Biphasic Activation of PKBalpha /Akt In Platelets
EVIDENCE FOR STIMULATION BOTH BY PHOSPHATIDYLINOSITOL 3,4-BISPHOSPHATE, PRODUCED VIA A NOVEL PATHWAY, AND BY PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE*

Hrvoje Banfic'Dagger , C. Peter Downes§, and Susan E. RittenhouseDagger

From the Dagger  Kimmel Cancer Institute and Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the § Department of Biochemistry, University of Dundee, Dundee DD1 4HN, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin alpha IIbbeta 3 activation. Such platelets rapidly and transiently form phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). After aggregation, a larger amount of PtdIns(3,4)P2 is generated. We report that this latter PtdIns(3,4)P2 arises largely through wortmannin-inhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon the activation of the Ca2+-dependent protease calpain. Elevation of cytosolic Ca2+ by ionophore, without integrin/ligand binding, is insufficient to activate the pathway. PtdIns3P 4-K is not the recently described "PIP5KIIalpha ." Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can be regulated negatively by the beta gamma subunit of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to Gbeta gamma and is, in addition, activated. Both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have been shown to stimulate PKBalpha /Akt phosphorylation and activation by phosphoinositide-dependent kinase 1. We find that activation of PKBalpha /Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P2 generated after aggregation. There is thus potential for both pre- and post-aggregation-dependent signaling by PKBalpha /Akt.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The activation of platelets via the thrombin receptor (THR-R)1 has been found to cause the rapid (within 60 s) stimulation of two phosphoinositide 3-kinases: PI3Kgamma and p85/PI3K (1). Each of these enzyme activities results in the generation of PtdIns(3,4,5)P3 (PtdInsP3) by phosphorylation of PtdIns(4,5)P2; shortly thereafter, a small amount of PtdIns(3,4)P2 is produced, either by hydrolysis of PtdInsP3 or phosphorylation of PtdIns4P (2). After 5-10 min, however, a large increase in PtdIns(3,4)P2 can be observed with THR-R agonists, as described below. Both PtdInsP3 and PtdIns(3,4)P2 have the potential to act as second messengers, stimulating in vitro the activities of some protein kinase C species (3-6), the protooncogene product PKB/Akt via PDK1 (7-9), and, in the case of PtdIns(3,4)P2, direct activation of PKB/Akt (10-12). Hence, elucidation of the routes by which these second messengers are synthesized and metabolized has potentially important implications for cell signaling.

PI3Kgamma is stimulated by Gbeta gamma , whereas activation of p85/PI3K appears to be down-stream of a protein kinase C (stimulated after THR-R-dependent activation of phospholipase C). Activation of p85/PI3K, but not of PI3Kgamma , can be achieved by the addition to platelets of protein kinase C-activating PMA (13). Apparently, it is p85/PI3K, rather than PI3Kgamma , whose activity contributes to the re-organization of integrin alpha IIbbeta 3 (13), allowing it to bind fibrinogen (FIB), which thereby facilitates platelet aggregation and subsequent signaling events. After platelets aggregate, the substantial burst of PtdIns(3,4)P2 accumulation that occurs is prevented by the integrin antagonist RGDS, removal of Ca2+, or the absence of integrin alpha IIbbeta 3 (14, 15). This increase has been found to be blocked by cell-permeable inhibitors of the Ca2+-dependent protease, calpain (16-18), an enzyme that is activated when platelets aggregate in an integrin alpha IIbbeta 3-dependent manner (19). Since one of several proteins cleaved by calpain is PtdIns(3,4)P2 4-phosphatase, leading to decreased activity, it had been suggested that inhibition of 4-phosphatase is responsible for the aggregation/calpain-dependent rise in PtdIns(3,4)P2 (17). Our recent data, however, point to another explanation (18). Integrin alpha IIbbeta 3 can be activated directly by LIBS (antibody Fab fragment to beta 3; Ref. 20), an event that causes FIB-dependent aggregation and PtdIns(3,4)P2 accumulation (21). We have found that LIBS+FIB-induced accumulation of PtdIns(3,4)P2, which is inhibited by calpain inhibitors such as calpeptin, cannot be accounted for by inhibition of 4-phosphatase (18). Instead, a new synthetic route is triggered, in which no PtdInsP3 is formed, but PtdIns3P is generated transiently by PtdIns 3-K in a calpeptin- and wortmannin-sensitive manner, and PtdIns (3,4)P2 arises through phosphorylation of PtdIns3P by Ptd Ins3P 4-K (18). The PtdIns(3,4)P2 that is formed after aggregation is generated primarily by this pathway and the increase in the activity of PtdIns3P 4-K is also inhibited by calpeptin. Moreover, the PtdInsP3-independent generation of PtdIns(3,4)P2 by this route leads to PKB/Akt activation in vivo. The purposes of the present study are to determine whether the new pathway is triggered in PMA+FIB and THR-R+FIB-activated platelets, whether PKB/Akt is activated only by PtdIns(3,4)P2 or by both PtdInsP3 and PtdIns(3,4)P2 in these cells, whether such activation(s) is/are dependent upon PKB/Akt phosphorylation, which PKB isoforms are stimulated, and to begin to address how PtdIns3P 4-K is regulated.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

