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• W1982893604 abstract "FcεRI-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 Cγ2 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 FcεRI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110β or p110δ catalytic isoforms of PI 3-kinase, but not antibodies to p110α, 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)P3receptor. 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 p110β and p110δ antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110α antibodies. These results suggest that the p110β and p110δ isoforms of PI 3-kinase support FcεRI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel. FcεRI-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 Cγ2 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 FcεRI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110β or p110δ catalytic isoforms of PI 3-kinase, but not antibodies to p110α, 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)P3receptor. 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 p110β and p110δ antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110α antibodies. These results suggest that the p110β and p110δ isoforms of PI 3-kinase support FcεRI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel. phospholipase C phosphatidylinositol 3-kinase phosphate-buffered saline bovine serum albumin SDS-polyacrylamide gel electrophoresis 4,5)P3, inositol 1,4,5-trisphosphate 4,5)P3, phosphatidylinositol 3,4,5-triphosphate transient receptor potential dinitrophenol Cross-linking of the high affinity receptor for IgE, FcεRI, on mast cells results in the activation of tyrosine kinases Lyn and Syk, followed by tyrosine phosphorylation and activation of PLCγ1 isoforms and other downstream effectors (1Park D.J. Min H.K. Rhee S.G. J. Biol. Chem. 1991; 266: 24237-24240Abstract Full Text PDF PubMed Google Scholar). Subsequent hydrolysis of phosphatidylinositol 4,5-diphosphate by PLCγ leads to the formation of Ins(1,4,5)P3, the release of Ca2+ from internal stores (2Streb H. Irvine R.F. Berridge M.J. Schulz I. Nature. 1983; 306: 67-69Crossref PubMed Scopus (1797) Google Scholar), and the influx of Ca2+ through store-operated channels (3Zhang L. McCloskey M.A. J. Physiol. ( Lond. ). 1995; 483: 59-66Crossref PubMed Scopus (68) Google Scholar). Calcium influx, in turn, supports degranulation and the release of inflammatory mediators (4Siraganian R.P. Kulczycki A.J. Mendoza G. Metzger H. J. Immunol. 1975; 115: 1599-1602PubMed Google Scholar). This scenario was complicated in recent years by observations that PLCγ activation and Ca2+ responses and secretion are all diminished in mast cells treated with inhibitors of PI 3-kinases (5Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar, 6Barker S.A. Caldwell K.K. Hall A. Martinez A.M. Pfeiffer J.R. Oliver J.M. Wilson B.S. Mol. Biol. Cell. 1995; 6: 1145-1158Crossref PubMed Scopus (124) Google Scholar). It was subsequently discovered that full activation of PLCγ proteins in mast cells requires both tyrosine phosphorylation, potentially mediated by Tec family kinases (7Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar), and interaction with the lipid products of PI 3-kinase (8Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 9: 483-496Crossref PubMed Scopus (95) Google Scholar). PtdIns(3,4,5)P3 mediates membrane recruitment and phosphorylation of PLCγ1 in RBL-2H3 cells (8Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 9: 483-496Crossref PubMed Scopus (95) Google Scholar) and increases the activity of both PLCγ1 and PLCγ2 against lipid micelle substratesin vitro (9Bae Y.S. Cantley L.G. Chen C.S. Kim S.R. Kwon K.S. Rhee S.G. J. Biol. Chem. 1998; 273: 4465-4469Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 10Barker S.A. Lujan D. Wilson B.S. J. Leukocyte Biol. 1999; 65: 321-329Crossref PubMed Scopus (73) Google Scholar). Despite demonstrated roles for PI 3-kinase in the activation of PLCγ, its specific contribution to the temporal regulation of FcεRI-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 (10Barker S.A. Lujan D. Wilson B.S. J. Leukocyte Biol. 1999; 65: 321-329Crossref PubMed Scopus (73) Google Scholar). The most dramatic effects of wortmannin treatment are on the amplitude and duration of the sustained phase of Ca2+ signaling (10Barker S.A. Lujan D. Wilson B.S. J. Leukocyte Biol. 1999; 65: 321-329Crossref PubMed Scopus (73) Google Scholar). One possible interpretation of this result is that tyrosine phosphorylation of the abundant PLCγ2 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 PLCγ, 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 (11Fierro L. Parekh A.B. J. Physiol. ( Lond. ). 