p110beta and p110delta Phosphatidylinositol 3-Kinases Up-regulate Fcepsilon RI-activated Ca2+ Influx by Enhancing Inositol 1,4,5-Trisphosphate Production*

Alexander J. SmithDagger , Zurab SurviladzeDagger , Elizabeth A. GaudetDagger , Jonathon M. Backer§, Christina A. Mitchell, and Bridget S. WilsonDagger ||

From the Dagger  Department of Pathology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, New Mexico, 87107, § Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, and  Department of Biochemistry and Molecular Biology, Monash University, Clayton 3168, Victoria, Australia

Received for publication, January 17, 2001, and in revised form, February 12, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fcepsilon RI-induced Ca2+ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase Cgamma 2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca2+ responses after Fcepsilon RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110beta or p110delta catalytic isoforms of PI 3-kinase, but not antibodies to p110alpha , lengthens the lag time to release of Ca2+ stores and blunts the sustained phase of the calcium response. Ca2+ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, a competitive inhibitor of the Ins(1,4,5)P3 receptor. This indicates a requirement for Ins(1,4,5)P3 to initiate and sustain Ca2+ responses even when PI 3-kinase is fully active. Antigen-induced cell ruffling, a calcium-independent event, is blocked by injection of p110beta and p110delta antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110alpha antibodies. These results suggest that the p110beta and p110delta isoforms of PI 3-kinase support Fcepsilon RI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cross-linking of the high affinity receptor for IgE, Fcepsilon RI, on mast cells results in the activation of tyrosine kinases Lyn and Syk, followed by tyrosine phosphorylation and activation of PLCgamma 1 isoforms and other downstream effectors (1). Subsequent hydrolysis of phosphatidylinositol 4,5-diphosphate by PLCgamma leads to the formation of Ins(1,4,5)P3, the release of Ca2+ from internal stores (2), and the influx of Ca2+ through store-operated channels (3). Calcium influx, in turn, supports degranulation and the release of inflammatory mediators (4). This scenario was complicated in recent years by observations that PLCgamma activation and Ca2+ responses and secretion are all diminished in mast cells treated with inhibitors of PI 3-kinases (5, 6). It was subsequently discovered that full activation of PLCgamma proteins in mast cells requires both tyrosine phosphorylation, potentially mediated by Tec family kinases (7), and interaction with the lipid products of PI 3-kinase (8). PtdIns(3,4,5)P3 mediates membrane recruitment and phosphorylation of PLCgamma 1 in RBL-2H3 cells (8) and increases the activity of both PLCgamma 1 and PLCgamma 2 against lipid micelle substrates in vitro (9, 10).

Despite demonstrated roles for PI 3-kinase in the activation of PLCgamma , its specific contribution to the temporal regulation of Fcepsilon RI-mediated Ca2+ signaling is unresolved. Although wortmannin treatment can slightly increase the lag time leading to the initial rise in calcium after antigen stimulation, it does not block the initial response (10). The most dramatic effects of wortmannin treatment are on the amplitude and duration of the sustained phase of Ca2+ signaling (10). One possible interpretation of this result is that tyrosine phosphorylation of the abundant PLCgamma 2 isoform, which is unaffected by wortmannin treatment, is sufficient stimulus for the initial Ins(1,4,5)P3 production required for rapid release of stores. In this study, we test the hypothesis that full activation of PLCgamma , mediated by PI 3-kinase, is particularly important to sustain Ca2+ responses. This is consistent with evidence in RBL-2H3 cells that activation of the store-operated Ca2+ influx current, ICRAC, requires concentrations of Ins(1,4,5)P3 higher than required to initiate store depletion (11-13). It is important to note that several alternative hypotheses could explain previous observations using wortmannin. First, it is conceivable that PtdIns(3,4,5)P3 can regulate Ca2+ influx through PLCgamma -independent mechanisms. Evidence in T cells suggests that PtdIns(3,4,5)P3 can directly activate Ca2+ channels (14), and work in platelets and megakaryocytes implicates the PtdIns(3,4,5)P3-sensitive Tec family kinase, Btk (15). Second, Choi et al. (16) proposed that the second messenger sphingosine-1-phosphate may regulate Fcepsilon RI-mediated store release, independent of Ins(1,4,5)P3 production. Third, PI 3-kinase-independent effects of wortmannin have been reported (17), raising the possibility that other targets of the inhibitor might be responsible for the inhibitory effects on the calcium responses.