All reagents and preparations of platelets were as described (18). Synthetic phosphoinositides were also purchased from Echelon Research Laboratories (Salt Lake City, UT). Antibodies to PKB (alpha , beta , and gamma  isoforms) and PDK1 were generously contributed by Dr. Dario Alessi (University of Dundee, Dundee, United Kingdom). Drs. Kath Hinchliffe and Robin Irvine kindly provided an affinity-purified antibody to "PIP5KIIalpha " that is immunoprecipitating and recognizes recombinant type IIalpha PIP kinase on Western blots. Gbeta gamma subunits of heterotrimeric GTP-binding proteins were isolated from bovine brain, as reported (22), and kindly provided by Dr. Xiu-wen Tang (University of Dundee). Protein phosphatase PP1gamma (human recombinant) and phosphatase inhibitor microcystin-LR were the generous gifts of Drs. P. T. W. Cohen and C. MacKintosh (University of Dundee). beta ARK-PH was expressed and purified as described (1).

Metabolic Studies with 32P-Labeled Platelets-- For studies with platelets labeled to equilibrium, washed and aspirin-treated platelets were prepared and labeled as described (18, 23), and incubated (2 × 109/ml) in the presence of Ca2+ and apyrase (18), with or without SFLLRN (25 µM) in the presence of RGDS (400 µM) or FIB (400 µg/ml), while stirring at 37 °C for up to 12 min. In other experiments, beta PMA (200 nM) with or without FIB or Ca2+ ionophore A23187 (2 µM) with RGDS was substituted for SFLLRN. For some 2-min and 10-min incubations, 20 nM wortmannin , 0-300 µM calpeptin, or 0-400 µg/ml FIB was included in incubations. All incubations were terminated with CHCl3/MeOH/HCl, and lipids extracted, resolved, and quantitated as described in Ref. 18.

For non-equilibrium labeling, platelets were exposed to [32P]Pi for 2 min with or without FIB prior to the addition of SFLLRN or PMA for 7 min, while stirring (20). When FIB was omitted, RGDS was included to block effects of secreted FIB caused by SFLLRN. Incubations were terminated as above, and lipids were extracted and incorporation of 32P into phosphoinositides assessed. [32P]PtdIns(3,4)P2 was digested for positional analysis of label as described (2, 18).

Platelet Fractions-- Triton X-100-insoluble CSK (1, 24) was isolated from platelets treated with SFLLRN+FIB or PMA+FIB (±calpeptin) after various periods of stirring. EGTA (20 mM) and calpeptin were included in Triton buffer for lysis. Washed CSK (50 µg) was incubated with 1 mg/ml mixtures (23) of diC16PtdIns3P, PtdIns, PtdIns4P, diC16PtdIns4P, PtdIns(4,5)P2, or diC16PtdIns(3,4)P2, and phosphatidylserine, for assays of lipid kinase activity (13, 18). In some cases, beta ARK-PH (5 µM; Ref. 1) or Gbeta gamma (0.5 µM) was included in assays. PKB was immunoprecipitated from Triton-soluble fractions using antibodies to alpha , beta , or gamma  isoforms, and its kinase activity was assayed with "Crosstide" peptide as described (18). In some experiments, PKB immunoprecipitates were incubated for 30 min at room temperature in Pi-free PKB assay buffer ± 50 milliunits/ml PP1gamma phosphatase (which does not hydrolyze PtdIns(3,4)P2 or PtdInsP3), followed by excess (2 µM) phosphatase inhibitor microcystin-LR, before assay of PKB activities. The presence of PKB and PDK1 in CSK and Triton-soluble fractions was also determined by Western blot (18).

