Protein kinase C mediates translocation of type II phosphatidylinositol 5-phosphate 4-kinase required for platelet α-granule secretion

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 α-granule secretion. SFLLRN-stimulated α-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 IIβ PtdIns 5-phosphate 4-kinase. In contrast, recombinant type IIβ PtdIns 5-phosphate 4-kinase augmented SFLLRN-stimulated α-granule secretion from SL-O-permeabilized platelets. SFLLRN-stimulated α-granule secretion was inhibited by a protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated α-granule secretion was inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the kinase-impaired point mutant of type IIβ PtdIns 5-phosphate 4-kinase and augmented by recombinant type IIβ 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 α-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.

␣-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 re-views see Refs. 1 and 2). The SNARE 1 protein isoforms SNAP-23, syntaxin 2 and 4, and VAMP-3 are found in platelets and have been demonstrated to mediate ␣-granule secretion (3)(4)(5)(6)(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)P 2 , 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.
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)P 2 . Regulated translocation of type I PIPK is directed by ADP-ribosylation factors (21)(22)(23). Investigators have hypothesized that focal sites of PtdIns(4,5)P 2 form in areas of PIPK activity following translocation to membranes. According to this model, the nidus of PtdIns(4,5)P 2 that is subsequently formed on the cytoplasmic face of the cell membrane binds components of the secretory machinery that contain PtdIns(4,5)P 2 -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)P 2 -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 partici-pate in MgATP-dependent, Ca 2ϩ -triggered ␣-granule secretion from platelets. In these studies, type II PIPK was shown to act at a priming step prior to Ca 2ϩ -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-Opermeabilized 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)P 2 -binding protein derived from gelsolin.

EXPERIMENTAL PROCEDURES
Chemicals and Reagents-All buffer constituents, Triton X-100, and CaCl 2 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)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)RFARKGALRQKNV), and 5,6-dichloro-1-␤-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 phosphatebuffered 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.
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 ϫ 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 ϫ 10 8 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 l of gel-filtered platelets (1-2 ϫ 10 8 /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 l of reaction mixture was transferred to 5 l of phycoerythrin-conjugated AC1.2 anti-P-selectin antibody. Phosphate-buffered saline (500 l) 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 sur-face 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 l of gel-filtered platelets (1-2 ϫ 10 8 /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 M Ca 2ϩ is indicated in the figure legends. The amount of CaCl 2 required to give a free Ca 2ϩ concentration of 10 M 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 Ca 2ϩ , 10 l of reaction mixture was transferred into 5 l of phycoerythrinconjugated 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.
Isolation of Platelet Cytoskeletal Fractions-For samples using SFLLRN-stimulated platelets, gel-filtered platelets (2-4 ml) were incubated with 25 M 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 g/ml leupeptin, 1 g/ml pepstatin, and 2 g/ml aprotinin). Cytoskeletons were isolated by centrifugation at 10,000 ϫ 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 M BAPTA-AM for 30 min and subsequently exposed to 100 M SFLLRN or buffer for 10 min. Noncytoskeletal, Triton X-100insoluble fractions were isolated by centrifugation of the 10,000 ϫ g supernatant (derived from the cytoskeletal preparation) at 100,000 ϫ g at 4°C for 3 h. Under conditions in which the 10,000 ϫ 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 ϫ 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 M 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)P 2 -binding domain and FITC-labeled control peptides to BAPTA-AM-treated, SL-O-permeabilized platelets, 20 l of gel-filtered platelets (1-2 ϫ 10 8 /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 M). Following an additional 20-min incubation, the sample was diluted in 500 l of phosphatebuffered 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)P 2 -binding domain to nonpermeabilized platelets was determined to be background. The amount of FITClabeled 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 2 K. A. Lamia, personal communication.
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 ␣-Granule
Secretion-We have previously demonstrated that type II PIPK participates in ␣-granule secretion in a MgATP-dependent, Ca 2ϩ -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. Pselectin 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 Ϯ 12% that of P-selectin expression from intact platelets exposed to SFLLRN. To assess the role of type II PIPK in SFLLRNinduced ␣-granule secretion, we used an antibody directed at the amino terminus of type II PIPK that inhibits PIP 2 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.
PKC Participates in Type II PIPK-mediated ␣-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 g/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 g/ml recombinant type II␤ PIPK (rType II␤ PIPK), or 15 g/ml kinaseimpaired 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. PIPK participates in PMA-stimulated ␣-granule secretion from SL-O-permeabilized platelets.