Intracellular phosphoinositide levels are also regulated by a family of inositol polyphosphate 5-phosphatases (18). Activation of p150SHIP, a member of this family that removes phosphate from the 5' position of PtdIns(3,4,5)P3 and the related inositol phosphate, inositol 1,3,4,5-phosphate 4, down-regulates Ca2+ signaling in RBL cells (19). In addition, bone marrow-derived mast cells from p150SHIP-/- mice show an increase in the amplitude of the Ca2+ response in comparison to bone marrow-derived mast cells from their wild type counterparts (20). Less well characterized is the role of the type I 5-phosphatase that regulates the levels of soluble phosphoinositides by removing phosphate from the 5' position of Ins(1,4,5)P3 and inositol 1,3,4,5-phosphate 4. In Chinese hamster ovary cells, overexpression of this enzyme causes an oscillatory Ca2+ response and blocks sustained increases in intracellular Ca2+ levels after purinergic receptor stimulation (21). Recent work with nonhydrolyzable Ins(1,4,5)P3 analogs suggests that this enzyme may limit activation of the store-operated Ca2+ influx current, ICRAC, in RBL cells (13). The type I 5-phosphatase is a potentially useful tool for examining the effect of lowering Ins(1,4,5)P3 levels in cells under conditions in which other phosphoinosotide signaling pathways are fully operational.

In addition to regulating Ca2+ signaling, PI 3-kinase activity regulates receptor-induced actin rearrangements in a number of cell types (22, 23). In the RBL-2H3 cell line, wortmannin-sensitive PI 3-kinase activity is required for the formation of large, actin-based, plasma membrane ruffles at the cell surface after Fcepsilon RI cross-linking (6). Although antigen-stimulated ruffling does not require extracellular Ca2+, it is mimicked by phorbol ester treatment (24) and is thus potentially dependent on PLCgamma activation.

In this report, we show that PtdIns(3,4,5)P3 enhances the activity of PLCgamma 2 that has already acquired the "uncapped" active conformation after in vivo tyrosine phosphorylation or in vitro exposure to low pH. Inhibitory antibodies to the class IA PI 3-kinase catalytic subunits, introduced by microinjection into RBL-2H3 cells, provide further proof that PI 3-kinases mediate the wortmannin-sensitive steps leading to Ca2+ signaling and membrane ruffling in antigen-stimulated RBL-2H3 cells. Consistent with recent evidence that the p110 subunits can have functionally distinct roles in intracellular signaling (22, 25, 26), we find that antibodies to p110beta and p110delta , but not to p110alpha , affect these two specific antigen-stimulated responses, despite the fact that all three subunits associate with tyrosine-phosphorylated proteins after receptor stimulation. Finally, we compare inhibition of Ca2+ signaling in cells injected with PI 3-kinase-inhibitory antibodies to that in cells microinjected with heparin, which blocks binding of Ins(1,4,5)P3 to its receptor (27), or with recombinant inositol polyphosphate 5-phosphatase I, which hydrolyzes Ins(1,4,5)P3. The results show that Fcepsilon RI-mediated calcium responses are absolutely dependent on Ins(1,4,5)P3-mediated gating of its receptors on intracellular stores. They are consistent with a model in which PI 3-kinase lipid products support maximal production of Ins(1,4,5)P3 required for store depletion and do not provide a PLCgamma -independent means to activate calcium influx in RBL-2H3 cells.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Blocking Antibodies and Reagents-- Isoform-specific inhibitory antibodies to the carboxyl termini of the p110alpha and p110beta catalytic subunits of class IA PI 3-kinase (26) were prepared as a 2.5 mg/ml solution in PBS. Commercial antibodies to the carboxyl terminus of the p110delta subunit (residues 1030-1044; PharMingen, San Diego, CA) were dialyzed against 0.1× PBS to remove azide and concentrated by freeze drying and resuspension at 2.5 mg/ml in double-distilled H20. For experiments in which p110beta and p110delta antibodies were coinjected, freeze-dried p110delta was resuspended in a solution of p110beta to yield a preparation containing 2.5 mg/ml of each antibody. Low molecular weight heparin (~3000 average molecular weight; Sigma) was injected as a 25 mg/ml solution in PBS. Fura-2 pentapotassium salt (Molecular Probes, Eugene, OR) was added to injection solutions to a final concentration of 5 mM.