Cytosol was prepared from unstimulated platelets (1). An antibody to PIP5KIIalpha (25-28) was used for immunoprecipitations from cytosol. After immunoprecipitation or after "mock" immunoprecipitation (resulting from incubation with antibody buffer and protein G-Sepharose), supernatants were assayed as above for PtdInsP kinase activities, and in some cases, synthetic diC16PtdIns5P or diC16PtdIns4P was used as a substrate. For separations of glycerophospho-Ins(3,5)P2 (derived from PtdIns(3,5)P2) and glycerophospho-Ins(3,4)P2 (derived from PtdIns(3,4)P2), HPLC fractions were collected every 10 s and monitored by scintillation spectrophotometry, rather than by Flo-One Beta detection (23).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Generation of Labeled 3-Phosphorylated Phosphoinositides in Intact Platelets-- As shown in Figs. 1 and 2, SFLLRN and PMA were each able to induce transient accumulations of labeled PtdInsP3, PtdIns3P, and sustained accumulations of PtdIns(3,4)P2 in 32P-labeled platelets. Only the increases in PtdIns3P and PtdIns(3,4)P2, however, were dependent upon the presence of FIB (Figs. 1 (B and C) and 2 (B and C)), the former totally dependent, and the latter largely FIB-dependent. The effects of FIB were linear up to 200 µg/ml (data not shown). These effects were very similar to those resulting from exposure of platelets to LIBS (which directly activates alpha IIbbeta 3)+FIB, under aggregating conditions (18). That we were indeed measuring changes in PtdIns(3,4)P2, and not in recently described PtdIns(3,5)P2 (29, 30), is illustrated in Fig. 3, which shows the results for a mixture of platelet PtdInsP2s that had been deacylated, resolved by HPLC, and counted. Separation to base line was achieved, and we observed that PtdIns(3,5)P2 contained a very minor fraction of the amount of 32P seen for PtdIns(3,4)P2. After exposure of platelets to PMA±FIB for 10 min, 32P in PtdIns(3,5)P2 increased 2-3-fold with FIB versus FIB-free controls, whereas [32P]PtdIns(3,4)P2 increased over 20-fold (Fig. 3). The addition of RGDS with PMA had no effect in comparison with PMA alone. Accumulations of PtdInsP3, which do not occur with LIBS+FIB (18), were rapid and unaffected by FIB (Figs. 1A and 2A), nor were they altered by inclusion of RGDS (13). All increases in 3-phosphorylated phosphoinositides were inhibited more than 90% by wortmannin (data not shown). After platelets were incubated with Ca2+ ionophore with stirring, but aggregation was prevented by omitting FIB and including RGDS to prevent binding of secreted FIB, a rapid increase in PtdInsP3 and a similarly rapid, but modest, increase in PtdIns(3,4)P2 levels occurred (Fig. 4). There was no increase, however, in PtdIns3P.


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Fig. 1.   Effects with time of SFLLRN±FIB on accumulations of 3-phosphorylated phosphoinositides in platelets. Platelets, labeled to equilibrium with 32P, were incubated with SFLLRN+RGDS (open symbols) or SFLLRN+FIB (filled symbols) with stirring for various periods. Lipids were extracted, digested, and resolved by HPLC with in-line isotopic detection. Data are shown for radiolabeled PtdInsP3 (A), PtdIns3P (B), and PtdIns(3,4)P2 (C) and are representative of two experiments.


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Fig. 2.   Effects with time of PMA±FIB on accumulations of 3-phosphorylated phosphoinositides in platelets. 32P-Labeled platelets, as in Fig. 1, were incubated for various periods with PMA (open symbols) or PMA+FIB (filled symbols). PtdInsP3 (A), PtdIns3P (B), and PtdIns(3,4)P2 (C).


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Fig. 3.   Resolution by HPLC of PtdInsP2 species from platelets. 32P-Labeled platelets were incubated for 10 min as in Fig. 2 with PMA (open symbols) or PMA+FIB (filled symbols). PtdInsP2 was digested and resolved on HPLC, and fractions were collected every 10 s and counted. The fraction designated PtdIns(3,5)P2 had the same retention time (consistent with those published; Ref. 28) as phosphoinositide products derived from in vitro incubations of cytosol with PtdIns3P and PtdIns5P (contributions of PtdIns(3,4)P2 and PtdIns(4,5)P2 excluded).


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Fig. 4.   Effects with time of cytosolic Ca2+ elevation on accumulations of 3-phosphorylated phosphoinositides in platelets. As in Fig. 1, 32P-labeled platelets were incubated with A23187+RGDS (RGDS alone had no effects) for various periods. Filled circles, PtdInsP3; open circles, PtdIns(3,4)P2; filled squares, PtdIns3P.