Type II PIPK Mediates an Agonist-dependent Step Prior to Ca 2ϩ -triggered Secretion-We have previously used a MgATPdependent, Ca 2ϩ -triggered platelet secretory model to demonstrate that type II PIPK acts at an ATP-dependent step prior to Ca 2ϩ -triggered secretion (15). Based on these studies, we hypothesized that type II PIPK acts prior to Ca 2ϩ -mediated triggering during agonist-induced ␣-granule secretion. To test this hypothesis, intact platelets were incubated with the cell-permeant Ca 2ϩ chelator BAPTA-AM to suppress rapid, Ca 2ϩ -mediated triggering of granule secretion from intact platelets. Platelets were subsequently exposed to SFLLRN. Following this exposure, platelets were permeabilized in the presence of Ca 2ϩ to elicit ␣-granule secretion. This strategy allows for the temporal separation of agonist-dependent events required for granule secretion from Ca 2ϩ -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 M free Ca 2ϩ 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 Ca 2ϩ -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 Ca 2ϩ 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 Ca 2ϩ -triggered P-selectin surface expression (Fig. 3B). Antibodies inhibited SFLLRNmediated 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- Platelets were then exposed to 0.2 M 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 g/ml nonimmune IgG (Non-immune IgG), or 40 g/ml anti-type II PIPK IgG (Anti-type II PIPK IgG). Platelets were then exposed to 0.2 M 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 g/ml recombinant type II␤ PIPK (rType II␤ PIPK), or 15 g/ml kinase-impaired type II␤ PIPK point mutant (Kinase-impaired rType II␤ PIPK). Platelets were then exposed to either buffer or 0.2 M 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. 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 Ca 2ϩ -induced triggering of secretion. Nonimmune antibodies had no effect on SFLLRN-responsive, Ca 2ϩ -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 Ca 2ϩ .
Like SFLLRN, PMA supported Ca 2ϩ -triggered ␣-granule release in BAPTA-AM-treated, SL-O-permeabilized platelets (Fig. 3C). In these studies, BAPTA-AM-treated platelets were exposed to 0.2 M PMA or buffer and then permeabilized with SL-O in the presence of Ca 2ϩ . The degree of P-selectin surface expression in BAPTA-AM-treated platelets exposed to PMA and permeabilized in the presence of Ca 2ϩ was 40 -60% of that in untreated intact platelets exposed to PMA. Thus, PMA was a stronger agonist than SFLLRN in rendering BAPTA-AMtreated platelets capable of secreting ␣-granules in response to Ca 2ϩ . Addition of anti-type II PIPK antibodies to permeabilized platelets prior to addition of PMA resulted in inhibition of PMA augmentation of Ca 2ϩ -triggered P-selectin surface expression (Fig. 3D). Anti-type II PIPK antibodies failed to inhibit PMAresponsive 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 Ca 2ϩ .
Agonist-induced Translocation of Type II PIPK-Translocation of PIPKs has been shown to be involved in the regulation of their activity (21)(22)(23)(24)(25)(26)(41)(42)(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, Ͼ50% of platelet type II PIPK remained associated with permeabilized platelets incubated with SFLLRN prior to permeabilization. These results suggest that type II PIPK trans-

FIG. 3. Type II PIPK mediates an agonist-dependent step prior to Ca 2؉ -triggered P-selectin surface expression in BAPTA-AMtreated, SL-O-permeabilized platelets.
A, gel-filtered platelets were incubated with 25 M 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 M Ca 2ϩ . 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 g/ml nonimmune IgG (Non-immune IgG), or 40 g/ml anti-type II PIPK IgG (anti-type II PIPK IgG) and subsequently exposed to 100 M SFLLRN and 10 M Ca 2ϩ . A second group of BAPTA-AM-treated platelets (white bars) was exposed to 100 M SFLLRN 10 min prior to permeabilization in the presence of buffer (Buffer), 40 g/ml nonimmune IgG (Non-immune IgG), or 40 g/ml anti-type II PIPK IgG (anti-type II PIPK IgG) and subsequently exposed to 10 M Ca 2ϩ . 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 Ca 2ϩ 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 M BAPTA-AM for 30 min and subsequently exposed to either buffer or 0.2 M 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 M Ca 2ϩ . 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 M 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 g/ml nonimmune IgG (Nonimmune IgG), or 40 g/ml anti-type II PIPK IgG (anti-type II PIPK IgG) and subsequently exposed to 0.2 M PMA and 10 M Ca 2ϩ . A second group of platelets (white bars) was exposed to 0.2 M PMA 10 min prior to permeabilization in the presence of buffer (Buffer), 40 g/ml nonimmune IgG (Nonimmune IgG), or 40 g/ml anti-type II PIPK IgG (anti-type II PIPK IgG) and subsequently exposed to 10 M Ca 2ϩ . 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. locates 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 SFLLRNand 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-AMtreated 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 ϫ 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 ϫ 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 SFLLRNexposed samples following centrifugation at 100,000 ϫ g (data not shown). Type II PIPK was also pelleted at 100,000 ϫ 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)P 2 -binding Domain-Studies in other secretory systems have suggested that translocation of PIPKs results in the recruitment of PtdIns(4,5)P 2 -binding proteins to membranes (24,42). We, therefore, sought to assess the hypothesis that type II PIPK mediates the recruitment of PtdIns(4,5)P 2 -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)P 2binding 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)P 2 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)P 2 -binding domain of gelsolin to platelets was analyzed by flow cytometry. Incubation of BAPTA-AM-treated platelets with SFLLRN increased the association of FITC-QR-LFQVKGRR with platelets following permeabilization with SL-O (Fig. 6A). Neomycin binds PtdIns(4,5)P 2 strongly (46) and has been used to study the role of PtdIns(4,5)P 2 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)P 2 -binding domain of gelsolin associates with platelet PtdIns(4,5)P 2 (Fig. 6A). Expo- 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 ϫ 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 ϫ 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 M PMA (PMA) for 10 min. Triton X-100-insoluble fractions were recovered by Triton X-100 extraction followed by centrifugation at 100,000 ϫ g. Proteins associated with the Triton X-100insoluble fraction were assayed for type II PIPK by immunoblotting.