Cell Culture and Stimulation-- For immunoprecipitation-based assays, RBL-2H3 cells were cultured on tissue culture-grade plastic in minimal essential medium (Life Technologies, Inc., Grand Island, NY) supplemented with 15% fetal calf serum (HyClone), penicillin-streptomycin, and L-glutamine. For microinjection experiments, cells were plated overnight onto glass coverslips mounted in Teflon dishes. In all cases, IgE receptors were primed with anti-DNP-IgE (1 µg/ml) for 12-20 h. Cells were washed to remove excess IgE and activated at 37 °C by the addition of polyvalent antigen (0.1 µg/ml DNP-BSA).

Phospholipase Activity-- Phospholipase activity was measured as described previously (10), except that EGTA was omitted from reaction buffers in experiments where pH was varied.

PI 3-Kinase Immunoprecipitation and Immunoblotting-- Adherent cells (107 cells/sample) were stimulated with antigen, washed with cold PBS, and scraped from plates into 1 ml of lysis buffer C (50 mM Tris-HCl, pH 7.2, 1% Brij 96, 150 mM NaCl, 1 mM NaVO4, and protease inhibitor mixture from Boehringer Mannheim (Indianapolis, IN)). Lysates were clarified by centrifugation (15,000 × g) for 5 min and rocked for 2 h at 4 °C with 40 µl of protein A/G-Sepharose bead mixture (1:1) (Pharmacia) prebound to 2 µg of rabbit polyclonal anti-p85 antibodies (Upstate Biotechnology) or p110 isoform-specific antibodies. Immunoprecipitates were washed three times in buffer C, solubilized by heating to 95 °C for 5 min in 1× Laemmli buffer, separated by SDS-PAGE on 7.5% acrylamide gels, and transferred to nitrocellulose. Membranes were blocked with 5% immunoglobulin-free BSA (Sigma) for 1 h at room temperature and incubated for 1 h at room temperature with 1 µg/ml horseradish peroxidase-conjugated PY20 antibody (Transduction Laboratories, Lexington, KY). Membranes were washed and incubated with enhanced chemiluminescence development substrate (Pierce). Blots were then stripped and reprobed with polyclonal p85 antibody, followed by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence development.

5-Phosphatase Purification and Assay-- The cDNA encoding murine Mr 45,000 5-phosphatase was subcloned into the pTrcHis expression plasmid (Invitrogen). His-tagged 5-phosphatase fusion proteins were produced in Escherichia coli after isopropyl-1-thio-beta -D-galactopyranoside induction and purified on Ni-NTAG-agarose (Qiagen) according to the manufacturer's instructions. Purified protein was dialyzed against phosphate buffer, yielding a preparation for microinjection containing 0.35 mg/ml (determined by the Bio-Rad Coomassie Blue protein assay kit) and a single prominent band of Mr 51,000 by SDS-PAGE. Phosphatase activity was confirmed in vitro, as described by Ono et al. (28).

Microinjection-- Microinjection was performed at room temperature with an Eppendorf semiautomated microinjector and micromanipulator (Eppendorf, Madison, WI) mounted on a Zeiss IM35 microscope equipped with a CO2 perfusion system. Injection needles were pulled with a vertical pipette puller (David Kopf Instruments, Tujunga, CA). Cells were allowed to recover for 30 min at room temperature after microinjection, before warming to 37 °C and the addition of antigen. Cells judged to contain morphological abnormalities after the recovery period were excluded from further analysis.

Ca2+ Imaging-- As described above, cells were microinjected with 5 mM solutions of fura-2-free acid plus or minus inhibitory reagents. After recovery, ratio imaging was performed at 37 °C on a Zeiss IM35 microscope equipped with a stage heater and a CO2 perfusion system (Medical Systems Corp., Greenvale, NY), a Ludl filter wheel, and a Dage-MTI intensified video charge-coupled device camera interfaced to Simca imaging software (Compix Inc., Cranberry Township, PA). Responses were measured from groups of three to eight injected cells, in a single field of view, per experiment. All Ca2+ experiments were performed in Hank's balanced salt solution. Calibration of background-corrected ratio values (350 nm/380 nm) against standard solutions in vitro was done as described previously (29). Analysis of data and graphing were performed with Graphpad Prism software.