2000; 522: 247-257Crossref PubMed Scopus (66) Google Scholar, 12Parekh A.B. Fleig A. Penner R. Cell. 1997; 89: 973-980Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 13Glitsch M.D. Parekh A.B. J. Physiol. ( Lond. ). 2000; 523: 283-290Crossref PubMed Scopus (35) Google Scholar). It is important to note that several alternative hypotheses could explain previous observations using wortmannin. First, it is conceivable that PtdIns(3,4,5)P3can regulate Ca2+ influx through PLCγ-independent mechanisms. Evidence in T cells suggests that PtdIns(3,4,5)P3 can directly activate Ca2+channels (14Hsu A.L. Ching T.T. Sen G. Wang D.S. Bondada S. Authi K.S. Chen C.S. J. Biol. Chem. 2000; 275: 16242-16250Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and work in platelets and megakaryocytes implicates the PtdIns(3,4,5)P3-sensitive Tec family kinase, Btk (15Pasquet J.M. Quek L. Stevens C. Bobe R. Huber M. Duronio V. Krystal G. Watson S.P. EMBO J. 2000; 19: 2793-2802Crossref PubMed Scopus (75) Google Scholar). Second, Choi et al. (16Choi O.H. Kim J.H. Kinet J.P. Nature. 1996; 380: 634-636Crossref PubMed Scopus (385) Google Scholar) proposed that the second messenger sphingosine-1-phosphate may regulate FcεRI-mediated store release, independent of Ins(1,4,5)P3 production. Third, PI 3-kinase-independent effects of wortmannin have been reported (17Downing G.J. Kim S. Nakanishi S. Catt K.J. Balla T. Biochemistry. 1996; 35: 3587-3594Crossref PubMed Scopus (104) Google Scholar), 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 (18Majerus P.W. Genes Dev. 1996; 10: 1051-1053Crossref PubMed Scopus (50) Google Scholar). 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 (19Malbec O. Fong D.C. Turner M. Tybulewicz V.L. Cambier J.C. Fridman W.H. Daeron M. J. Immunol. 1998; 160: 1647-1658PubMed Google Scholar). 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 (20Huber M. Helgason C.D. Damen J.E. Liu L. Humphries R.K. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11330-11335Crossref PubMed Scopus (285) Google Scholar). 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 (21De Smedt F. Missiaen L. Parys J.B. Vanweyenberg V. De Smedt H. Erneux C. J. Biol. Chem. 1997; 272: 17367-17375Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 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 (13Glitsch M.D. Parekh A.B. J. Physiol. ( Lond. ). 2000; 523: 283-290Crossref PubMed Scopus (35) Google Scholar). 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 (22Siddhanta U. McIlroy J. Shah A. Zhang Y. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Crossref PubMed Scopus (138) Google Scholar, 23Johanson S.O. Naccache P.A. Crouch M.F. Exp. Cell Res. 1999; 248: 223-233Crossref PubMed Scopus (16) Google Scholar). 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 FcεRI cross-linking (6Barker S.A. Caldwell K.K. Hall A. Martinez A.M. Pfeiffer J.R. Oliver J.M. Wilson B.S. Mol. Biol. Cell. 1995; 6: 1145-1158Crossref PubMed Scopus (124) Google Scholar). Although antigen-stimulated ruffling does not require extracellular Ca2+, it is mimicked by phorbol ester treatment (24Pfeiffer J.R. Seagrave J.C. Davis B.H. Deanin G.G. Oliver J.M. J. Cell Biol. 1985; 101: 2145-2155Crossref PubMed Scopus (184) Google Scholar) and is thus potentially dependent on PLCγ activation. In this report, we show that PtdIns(3,4,5)P3 enhances the activity of PLCγ2 that has already acquired the “uncapped” active conformation after in vivo tyrosine phosphorylation orin 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 (22Siddhanta U. McIlroy J. Shah A. Zhang Y. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Crossref PubMed Scopus (138) Google Scholar, 25Hooshmand-Rad R. Hajkova L. Klint P. Karlsson R. Vanhaesebroeck B. Claesson-Welsh L. Heldin C.H. J. Cell Sci. 2000; 113: 207-214Crossref PubMed Google Scholar, 26Hill K. Welti S., Yu, J. Murray J.T. Yip S.C. Condeelis J.S. Segall J.E. Backer J.M. J. Biol. Chem. 2000; 275: 3741-3744Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), we find that antibodies to p110β and p110δ, but not to p110α, 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 (27Hill T.D. Berggren P.O. Boynton A.L. Biochem. Biophys. Res. Commun. 1987; 149: 897-901Crossref PubMed Scopus (112) Google Scholar), or with recombinant inositol polyphosphate 5-phosphatase I, which hydrolyzes Ins(1,4,5)P3. The results show that FcεRI-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 PLCγ-independent means to activate calcium influx in RBL-2H3 cells. Isoform-specific inhibitory antibodies to the carboxyl termini of the p110α and p110β catalytic subunits of class IA PI 3-kinase (26Hill K. Welti S., Yu, J. Murray J.T. Yip S.C. Condeelis J.S. Segall J.E. Backer J.M. J. Biol. Chem. 2000; 275: 3741-3744Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) were prepared as a 2.5 mg/ml solution in PBS. Commercial antibodies to the carboxyl terminus of the p110δ 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 p110β and p110δ antibodies were coinjected, freeze-dried p110δ was resuspended in a solution of p110β 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. 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 was measured as described previously (10Barker S.A. Lujan D. Wilson B.S. J. Leukocyte Biol. 1999; 65: 321-329Crossref PubMed Scopus (73) Google Scholar), except that EGTA was omitted from reaction buffers in experiments where pH was varied. 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 mmNaCl, 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. 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-β-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. (28Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (648) Google Scholar). 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. 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 (29Lee R.J. Oliver J.M. Mol. Biol. Cell. 1995; 6: 825-839Crossref PubMed Scopus (41) Google Scholar). Analysis of data and graphing were performed with Graphpad Prism software. 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. To confirm and extend our previous results demonstrating enhancement of PLCγ activity by the inclusion of PtdIns(3,4,5)P3 in lipid micelle substrates (8Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 9: 483-496Crossref PubMed Scopus (95) Google Scholar), we have investigated the effect of PtdIns(3,4,5)P3 on PLCγ2 activated in vivo by receptor stimulation andin vitro by pH-dependent conformational change. As demonstrated in Fig. 1 A,PtdIns(3,4,5)P3 is a weak stimulator of nonphosphorylated PLCγ2 that has been immunoprecipitated from resting RBL-2H3 cells. However, even small amounts of PtdIns(3,4,5)P3 markedly increase the activity of PLCγ2 that was tyrosine-phosphorylated after FcεRI 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 PLCγ (30Zhou C. Horstman D. Carpenter G. Roberts M.F. J. Biol. Chem. 1999; 274: 2786-2793Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), we next tested the effect of adding PtdIns(3,4,5)P3 to phospholipase reactions conducted at pH 5. Results in Fig.1 B show that PtdIns(3,4,5)P3 also augments PLCγ2 activated by low pH and suggest that PtdIns(3,4,5)P3 activates PLCγ2 by a mechanism distinct from uncapping. Results in Fig. 1 confirm a role for PI 3-kinase lipid products in the direct activation of PLCγ but do not identify the specific form of PI 3-kinase involved in FcεRI 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.2 A, 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, andMr 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. 