in untreated intact platelets exposed to PMA. Thus, PMA was a stronger
agonist than SFLLRN in rendering BAPTA-AM-treated platelets capable of
secreting
-granules in response to Ca2+. Addition of
anti-type II PIPK antibodies to permeabilized platelets prior to
addition of PMA resulted in inhibition of PMA augmentation of
Ca2+-triggered P-selectin surface expression (Fig.
3D). Anti-type II PIPK antibodies failed to inhibit
PMA-responsive P-selectin surface expression in samples that were
exposed to PMA prior to permeabilization in the presence of antibodies
(Fig. 3D). Thus, type II PIPK is required for a
PMA-dependent priming event that is necessary for
-granule membrane fusion in response to Ca2+.
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Fig. 4.
PKC mediates SFLLRN-induced translocation of
type II PIPK in BAPTA-AM-treated, SL-O-permeabilized platelets.
A, gel-filtered platelets were incubated in the presence of
25 µM BAPTA-AM for 30 min. Platelets were subsequently
incubated in the presence or absence of 100 µM SFLLRN for
10 min and the indicated samples were permeabilized with 4 units/ml
SL-O for 15 min. Platelets were then pelleted. Proteins from platelet
pellets were assayed for type II PIPK by immunoblotting. B,
gel-filtered platelets were incubated in BAPTA-AM for 30 min. Platelets
were then permeabilized in the presence of 4 units/ml SL-O in the
presence or absence of the PKC inhibitor peptide. Platelets were
subsequently incubated in the presence or absence of 100 µM SFLLRN. Following a 15-min incubation, platelets were
pelleted. Proteins from platelet pellets were assayed for type II PIPK
by immunoblotting.
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Fig. 5.
Aggregation-independent translocation of type
II PIPK to a Triton X-100-insoluble platelet fraction. A,
gel-filtered platelets were incubated with BAPTA-AM for 30 min.
Platelets were then incubated in the presence (SFLLRN) or
absence (Buffer) of 100 µM SFLLRN for 10 min.
To assess the effect of platelet aggregation on the translocation of
type II PIPK, a third group of gel-filtered platelets was incubated
with 1 unit/ml thrombin at 37 °C and stirred to facilitate platelet
aggregation. Platelets were subsequently permeabilized with SL-O and
pelleted. An aliquot of platelet pellets was assayed for type II PIPK
by immunoblotting (Pellets). Platelet cytoskeletons were
prepared from the remaining platelet pellets by extraction in 2%
Triton X-100 followed by centrifugation at 10,000 × g.
Proteins associated with the cytoskeleton were then assayed for type II
PIPK by immunoblotting (Cytoskeleton). B,
BAPTA-AM-treated platelets were exposed to either buffer
(Buffer) or SFLLRN (SFLLRN) for 10 min. Triton
X-100-insoluble fractions were recovered by Triton X-100 extraction
followed by centrifugation at 100,000 × g as described
above. Proteins associated with the Triton X-100-insoluble fraction
were assayed for type II PIPK by immunoblotting. C,
gel-filtered platelets were exposed to either buffer
(Buffer) or 0.2 µM PMA (PMA) for 10 min. Triton X-100-insoluble fractions were recovered by Triton X-100
extraction followed by centrifugation at 100,000 × g.
Proteins associated with the Triton X-100-insoluble fraction were
assayed for type II PIPK by immunoblotting.
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Fig. 6.
Agonist-dependent association of
FITC-labeled gelsolin PtdIns(4,5)P2-binding domain to
BAPTA-AM-treated, SL-O-permeabilized platelets. A,
gel-filtered platelets (20 µl) were incubated with 25 µM BAPTA-AM for 30 min in the presence of buffer
(Buffer), 500 µM neomycin
(Neomycin), or 1.5 µM bisindolylmaleimide I
(BIM I). Samples were then incubated in the presence
(black bars) or absence (white bars) of 100 µM SFLLRN as indicated. Following a 10-min incubation,
samples were permeabilized with 4 units/ml of SL-O in the presence of 5 µM FITC-QRLFQVKGRR. Following an additional 20-min
incubation, 10-µl samples were transferred into 500 µl of
phosphate-buffered saline and analyzed by flow cytometry. For these
experiments, the amount of FITC-QRLFQVRKGRR fluorescence above
background observed in BAPTA-AM-treated platelets exposed to SFLLRN was
set to 100% and all other secretions are expressed as a percent of
this control. Error bars represent the S.D. of three
independent experiments. B, gel-filtered platelets were
incubated in the presence of 50 µM BAPTA-AM for 30 min.