Fluorescence Microscopy-- Microinjected, IgE-primed cells were activated for 10 min with 100 ng/ml DNP-BSA and then fixed with 2% paraformaldehyde for 20 min. Cells were permeabilized with 0.1% Triton X-100, followed by sequential staining with 1 µg/ml fluorescein isothiocyanate-conjugated anti-rabbit IgG (to identify injected cells; Cappell, Westchester, PA) and rhodamine phalloidin (Molecular Probes) in PBS with 0.1% BSA. Cells were mounted in Vectashield (Vector Laboratories, Burlington, CA) and viewed on a Zeiss IM35 microscope. Images were captured with a Photometrics CH250 cooled charge-coupled device camera interfaced to Compix imaging software. Figures were assembled with Adobe Photoshop software.

    RESULTS
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INTRODUCTION
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To confirm and extend our previous results demonstrating enhancement of PLCgamma activity by the inclusion of PtdIns(3,4,5)P3 in lipid micelle substrates (8), we have investigated the effect of PtdIns(3,4,5)P3 on PLCgamma 2 activated in vivo by receptor stimulation and in vitro by pH-dependent conformational change. As demonstrated in Fig. 1A, PtdIns(3,4,5)P3 is a weak stimulator of nonphosphorylated PLCgamma 2 that has been immunoprecipitated from resting RBL-2H3 cells. However, even small amounts of PtdIns(3,4,5)P3 markedly increase the activity of PLCgamma 2 that was tyrosine-phosphorylated after Fcepsilon RI cross-linking and immunoprecipitated from activated cells. Based on recent models that propose "uncapping" as the means by which either tyrosine phosphorylation or low pH activates PLCgamma (30), we next tested the effect of adding PtdIns(3,4,5)P3 to phospholipase reactions conducted at pH 5. Results in Fig. 1B show that PtdIns(3,4,5)P3 also augments PLCgamma 2 activated by low pH and suggest that PtdIns(3,4,5)P3 activates PLCgamma 2 by a mechanism distinct from uncapping.


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Fig. 1.   Activation of immunoprecipitated PLCgamma 2 by PtdIns(3,4,5)P3. A, PLCgamma 2 immunoprecipitates prepared from resting and activated RBL-2H3 cells were incubated with 200 µM [3H]phosphatidylinositol 4,5-diphosphate in the presence of the indicated concentrations of PtdIns(3,4,5)P3. B, PLCgamma 2 immunoprecipitates were incubated at either pH 5 or pH 6.8 with 200 µM [3H]phosphatidylinositol 4,5-diphosphate and the indicated concentrations of PtdIns(3,4,5)P3. Data represent the average of duplicate assays in a single, representative experiment. Experiments were repeated at least three times using cell lysates prepared on separate occasions.

Results in Fig. 1 confirm a role for PI 3-kinase lipid products in the direct activation of PLCgamma but do not identify the specific form of PI 3-kinase involved in Fcepsilon RI signaling. Because RBL-2H3 cells express all three catalytic p110 isoforms of class IA PI 3-kinase, commercial isoform-specific antibodies were used to immunoprecipitate PI 3-kinase heterodimers from resting and antigen-stimulated cells. Tyrosine-phosphorylated proteins associated with these immune complexes were identified after SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 2A, all three PI 3-kinases isolated from resting cells associate with a prominent tyrosine-phosphorylated protein migrating at an approximate molecular weight of 100,000. Within 2 and 5 min after stimulation, new tyrosine-phosphorylated species appear at Mr ~85,000, Mr 42,000, and Mr 27,000. The profile of tyrosine-phosphorylated proteins is similar but not identical in p85 immunoprecipitates. Blots were stripped and reprobed with anti-p85 antibodies (Fig. 2B). The amount of p85 precipitated with anti-p110alpha antibodies was consistently less than that precipitated with the other two isoform-specific antibodies, suggesting that p110alpha is the least abundant isoform in RBL-2H3 cells.