When the relative specific activities of 32P at the 1-, 3-, and 4-positions of the inositol ring of PtdIns(3,4)P2 were analyzed after non-equilibrium labeling of platelets and exposure of stirred platelets to either SFLLRN±FIB or PMA±FIB for 7 min, it was found that the presence of FIB affected the relative positional labeling (Fig. 5). As was true for LIBS+FIB-stimulated platelets, position 4 was hotter than position 3, and labeling of position 1 was negligible after platelets were aggregated in response to either SFLLRN or PMA. This indicated that 32P was added to position 4 after 3, i.e. that the predominant synthetic pathway for PtdIns(3,4)P2 is PtdIns3P right-arrow PtdIns(3,4)P2. In contrast, as is true for platelets exposed briefly (20 s or 60 s) to thrombin or SFLLRN (2, 18), platelets incubated for 7 min with PMA or SFLLRN+RGDS in the absence of FIB, and therefore without aggregation, contained "hotter" position 3 than 4 in PtdIns(3,4)P2. Under these conditions, the predominant pathway for PtdIns(3,4)P2 synthesis could be either PtdIns(4,5)P2 right-arrow PtdIns(3,4,5)P3 right-arrow PtdIns(3,4)P2 or PtdIns4P right-arrow PtdIns(3,4)P2.


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Fig. 5.   Distribution of 32P on the inositol ring of PtdIns(3,4)P2. Non-equilibrium 32P-labeled, stirred platelets were exposed to PMA or SFLLRN for 7 min under conditions which permitted (filled bars) or prevented (open bars) binding of FIB. The amount of 32P at each position of the inositol ring was analyzed and shown as a percent of the total. Data are the means ± S.D. of two or three experiments.

Activation of PKB/Akt in Stimulated Platelets-- Immunoprecipitations with antibodies specific for PKBalpha , -beta , or -gamma revealed that only PKBalpha had significant activity, and only this activity was stimulated when platelets were activated. PKBalpha and PDK1 were found to be present in both Triton-soluble and CSK fractions, as detected by Western blotting; however, when the same amount of CSK protein from resting and activated platelets was compared, detectable PKBalpha and PDK1 did not increase. Total CSK protein did increase, however, in activated platelets, rising 2-3-fold; therefore, the amounts of PKBalpha and PDK1 increased as CSK-associated proteins. The majority of PKBalpha and PDK1 was present in the Triton-soluble fraction of platelets (data not shown).

In contrast to the case for LIBS+FIB (18), in which PKBalpha activity increased (3-fold) rather late (maximally at 10-12 min), following platelet aggregation and paralleling PtdIns(3,4)P2 levels (20-30-fold), PKBalpha activity in response to SFLLRN±FIB increased much more rapidly, and to a greater degree (8-9-fold), following the increases in PtdInsP3 (8-fold) after a 15-s lag (Fig. 6). This was totally inhibited by wortmannin (data not shown). After 10 min, however, when PtdInsP3 had decreased to control (agonist-free) levels, PKBalpha activity had also returned to base-line levels, except where FIB was present (Fig. 7A). The presence of FIB had no effect on accumulations of PtdInsP3 (shown as well in Fig. 1A), including the return to base-line levels, but had a marked enhancing effect on PtdIns(3,4)P2 accumulations (26-fold; Figs. 1C and Fig. 7A) and PKBalpha activity (3-fold; Fig. 7A). Calpeptin (IC50 1 µM) did not inhibit increases in PtdInsP3 or early activation of PKBalpha (Fig. 7A; 2 min), but totally inhibited the rises in PtdIns(3,4)P2 and PKBalpha activity associated with later incubations in the presence of FIB (Fig. 7A; 10 min). The same phenomena were observed when PMA was used as an agonist, in place of SFLLRN (Fig. 7B). "Second phase" stimulations of PKBalpha and accumulations of PtdIns(3,4)P2 in response to either SFLLRN+FIB or PMA+FIB were thus similar to the increases observed with LIBS+FIB (18).


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Fig. 6.   Initial formation of PtdInsP3 and activation of PKBalpha in platelets exposed to SFLLRN. As in Fig. 1, 32P-labeled platelets, or non-labeled platelets, were incubated with SFLLRN for short periods. Incubations were terminated with organic solvents (32P-labeled platelets) or ice-cold Triton lysis buffer (unlabeled platelets). Lipids were resolved and [32P]PtdInsP3 (filled circles) quantitated, or PKBalpha was immunoprecipitated from Triton-soluble fractions, and its activity assayed (open circles). There was negligible production of PtdIns(3,4)P2 within the first 30 s.