Platelets were subsequently treated as described above except that 0.2 µM PMA was used as the agonist instead of SFLLRN.
Error bars represent the S.D. of six independent
experiments.
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Fig. 7.
Effect of anti-type II PIPK antibodies on
agonist-dependent association of FITC-labeled gelsolin
PtdIns(4,5)P2-binding domain to BAPTA-AM-treated,
SL-O-permeabilized platelets. A, gel-filtered platelets
were incubated in the presence of 25 µM BAPTA-AM for 30 min. One group of platelets (black bars) was permeabilized
with 6 units/ml SL-O in the presence of buffer (Buffer), 40 µg/ml nonimmune IgG (Non-immune IgG), or 40 µg/ml
anti-type II PIPK IgG (anti-type II PIPK IgG) and
subsequently exposed to 100 µM SFLLRN and 5 µM FITC-QRLFQVKGRR. A second group of BAPTA-AM-treated
platelets (white bars) were exposed to 100 µM
SFLLRN 10 min prior to permeabilization in the presence of buffer
(Buffer), 40 µg/ml nonimmune IgG (Non-immune
IgG), or 40 µg/ml anti-type II PIPK IgG (anti-type II PIPK
IgG). FITC-QRLFQVKGRR (5 µM) was subsequently added
to the permeabilized platelets. Following an additional 20-min
incubation, platelets were assayed for FITC-QRLFQVKGRR binding by flow
cytometry. Error bars represent the S.D. of three to six
independent experiments. p value of nonimmune IgG
versus anti-type II PIPK IgG is 0.05 in samples in which IgG
was added prior to SFLLRN exposure. B, gel-filtered
platelets were incubated in the presence of 50 µM
BAPTA-AM for 30 min. Platelets were subsequently treated as described
above except that 0.2 µM PMA was used as the agonist
instead of SFLLRN. Error bars represent the S.D. of three to
six independent experiments. p value of nonimmune IgG
versus anti-type II PIPK IgG is 0.001 in samples in which
IgG was added prior to PMA exposure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-granule
secretion (15). Several lines of evidence lend credence to the
possibility that type II PIPK contributes to
activation-dependent platelet
-granule secretion. Platelets contain unusually high levels of type II PIPK (49). Type II
PIPK immunoprecipitated from thrombin-stimulated platelets demonstrates
more than twice the PIPK activity of type II PIPK immunoprecipitated
from resting platelets (50). Thus, type II PIPK activity is
up-regulated in an activation-dependent manner. PtdIns
5-phosphate levels are also increased upon exposure of intact platelets
to thrombin (51). The most direct evidence suggesting a functional role
for type II PIPK in priming platelet granule secretion, however, is
derived from experiments demonstrating that anti-type II PIPK
antibodies inhibit an ATP-dependent, but not a
Ca2+-dependent, step required for platelet
-granule secretion in an ATP-primed, Ca2+-triggered
secretory model (15). In the present study, we demonstrate that type II
PIPK kinase participates in agonist-induced
-granule secretion. This
conclusion is based on the fact that anti-type II PIPK antibodies and a
kinase-impaired type II
PIPK point mutant inhibit both SFLLRN- and
PMA-induced platelet activation. Furthermore, recombinant type II
PIPK augments
-granule secretion in an agonist-dependent manner. Recombinant type II
PIPK itself does not stimulate
-granule secretion. Rather, it augments SFLLRN- and PMA-stimulated
-granule secretion. This observation suggests that type II PIPK is
responsive to activation-dependent signals. Thus, type II
PIPK serves as a downstream effector of PKC activity during
agonist-induced platelet secretion.
-granule
secretion is both responsive to PMA exposure and inhibited by a PKC
inhibitor peptide (38) and bisindolylmaleimide I (39). Taken together,
these observations provide strong support for a role for PKC in our
-granule secretion model. PKC appears to mediate its effect on platelet granule secretion via several mechanisms including
phosphorylation of myristoylated alanine-rich C kinase substrate (16)
and phosphorylation of Munc-18c (56). Our data suggest that PKC also
influences platelet granule secretion by regulating the translocation
of type II PIPK. It is possible that PKC stimulates translocation of
type II PIPK by direct phosphorylation. Platelet activation results in
complex changes in the phosphorylation state of type II PIPK (50).