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Fig. 2.   Class IA PI 3-kinase catalytic subunits associate with similar tyrosine-phosphorylated proteins in RBL cells after Fcepsilon RI cross-linking. Cells were activated for the indicated times with 0.1 µg/ml DNP-BSA and then lysed. Lysates were immunoprecipitated with the indicated antibody to the p110 or p85 subunits of class IA PI 3-kinase, separated by SDS-PAGE, and then Western blotted for phosphotyrosine (A) or p85 (B).

Several reports have described effective inhibition of catalytic activity by antibodies raised to specific sequences in the carboxyl termini of p110alpha , p110beta , and p100delta (22, 26, 31). We introduced anti-carboxyl-terminal inhibitory antibodies into RBL-2H3 cells to identify the class IA PI 3-kinase p110 isoforms that regulate Ca2+ signaling after Fcepsilon RI cross-linking. Inhibitory antibodies to p110alpha and p110beta used here have been described previously (22, 26). Antibodies to p110delta , which were raised against the same peptide as antibodies used to inhibit p110delta in other studies (31), were demonstrated to inhibit p110delta activity in vitro (data not shown). Fig. 3, A---E, shows representative traces from control cells (Fig. 3A, cells injected with fura-2 alone) compared with cells microinjected with the isoform-specific, anti-p110 antibodies (Fig. 3, B-E). Whereas the blocking antibodies to p110alpha have no significant effect on the overall Ca2+ response (Fig. 3B), the presence of either p110beta (Fig. 3C) or p110delta (Fig. 3D) blocking antibodies results in diminished Ca2+ responses, including a delayed onset after stimulus, a blunted sustained phase and oscillations. Injection of the combination of both p110beta and p110delta antibodies dramatically reduces antigen-stimulated Ca2+ mobilization, resulting in a weak, transient response (Fig. 3E). Fig. 3F reports the average area under the curve for multiple experiments, where Ca2+i is plotted against time after activation.


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Fig. 3.   Inhibition of Ca2+ signaling in cells microinjected with PI 3-kinase class IA isotype-specific blocking antibodies. A-E, representative traces from three cells microinjected with fura-2 alone (control) or fura-2 and the indicated antibodies as described under "Experimental Procedures." F, area under the curve (from at least 17 cells for each condition), defined as the change above baseline over a 10-min period. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.0001 versus control by t test.

Coverslips containing antibody-injected cells were fixed immediately after Ca2+ imaging had been performed. To determine the effects of the microinjected antibodies on plasma membrane ruffling, cells were permeabilized by a brief detergent treatment and stained with rhodamine phalloidin (which labels filamentous actin in all cells) and fluorescein isothiocyanate-conjugated anti-rabbit IgG (which identifies injected cells). Fluorescence micrographs in Fig. 4 show that antibodies to p110beta and p110delta both inhibited the formation of actin-based plasma membrane ruffles in activated cells, whereas antibodies to p110alpha had little or no effect. Injection of inhibitory antibodies did not prevent the formation of actin plaques on the ventral surface of activated cells (data not shown), in agreement with results obtained using the PI 3-kinase inhibitor wortmannin (6).


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Fig. 4.   Inhibition of Fcepsilon RI-induced actin ruffling in cells microinjected with class IA PI 3-kinase isotype-specific blocking antibodies. Left panels show rhodamine phalloidin staining of cells fixed 10 min after Fcepsilon RI cross-linking; right panels show fluorescein isothiocyanate anti-rabbit staining, indicating injected cells. A, cells injected with anti-p110alpha ; B, cells injected with anti-p110beta ; C, cells injected with anti-p110delta . *, injected cells that failed to form actin ruffles after activation. Results were confirmed in at least three separate experiments.