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Fig. 7.   Effects of FIB binding or calpeptin exposure on platelet production of PtdInsP3, PtdIns(3,4)P2, and PKBalpha activation in response to agonists. As in Fig. 6, formation of [32P]PtdIns(3,4)P2 and [32P]PtdInsP3 were quantitated after platelets were exposed to SFLLRN (A) or PMA (B). Two incubation periods were examined and conditions varied with respect to the presence of FIB, RGDS, and prior exposure of platelets to calpeptin. The data with calpeptin are for its maximum effective concentration. The IC50 for calpeptin was 1 µM. Calpeptin, FIB, and RGDS had no effect on basal activity of PKBalpha or radiolabeling of phosphoinositides. Data shown are presented as a multiple of agonist-free (basal) values and are representative of three or four experiments.

In order to determine whether phosphorylation of PKBalpha , presumably by PDK1, was required for its early stage and late stage activations, immunoprecipitated PKBalpha from platelets incubated with or without SFLLRN (2 min) or with LIBS+FIB (stirring, 10 min) were incubated with or without protein phosphatase and then phosphatase inhibitor prior to assay of PKBalpha activity. We observed (Table I) that incubation with phosphatase inhibited SFLLRN-activated PKBalpha by 100% and LIBS+FIB-activated PKBalpha by 95%.

                              
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Table I
Inhibition of activated PKBalpha by dephosphorylation
PKBalpha was immunoprecipitated after incubations of platelets with buffer or SFLLRN+RGDS or LIBS+FIB+stirring. Immunoprecipitates were incubated with or without protein phosphatase; incubations were terminated with excess phosphatase inhibitor, and PKBalpha activities were assayed. Background dpm (assays without PKBalpha immunoprecipitates) are subtracted from all results.

Regulation of CSK-associated PtdIns3P 4-K and PtdIns4P 3-K from Activated Platelets-- Exposure of platelets to SFLLRN+FIB led to an initial (within 3 min) inhibition of CSK-associated PtdIns3P 4-K activity, followed (5-10 min) by increased activity (Fig. 8A). The initial inhibition was overcome by the addition to the assay incubation mixture of beta ARK-PH, which binds Gbeta gamma , known to be present in CSK of SFLLRN/THR-activated platelets (1). Incubation of platelets with calpeptin had no effect on the early PtdIns3P 4-K activities in CSK, whether or not beta ARK-PH was added to assay mixtures. At late times, however, the stimulated PtdIns3P 4-K activity could not be increased further by beta ARK-PH, but it was blocked by exposure of platelets to calpeptin prior to SFLLRN+FIB. That Gbeta gamma can indeed inhibit PtdIns3P 4-K activity at early time points, and this is the likeliest explanation for the effects of beta ARK-PH shown in Fig. 8A, is illustrated in Fig. 9A. There, Gbeta gamma added to assay mixtures is shown to have inhibited PtdIns3P 4-K in CSK of resting platelets or of platelets exposed for 2 min to PMA+FIB, whereas beta ARK-PH was without effect (PMA does not cause liberation of Gbeta gamma to platelet CSK; Ref. 13). After 10 min, however, PtdIns3P 4-K lost its sensitivity to Gbeta gamma . Prior treatment of platelets with calpeptin, however, allowed PtdIns3P 4-K to remain susceptible to inhibition by Gbeta gamma , even after 10 min. It is known that CSK of SFLLRN/THR-activated platelets contains increased activities of both p85/PI3K and PI3Kgamma , the latter due to Gbeta gamma , and that this Gbeta gamma -dependent activation is blocked by beta ARK-PH added to isolated CSK (1). We have confirmed this in Fig. 8B, for 0-3 min. Early activation of PtdIns4P 3-K (which utilized either commercial PtdIns4P or synthetic diC16PtdIns4P with similar results) was inhibited about 50% by beta ARK-PH, but not by calpeptin treatment of platelets. The sensitivity to beta ARK-PH also decreased with time, but this was not improved by calpeptin treatment. Calpeptin was inhibitory at late time points, and the combination of beta ARK-PH + calpeptin was more inhibitory than either alone, implying the presence of both a calpain-activated PtdIns4P 3-K and some remaining Gbeta gamma -sensitive PtdIns4P 3-K in CSK of "late stage" SFLLRN-activated platelets. In Fig. 9B, the results for PtdIns4P 3-K activity in CSK of platelets activated for 10 min with PMA+FIB (where released Gbeta gamma is not a significant factor, and thus beta ARK-PH has no effect) show that calpeptin treatment was inhibitory here, as well. In contrast, activating effects of Gbeta gamma were preserved by calpeptin treatment. Thus, there appears to be a calpain-promoted PtdIns4P 3-K activity in the CSK of both SFLLRN- and PMA-stimulated platelets, as well as stimulated PtdIns3P 4-K activity. Regulation by Gbeta gamma of both PtdIns4P 3-K (activating) and PtdIns3P 4-K (inhibiting) activities, however, appears to be decreased by calpain action.