Although protein kinase CK2 was found to be a major platelet type II
PIPK kinase (26), the potent protein CK2 inhibitor,
5,6-dichloro-1-
-D-ribofuranosyl benzimidazole, does not
inhibit either PMA-mediated
-granule secretion or type II PIPK
translocation in our system (data not shown). Thus, PMA does not
mediate its effects through protein CK2. Of course, it remains possible
that PKC mediates type II PIPK translocation via an alternative
indirect mechanism that does not involve direct phosphorylation by
PKC.
to locations of membrane ruffling (41) and actin-coated vacuoles (21) is mediated by the small G protein ADP-ribosylation factor 6. ADP-ribosylation factor 1 has been demonstrated to direct recruitment of PtdIns 4-kinase
and type I PIPK to a Golgi
compartment (22, 23). In live macrophages, colocalization of
PtdIns(4,5)P2 and type I PIPK
at sites of focal
exocytosis has been demonstrated (42). Inhibition of
PtdIns(4,5)P2 focally localized on the plasma membrane of
chromaffin cells inhibits agonist-induced secretion (24), suggesting
that focal PtdIns(4,5)P2 is important for granule secretion. This literature supports the concept that
activation-dependent translocation of PIPKs leads to the
formation of PtdIns(4,5)P2 microdomains that participate in
granule fusion. In a study by Hinchliffe et al. (43),
co-expression of type II
PIPK with type I PIPKs in transfected HeLa
cells results in translocation of type II
to the cell periphery. In
a separate study, expression of mutant forms of type II
PIPK also
translocate to membranes. Mutagenesis of Ser304 to alanine
or aspartate, but not threonine, resulted in the translocation of type
II
PIPK from the cytosol to the plasma membrane in HeLa cells and
did not affect kinase function (26). Based on these results, the
authors suggested that phosphorylation of Ser304 results in
the unmasking of a membrane localization sequence on type II
PIPK
(26). In platelets that did not undergo aggregation, type II PIPK
translocated to a Triton X-100-insoluble fraction but not to the
platelet actin cytoskeleton upon stimulation of platelets with agonists
(Fig. 5). Type II PIPK may associate with a protein of the Triton
X-100-insoluble membrane skeleton that is not precipitated with the
actin cytoskeleton following low speed centrifugation. Alternatively,
it is possible that type II PIPK is associated with a Triton
X-100-insoluble membrane fraction. Indeed, other phosphatidylinositol
kinases have been demonstrated to cluster in lipid rafts (57, 58).
Future studies will focus on the molecular identification of the type
II PIPK-binding site.
-granule secretory system
suggest a model for agonist-induced PtdIns(4,5)P2 synthesis in the process of
-granule secretion. According to this model, engagement of a ligand with its platelet surface receptor results in
activation of PKC. PKC, either by direct phosphorylation or via an
indirect mechanism, facilitates the translocation of type II PIPK to
binding sites adjacent to membrane. Translocation of type II PIPK may
lead to synthesis of PtdIns(4,5)P2 in the vicinity of the
kinase. Consistent with this hypothesis,
PtdIns(4,5)P2-binding peptides are recruited to
agonist-stimulated permeabilized platelets in a PKC- and type II
PIPK-dependent manner (Figs. 6 and 7). Whether translocation of type II PIPK in platelets leads to the formation of
PtdIns(4,5)P2 microdomains capable of recruiting
PtdIns(4,5)P2-binding proteins remains to be proven.
Furthermore, the role and identity of putative
PtdIns(4,5)P2-binding proteins in
-granule secretion involved in platelet granule secretion remain unknown.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Christopher Carpenter for critical
reading of the paper. We thank Paul A. Janmey for the gift of the
FITC-QRLFQVKGRR used in preliminary studies, Margaret Jacobs for
synthesis of the FITC-QRLFQVKGRR and FITC-QALFQVAGAA peptides, and
Katia A. Lamia for the type II PIPK D216A kinase-impaired mutant cDNA.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Institutes of Health Grant HL63250 and a
Burroughs Wellcome Fund Career Award. To whom correspondence should be
addressed: RE 318, Research East, P.O. Box 15732, Boston, MA 02215. Tel.: 617-667-0627; Fax: 617-975-5505; E-mail:
rflaumen@caregroup.harvard.edu.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M206493200
2 K. A. Lamia, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SNARE, soluble NSF attachment protein receptor; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetoxymethyl)ester; FITC, fluorescein isothiocynate; PIPES, 1,4-piperazinediethanesulfonic acid; PMA, phorbol 12-myristate 13-acetate; PtdIns, phosphatidylinositol; PtdIns(4, 5)P2, phosphatidylinositol (4,5)-bisphosphate; PKC, protein kinase C; SL-O, streptolysin-O; PIPK, phosphatidylinositol 4-phosphate 5-kinase.
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