To investigate the Ins(1,4,5)P3 dependence of antigen-stimulated calcium responses, RBL-2H3 cells were microinjected with recombinant inositol polyphosphate 5-phosphatase I. The 5-phosphatase was produced as a His-tagged protein in E. coli and purified by nickel chromatography. The purity of the enzyme was greater than 90%, based on analysis of Coomassie Blue-stained SDS-PAGE gels, and inositol 5-phosphatase activity was confirmed using [3H]inositol 1,3,4,5-tetrakis-phosphate as a substrate (data not shown). Representative traces of Ca2+ responses after Fcepsilon RI cross-linking in control-injected and 5-phosphatase I-injected cells are shown in Fig. 5, A and B. These results demonstrate that high levels of 5-phosphatase inhibit the sustained phase of Fcepsilon RI-induced Ca2+ signaling, resulting in a single initial spike of calcium release (Fig. 5B). The overall magnitude of the Ca2+ response in 5-phosphatase-injected cells was significantly inhibited compared with the response of control cells (Fig. 5C). The inhibition of Ca2+ responses caused by 5-phosphatase injection was similar to that seen by injection of inhibitory antibodies to p110beta and p110delta (Fig. 3). However, direct observation of injected cells after Ca2+ imaging revealed that 5-phosphatase injection did not affect the plasma membrane ruffling response (data not shown).


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Fig. 5.   Microinjection of recombinant Mr 43,000 5-phosphatase inhibits the sustained phase of Fcepsilon RI-induced Ca2+ signaling. Representative traces are shown from control-injected cells (A) or Mr 43,000 5-phosphatase-injected cells (B). The average area under the curve from at least 20 cells from each condition, defined as the change above baseline over a 10-min period, is shown in C. *, p < 0.001 versus control by t test.

Heparin is a competitive inhibitor of the Ins(1,4,5)P3 receptor that blocks Ins(1,4,5)P3-mediated store release (27). Ca2+ responses in heparin-microinjected RBL-2H3 cells were either completely ablated (Fig. 6B, top panel) or reduced to a single initial spike (Fig. 6B, bottom panels), presumably reflecting a brief pulse of intracellular store release. In this series of experiments, complete inhibition of the Ca2+ signal was seen in ~30% of heparin-injected cells. The SERCA inhibitor thapsigargin was added 10 min after stimulation with antigen to passively deplete internal stores and rule out possible direct effects of heparin on the store-operated influx pathway. Thapsigargin induced a sustained Ca2+ response in heparin-injected cells, demonstrating that the store-operated pathway was functionally intact and apparently ruling out any direct role for binding of Ins(1,4,5)P3 to its receptor in the store-operated influx pathway. Thapsigargin induced a secondary response in antigen-stimulated control cells, suggesting that some refilling of internal Ca2+ stores can occur during continuous antigen stimulation. Direct observation of injected cells after Ca2+ imaging revealed that heparin did not affect the ruffling response induced by Fcepsilon RI cross-linking, even in cells displaying no Ca2+ response (data not shown). These results demonstrate that neither heparin nor 5-phosphatase I interferes with global events downstream of PI 3-kinase and support earlier observations that membrane ruffling is not dependent on elevations in intracellular Ca2+ (24).


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Fig. 6.   Inhibition of antigen-induced Ca2+ signaling by heparin microinjection. Cells were microinjected with fura-2 alone (control, A) or fura-2 and heparin (B) as described under "Experimental Procedures." Cells were then activated by the addition of 100 ng/ml DNP-BSA as indicated, followed by the addition of 100 nM thapsigargin 10 min later. Traces are representative of results obtained from at least 20 cells in four separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tyrosine phosphorylation of PLCgamma isoforms was first demonstrated to occur downstream of epidermal growth factor receptor activation (32). This led to a simple model for PLCgamma activation downstream of tyrosine kinase signaling cascades and, in the Fcepsilon RI system, was supported by early evidence that PLCgamma proteins were rapidly tyrosine-phosphorylated after antigen stimulation (1) (33). Unexpectedly, we showed that PI 3-kinase inhibitors markedly reduced the antigen-stimulated production of Ins(1,4,5)P3 (6), an observation explained in part by inhibition of the translocation and tyrosine phosphorylation of the less abundant PLCgamma 1 isoform (8) and by direct activation of both PLCgamma isoforms by the PI 3-kinase lipid product PtdIns(3,4,5)P3 (9, 10). Here, we compared the ability of PtdIns(3,4,5)P3 to enhance enzymatic activity of PLCgamma 2 immunoprecipitated from resting and stimulated cells, demonstrating that PtdIns(3,4,5)P3 is a more effective stimulator of tyrosine-phosphorylated PLCgamma than of enzyme in its basal state (Fig. 1A). We also tested the ability of PtdIns(3,4,5)P3 to stimulate PLCgamma activity measured at pH 5.0, where a pH-dependent conformational change "uncaps" the active site of the enzyme (30). Consistent with the model that tyrosine phosphorylation also uncaps the active site, enzymatic activity of basal state PLCgamma is elevated at pH 5.0 and comparable to that of phosphorylated enzyme at either pH 5.0 or pH 6.8. The addition of PtdIns(3,4,5)P3 to the mixed micelle reaction results in enhanced activity at both pH conditions (Fig. 1B), suggesting that PtdIns(3,4,5)P3 regulates PLCgamma 2 through a distinct mechanism.