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Fig. 8.   Effects of calpeptin and beta ARK-PH on CSK-associated phosphoinositide kinase activities of SFLLRN-activated platelets. Unlabeled platelets were incubated as in Fig. 7, were incubated with (open or filled diamonds) or without calpeptin (circles) prior to exposure to buffer or SFLLRN+FIB for varied periods. Incubations were terminated with ice-cold Triton lysis buffer. Washed, Triton-insoluble CSKs at the same concentration were incubated with kinase assay buffer, using PtdIns3P (for 4-K; A) or PtdIns4P (for 3-K; B) substrates in the presence (open circles, open diamonds) or absence (filled circles, filled diamonds) of beta ARK-PH. [32P]PtdIns(3,4)P2 was quantitated, and the results expressed as a multiple of control (unstimulated platelet) values. Data are representative of two incubations, in duplicate.


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Fig. 9.   Effects of calpeptin, Gbeta gamma , and beta ARK-PH on CSK-associated phosphoinositide kinase activities of PMA-activated platelets. Platelets were incubated as in Fig. 8, substituting PMA for SFLLRN. In some cases, Gbeta gamma was added to kinase assay mixtures. Substrates were PtdIns3P (for 4-K; A) and PtdIns4P (for 3-K; B). calp, calpeptin; beta ARK, beta ARK-PH. Results are expressed as a multiple of control (no agonist or inhibitors) values as means ± S.D. for two or three experiments.

Depletion of PtdInsP Kinase Activities with Antibody to PIP5KIIalpha -- Most PtdIns3P 4-K activity in unstimulated platelets was cytosolic, and inhibitable by Gbeta gamma , as above, but unaffected by GTPgamma S (data not shown). In order to gain additional information about the identity of this platelet PtdIns3P 4-K, immunodepletion experiments were performed. Immunodepletions of platelet cytosol, using an antibody to PIP5KIIalpha (which has been cloned and expressed; Refs. 25 and 26), led to several findings (Fig. 10). 1) When PtdIns4P (either synthetic or natural) was used as a substrate, PtdIns 4P 5-K (i.e. "type I" activity), yielding PtdIns(4,5)P2, was depleted by 80%, indicating cross-reactivity of the antibody with type I 5-K. 2) When PtdIns5P was used, PtdIns5P 4-K (erroneously, according to recent literature, referred to as type II "5-K"; Ref. 28), which also yielded PtdIns(4,5)P2, was depleted by 65%. 3) No significant depletion was seen for 3-K activities, i.e. PtdIns4P 3-K or PtdIns5P 3-K. 4) PtdIns3P 5-K activity was depleted 50%. 5) Very importantly, no significant depletion was seen for platelet PtdIns3P 4-K activity (and no PtdInsP3 was formed in these assays). The finding for PtdIns3P 4-K was rather surprising, in that it has been reported (28) that type II "5-K" (actually PtdIns5P 4-K) also acts on PtdIns3P, albeit less efficiently, forming PtdIns(3,4)P2. Platelet PtdIns3P 4-K, by these immuno-criteria, therefore, qualifies as neither type I nor type II "5-K."