2 B). The amount of p85 precipitated with anti-p110α antibodies was consistently less than that precipitated with the other two isoform-specific antibodies, suggesting that p110α is the least abundant isoform in RBL-2H3 cells. Several reports have described effective inhibition of catalytic activity by antibodies raised to specific sequences in the carboxyl termini of p110α, p110β, and p100δ (22Siddhanta U. McIlroy J. Shah A. Zhang Y. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Crossref PubMed Scopus (138) Google Scholar, 26Hill K. Welti S., Yu, J. Murray J.T. Yip S.C. Condeelis J.S. Segall J.E. Backer J.M. J. Biol. Chem. 2000; 275: 3741-3744Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Crossref PubMed Scopus (201) Google Scholar). 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 FcεRI cross-linking. Inhibitory antibodies to p110α and p110β used here have been described previously (22Siddhanta U. McIlroy J. Shah A. Zhang Y. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Crossref PubMed Scopus (138) Google Scholar, 26Hill K. Welti S., Yu, J. Murray J.T. Yip S.C. Condeelis J.S. Segall J.E. Backer J.M. J. Biol. Chem. 2000; 275: 3741-3744Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Antibodies to p110δ, which were raised against the same peptide as antibodies used to inhibit p110δ in other studies (31Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Crossref PubMed Scopus (201) Google Scholar), were demonstrated to inhibit p110δ activity in vitro(data not shown). Fig. 3,A—E, shows representative traces from control cells (Fig.3 A, 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 p110α have no significant effect on the overall Ca2+ response (Fig.3 B), the presence of either p110β (Fig. 3 C) or p110δ (Fig. 3 D) 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 p110β and p110δ antibodies dramatically reduces antigen-stimulated Ca2+ mobilization, resulting in a weak, transient response (Fig. 3 E). Fig. 3 F reports the average area under the curve for multiple experiments, where Ca2+ i is plotted against time after activation. 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 p110β and p110δ both inhibited the formation of actin-based plasma membrane ruffles in activated cells, whereas antibodies to p110α 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 (6Barker S.A. Caldwell K.K. Hall A. Martinez A.M. Pfeiffer J.R. Oliver J.M. Wilson B.S. Mol. Biol. Cell. 1995; 6: 1145-1158Crossref PubMed Scopus (124) Google Scholar). 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 FcεRI 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 FcεRI-induced Ca2+ signaling, resulting in a single initial spike of calcium release (Fig.5 B). The overall magnitude of the Ca2+ response in 5-phosphatase-injected cells was significantly inhibited compared with the response of control cells (Fig. 5 C). The inhibition of Ca2+ responses caused by 5-phosphatase injection was similar to that seen by injection of inhibitory antibodies to p110β and p110δ (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). Heparin is a competitive inhibitor of the Ins(1,4,5)P3receptor that blocks Ins(1,4,5)P3-mediated store release (27Hill T.D. Berggren P.O. Boynton A.L. Biochem. Biophys. Res. Commun. 1987; 149: 897-901Crossref PubMed Scopus (112) Google Scholar). Ca2+ responses in heparin-microinjected RBL-2H3 cells were either completely ablated (Fig.6 B, top panel) or reduced to a single initial spike (Fig. 6 B, 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 FcεRI 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+ (24Pfeiffer J.R. Seagrave J.C. Davis B.H. Deanin G.G. Oliver J.M. J. Cell Biol. 1985; 101: 2145-2155Crossref PubMed Scopus (184) Google Scholar). Tyrosine phosphorylation of PLCγ isoforms was first demonstrated to occur downstream of epidermal growth factor receptor activation (32Wahl M.I. Jones G.A. Nishibe S. Rhee S.G. Carpenter G. J. Biol. Chem. 1992; 267: 10447-10456Abstract Full Text PDF PubMed Google Scholar). This led to a simple model for PLCγ activation downstream of tyrosine kinase signaling cascades and, in the FcεRI system, w" @default.
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