sure to bisindolylmaleimide I also inhibited SFLLRN-induced binding of the PtdIns(4,5)P 2 -binding peptide to BAPTA-AMtreated platelets (Fig. 6A). In contrast, neither neomycin nor bisindolylmaleimide I affected the levels of FITC-QR-LFQVKGRR 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)P 2 -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)P 2 -binding domain are replaced with alanine. The relative fluorescence of unstimulated permeabilized platelets incubated in the presence of 5 M of the mutant FITC-binding protein was 49 Ϯ 14% of that of permeabilized platelets incubated in the presence of 5 M native FITC-binding protein. Binding of FITC-labeled alanine mutant to permeabilized BAPTA-AM-treated, SFLLRN-exposed platelets was 114 Ϯ 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-AMtreated 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 Ϯ 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)P 2 -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 agonistinduced increases in binding of the gelsolin PtdIns(4,5)P 2 -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)P 2 -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 agonistinduced recruitment of the gelsolin PtdIns(4,5)P 2 -binding domain is mediated through type II PIPK. DISCUSSION PtdIns(4,5)P 2 is required for regulated granule secretion in numerous secretory systems. In most cells, PtdIns(4,5)P 2 required for granule secretion is synthesized via a type I PIPKdependent pathway (18,48). A role for type II PIPK, however, has been proposed for platelet ␣-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 thrombinstimulated 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 Ca 2ϩ -dependent, step required for platelet ␣-granule secretion in an ATP-primed, Ca 2ϩ -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 activationdependent signals. Thus, type II PIPK serves as a downstream effector of PKC activity during agonist-induced platelet secretion.
A role for PKC in Ca 2ϩ -triggered granule secretion has been demonstrated in several secretory systems. Phorbol esters enhance Ca 2ϩ -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 Ca 2ϩ -triggered secretion in PC12 cells (55) and in platelets (13). In our permeabilized platelet model, ␣-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.
Regulated translocation of PIPKs to membrane compartments has previously been observed in other cell models. Recruitment of type I PIPK␣ 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)P 2 and type I PIPK␣ at sites of focal exocytosis has been demonstrated (42). Inhibition of PtdIns(4,5)P 2 focally localized on the plasma membrane of chromaffin cells inhibits agonist-induced secretion (24), suggesting that focal PtdIns(4,5)P 2 is important for granule secretion. This literature supports the concept that activation-dependent translocation of PIPKs leads to the formation of PtdIns(4,5)P 2 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 Ser 304 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 Ser 304 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-100insoluble 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.
Our studies using the agonist-responsive ␣-granule secretory system suggest a model for agonist-induced PtdIns(4,5)P 2 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 phosphoryl- One group of platelets (black bars) was permeabilized with 6 units/ml SL-O in the presence of buffer (Buffer), 40 g/ml nonimmune IgG (Non-immune IgG), or 40 g/ml anti-type II PIPK IgG (anti-type II PIPK IgG) and subsequently exposed to 100 M SFLLRN and 5 M FITC-QRLFQVKGRR. A second group of BAPTA-AM-treated platelets (white bars) were exposed to 100 M SFLLRN 10 min prior to permeabilization in the presence of buffer (Buffer), 40 g/ml nonimmune IgG (Non-immune IgG), or 40 g/ml anti-type II PIPK IgG (anti-type II PIPK IgG). FITC-QRLFQVKGRR (5 M) was subsequently added to the permeabilized platelets. Following an additional 20-min incubation, platelets were assayed for FITC-QR-LFQVKGRR 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 M BAPTA-AM for 30 min. Platelets were subsequently treated as described above except that 0.2 M 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. ation 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)P 2 in the vicinity of the kinase. Consistent with this hypothesis, PtdIns(4,5)P 2 -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)P 2 microdomains capable of recruiting PtdIns-(4,5)P 2 -binding proteins remains to be proven. Furthermore, the role and identity of putative PtdIns(4,5)P 2 -binding proteins in ␣-granule secretion involved in platelet granule secretion remain unknown.