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Fig. 10.   Effect of depletion with PIP5KIIalpha antibody on platelet cytosolic PtdInsP kinase activities. Platelet cytosolic fractions were incubated with or without antibody and then protein G-Sepharose. After immunoprecipitation (or after mock immunoprecipitation), supernatants were assayed for kinase activity with synthetic or natural PtdIns4P, synthetic PtdIns3P, and synthetic PtdIns5P substrate, in the linear range of the time course, and the PtdInsP2 products resolved, after digestion, by HPLC. Glycerophospho-Ins(3,5)P2 was separated from glycerophospho-Ins(3,4)P2 as in Fig. 3. Results are the means ± S.D. of two to four experiments, in duplicate.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have demonstrated (Figs. 6 and 7 (A and B)) that stimulation of platelets by either SFLLRN or PMA, in the absence of FIB, leads to monophasic activation of PKBalpha /Akt due to the generation of PtdInsP3 (see also Figs. 1A and 2A). This is the first indication, to our knowledge, of PtdInsP3-dependent activation in vivo of PKBalpha /Akt in the absence of a stimulated increase in PtdIns(3,4)P2. When FIB (which binds to activated integrin alpha IIbbeta 3) is present, however, a second phase of PKBalpha activation occurs. This phase is dependent upon the accumulation of PtdIns(3,4)P2 (see also Figs. 1C and 2C), consistent with findings that we have obtained in studies in which alpha IIbbeta 3 was activated directly by LIBS (18). The PtdIns(3,4)P2-dependent activation of PKBalpha can occur in the absence of PtdInsP3 (Fig. 7, A and B; Ref. 18), and is not due to potentiation by PtdIns(3,4)P2 of residual effects of PtdInsP3 that had accumulated earlier since, in LIBS+FIB-activated platelets, no generation of PtdInsP3 occurs (18). It appears that PtdInsP3 is more potent than is PtdIns(3,4)P2 as an activator of PKBalpha , which might be predicted from studies (8, 9) that have demonstrated that PtdInsP3 regulates PDK1 more efficiently than does PtdIns(3,4)P2. Since PDK1 is present in platelets, and is required (via its phosphoinositide-stimulated phosphorylation of PKBalpha ) as the intermediate stage in the activation of PKBalpha by PtdInsP3, this is the likely route by which PKBalpha is regulated in platelets. Indeed, the exposure of immunoprecipitated, activated PKBalpha to protein phosphatase prior to assay of activity was found to decrease early (PtdInsP3-dependent) or late (PtdIns(3,4)P2dependent) stage PKBalpha activities by 95-100%, whereas basal PKBalpha activity was minimally affected (Table I). Although this argues in favor of regulation of PKBalpha by phosphorylation, presumably by PDK1, it cannot rule out additional direct regulation of PKBalpha by PtdIns(3,4)P2 (10-12) in vivo, which association might not have been preserved after immunoprecipitation of PKBalpha from Triton lysates. Nonetheless, the early and late phase activations of PKBalpha , the latter dependent on a post-aggregatory event, point to possible roles for PKBalpha in both pre- and post-aggregatory signaling.

Our data (Figs. 1B, 2B, and 5) indicate that the majority of post-aggregatory PtdIns(3,4)P2 is synthesized by a route that involves the generation of PtdIns3P, and its phosphorylation by PtdIns3P 4-K. In the absence of FIB, however, PtdIns(3,4)P2 is formed primarily from the hydrolysis of PtdIns(3,4,5)P3 or by PtdIns4P 3-K. In the presence of FIB, the large accumulation of PtdIns(3,4)P2 is dependent upon the activation of alpha IIbbeta 3, the binding of FIB to this integrin (as indicated above), aggregation (18), and the activation of the Ca2+-dependent protease, calpain (Fig. 7; Ref. 18). The binding of FIB to integrin alpha IIbbeta 3 has been reported to cause an influx of extracellular Ca2+ (31, 32), but in the absence of FIB binding to integrin, the elevation of cytosolic Ca2+ is insufficient to activate this pathway (Fig. 4) or to activate calpain (19). It may be sufficient, however, to activate calmodulin, and thereby p85/PI3K (33), which would account for the increase in PtdInsP3 and the small increase in PtdIns(3,4)P2 that we observed, in the absence of a rise in PtdIns3P.

Data that we present here for CSK fractions point to a role for calpain in the activation of PtdIns3P 4-K (Figs. 8A and 9A; Ref. 18), as is also true for PtdIns 3-K (18). Both of these increased activities are necessary for integrin signaling leading to maximum PtdIns(3,4)P2 generation and PKBalpha activation (18). Our data further indicate that PtdIns3P 4-K is negatively regulated by Gbeta gamma , which can be released when THR-R is stimulated. Presumably, this might minimize the formation of PtdIns(3,4)P2 from endogenous PtdIns3P during early phases of platelet activation by THR-R. It seems likely that some common calpain target is involved in the negative regulation of PtdIns3P 4-K and positive regulation of PtdIns4P 3-K (Fig. 9B) by Gbeta gamma since, once calpain is activated, the ability of Gbeta gamma to regulate both of these activities is diminished. Therefore, calpain appears to be important not only for eliminating regulatability of PtdIns3P 4-K and PtdIns4P 3-K by Gbeta gamma , but also for activating these phosphoinositide kinases by another mechanism. Our "positional labeling" experiments (Fig. 5) do not rule out some participation of PtdIns4P 3-K in integrin-linked production of PtdIns(3,4)P2. Rather, they indicate that this is not the predominant pathway. The data for CSK may thus have some bearing on post-integrin generation of a minor portion of PtdIns(3,4)P2 via PtdIns4P 3-K, as they do for pre-integrin activation of PI3Kgamma and/or p85/PI3K when SFLLRN/THR or PMA is the agonist (1). Further studies are needed to address this issue.