Having established direct effects of PI 3-kinase lipid products on PLCgamma activity, we next focused on the consequences of PI 3-kinase activation on overall Ca2+ responses in antigen-stimulated RBL-2H3 cells. We found that Ca2+ profiles in cells injected with antibodies to p110beta and p110delta are remarkably similar to the response of cells treated with low nanomolar concentrations of the PI 3-kinase inhibitor wortmannin (10). These data rule out the possibility that wortmannin inhibits Ca2+ mobilization by PI 3-kinase-independent means. Both approaches lead to a reduction in the amplitude of the sustained phase of the Ca2+ response and to an increase in the lag time to response after antigen addition.

Previous data suggested that the PI 3-kinase D3-phosphatidylinositol product, Ptd(3,4,5)P3, might independently regulate Ca2+ influx in RBL-2H3 cells (10). However, our earlier experiments assessing the state of calcium stores in wortmannin-treated RBL-2H3 cells were performed after a solution exchange to calcium-free medium, a condition that did not permit the refilling of stores (10). Here, we compared antigen-stimulated calcium responses in cells that were injected with either inhibitory PI 3-kinase antibodies (to inhibit PI 3-kinase contributions to PLCgamma activation), heparin (to block Ins(1,4,5)P3 receptor binding), or 5-phosphatase I (to promote Ins(1,4,5)P3 degradation), all in the presence of extracellular calcium, to facilitate store refilling. It is particularly revealing that microinjection of 5-phosphatase I markedly inhibits the extent and duration of store depletion necessary to support Ca2+ influx, giving results that are very similar to those seen after inhibition of PI 3-kinase. The best explanation is that the sustained store-operated calcium influx phase, and not the initial spike of stores release, is dependent on maintaining high rates of Ins(1,4,5)P3 synthesis and/or low rates of degradation.

This conclusion agrees with recent work on the properties of the Ca2+ influx current, ICRAC, in RBL cells. First identified as store-operated Ca2+ channels in mast cells by Hoth and Penner (34), ICRAC is highly selective for calcium and is activated in mast cells when intracellular Ca2+ stores are depleted by dialysis with Ca2+ chelators or Ins(1,4,5)P3, by treatment of cells with ionomycin or thapsigargin, or by antigen stimulation (3, 34). Essentially identical ICRAC currents can be recorded in both Jurkat T cells (35, 36) and RBL cells (37). Importantly, ICRAC current induced by Ins(1,4,5)P3 dialysis via the patch clamp requires relatively high concentrations of Ins(1,4,5)P3 (12). The sensitivity of ICRAC current to Ins(1,4,5)P3 levels is not linear but rather resembles an all or nothing phenomenon (12). In the nanomolar to low micromolar range, calcium store release can be measured in the absence of influx through ICRAC. Recent work from the Parekh laboratory suggests that the threshold reflects the state of the Ins(1,4,5)P3-sensitive stores, which can initially be mobilized by submaximal concentrations of Ins(1,4,5)P3 but are subsequently limited by negative feedback regulation or spatial restrictions, curtailing Ca2+ efflux through the Ins(1,4,5)P3-gated channel (11). Recent experiments of Glitsch and Parekh (13) using the nonmetabolizable analogue inositol 2,4,5-triphosphate suggest that dephosphorylation of Ins(1,4,5)P3 by endogenous 5-phosphatase I is likely to be one important negative feedback pathway that limits store-operated ICRAC current.