In hopes of elucidating the nature of the PtdIns3P 4-K activity that we have observed in the cytosol and in the CSK of platelets, we have utilized an antibody that can immunoprecipitate and recognize on Western blots PIP5KIIalpha (25-28). This is an enzyme that has been expressed and found to phosphorylate synthetic PtdIns5P, as well as PtdIns5P found as an impurity in PtdIns4P isolated from natural sources (28). To a much lesser degree, it can also phosphorylate PtdIns3P, and all phosphorylations occur at the 4-OH position of the inositol ring, to produce PtdIns(4,5)P2 and PtdIns(3,4)P2; PtdIns(4,5)P2 is not produced from pure (synthesized) PtdIns4P (28) by this enzyme. Technically, therefore, the type II "5-K" is not a "5-K", but a "4-K." We have found that the antibody not only recognizes and removes PtdIns5P 4-K from platelet cytosol, but also PtdIns4P 5-K (utilizing either natural or synthetic PtdIns4P substrate) and PtdIns3P 5-K (Fig. 10). Thus, the antibody apparently can recognize true 5-K activities, as well. Despite removing 50-80% of these activities, however, the immunoprecipitation procedure does not remove PtdIns3P 4-K or 3-K (PtdIns4P 3-K or PtdIns5P 3-K) activities. This observation with respect to PtdIns3P 4-K activity is unexpected, since it has been reported recently that type II kinases display PtdIns3P 4-K (27, 28) and concerted PtdIns3P right-arrow PtdIns(3,4)P2 right-arrow PtdIns(3,4,5)P3 activities (27). The PtdIns3P 4-K activity that we observe in platelet lysates is thus not the type II or PtdIns5P 4-K enzyme described in the literature. Only purification and sequencing of the platelet enzyme will provide information on how these families of 4-kinases are related.

Our findings with respect to the activation of alpha IIbbeta 3 integrin, PI3Ks, and PKBalpha /Akt are summarized in Fig. 11. As indicated, the targets for PKBalpha /Akt (pre- and post-integrin) are as yet unknown. Additional targets for post-integrin-generated PtdIns(3,4)P2 may also exist. It is possible, for example, that such PtdIns(3,4)P2 may be involved in late (post-aggregation) filopod formation (34). The involvement of 3-phosphorylated phosphoinositides in both early and late phases of agonist-induced platelet signaling, and the nature of the phosphoinositide kinases that are involved, should be important topics for future exploration.


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Fig. 11.   Summary of pre-integrin and post-integrin pathways leading to activation of PKBalpha /Akt. For clarity, activation of PI3Kgamma via THR-R has been omitted. Whereas PI3Kgamma does not contribute to the activation of alpha IIbbeta 3 (13), it may, however, affect PKBalpha through its contribution to PtdInsP3.

    ACKNOWLEDGEMENTS

We thank Drs. Dario Alessi, Kath Hinchliffe, and Robin Irvine for the generous contribution of antibodies, and Drew Likens of the Cardeza Foundation and Michelle Levinski for artwork.

    FOOTNOTES

* This work was supported by National Institutes of Health NHLBI Grant HL 38622 (to S. E. R.), NATO Grant 950672 (to S. E. R.), and awards from the Medical Research Council and Leukaemia Research Fund (to C. P. D.).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.

To whom correspondence should be addressed: Kimmel Cancer Institute and Cardeza Foundation for Hematologic Research, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. Fax: 215-923-7145.

1 The abbreviations used are: THR-R, thrombin receptor (activated by alpha -thrombin and the protease-activated receptor 1 agonist peptide SFLLRN); Gbeta gamma , heterodimer derived from GTP-binding protein; beta ARK-PH, C-terminal pleckstrin homology-containing domain of beta -adrenergic receptor kinase that binds Gbeta gamma ; PI3K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol (locants of other phosphates on the myoinositol ring are indicated); P, phosphate; PtdInsP3, PtdIns(3,4,5)P3; diC16PtdIns, 1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-phosphoryl)-D-myoinositol (other locants as above); PDK1, phosphoinositide-dependent kinase 1; FIB, fibrinogen; LIBS, anti-ligand-induced binding site 6 antibody Fab fraction; CSK, cytoskeleton; PKB/Akt, protein kinase B related to AKR mouse T-cell lymphoma-derived oncogenic product; HPLC, high performance liquid chromatography; PMA, beta -phorbol myristate acetate; PIP, phosphatidylinositol phosphate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PIP5KIIalpha , phosphatidylinositol phosphate 5-kinase, type II alpha ; 3-K, 3-kinase; 4-K, 4-kinase, 5-K, 5-kinase.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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