Calcium responses are either abolished or consist of only a brief calcium spike in cells microinjected with heparin (Fig. 6). These data suggest that Ins(1,4,5)P3 may be solely responsible for Fcepsilon RI-mediated release of internal Ca2+ stores. Thapsigargin challenge demonstrated that calcium influx, per se, is not inhibited by heparin injection because thapsigargin-mediated responses are indistinguishable from controls. A potential explanation for the Ins(1,4,5)P3 dependence of Ca2+ influx is provided by recent work on the transient receptor potential (trp) family of Ca2+ channels. Six mammalian trp isoforms have been identified as potential store-operated channels. Although the electrophysiological properties of overexpressed, recombinant trp channels do not resemble those of ICRAC (38), it remains possible that heterotetramers of different family members, expressed at a low level, may form the ICRAC channel. An early report detected trp2 and trp5 expression in RBL-2H3 cells by reverse transcription-polymerase chain reaction analysis (39). Recent studies have demonstrated that trp1 and trp3 couple to Ins(1,4,5)P3 receptors (40, 41) and that this coupling is required for activation of trp3 (40). Others have observed that Ins(1,4,5)P3 receptors may detect the state of calcium stores in a manner that regulates the formation of this complex (42). We demonstrate here that heparin microinjection does not inhibit thapsigargin-mediated Ca2+ influx. This suggests that if the Ins(1,4,5)P3 receptor acts as the sensor of store depletion, it does so independently of Ins(1,4,5)P3 binding.

We showed previously that the type II Ins(1,4,5)P3 receptors of RBL cells cluster in the endoplasmic reticulum within minutes of calcium mobilization induced by antigen, inonomycin, or thapsigargin treatment (43). We speculate that this dramatic redistribution results in the formation of Ca2+ pools within the ER whose efflux is limited by distance from the Ins(1,4,5)P3 receptor clusters. Although it does not preclude the influence of other feedback mechanisms operating on Ins(1,4,5)P3 receptors (44), it offers one potential explanation for increased dependence on high Ins(1,4,5)P3 levels during the latter, but not the early, phase of the calcium response.

Based on antigen-stimulated increases in tyrosine-phosphorylated proteins found in immune complexes prepared using each of the three class IA PI 3-kinase-specific antibodies, all of the p110/p85 heterodimers are likely to function in some aspect of Fcepsilon RI signaling. Nevertheless, only antibodies to the p110beta and p110delta catalytic subunits blocked Ca2+ responses and ruffling after antigen stimulation. Absence of inhibition with antibodies to p110alpha may have several explanations. Based on semiquantitative analysis by immunoprecipitation and immunoblotting comparisons (Fig. 2), p110alpha appears to be the least abundant class IA isoform in RBL-2H3 cells. Thus, the low expression levels may be a simple explanation for the lack of inhibition using blocking p110alpha antibodies. Alternatively, distinct functions of the class IA PI 3-kinases may reflect intrinsic differences in the isoforms that mediate differential coupling to specific receptors and/or downstream effectors. An important component of coupling specificity may be the p85 isoform present in the PI 3-kinase heterodimer, as suggested the recent report that cKit signaling, but not that of Fcepsilon RI, is blocked in p85alpha -deficient bone marrow-derived mast cells (45).

In conclusion, our results demonstrate that class IA PI 3-kinase isoforms p110beta and p110delta act synergistically to regulate Fcepsilon RI-induced Ca2+ signaling and plasma membrane ruffling. The principal role for these PI 3-kinases in mast cell Ca2+ responses is likely to be the enhancement of PLCgamma activity by PtdIns(3,4,5)P3-mediated translocation and phosphorylation of PLCgamma 1 (8) and the direct activation of both PLCgamma isoforms (10).

    ACKNOWLEDGEMENTS

We thank Dr. Janet Oliver for critical reading of the manuscript. Use of the Fluorescence Microscopy Facility at the University of New Mexico Cancer Research Facility is gratefully acknowledged.

    FOOTNOTES

* This work was supported by American Cancer Society Grant RPG-99-233-01-CIM (to B. S. W.).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: University of New Mexico Cancer Research Facility, Rm. 205, Albuquerque, NM 87131. Tel.: 505-272-8852; Fax: 505-272-1435; E-mail: bwilson@thor.unm.edu.

Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M100417200

    ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; PI 3-kinase, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; trp, transient receptor potential; DNP, dinitrophenol.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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