The Phospholipase Cγ1-dependent Pathway of FcϵRI-mediated Mast Cell Activation Is Regulated Independently of Phosphatidylinositol 3-Kinase
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m301350200
ISSN1083-351X
AutoresChristine Tkaczyk, Michael A. Beaven, Saskia M. Brachman, Dean D. Metcalfe, Alasdair M. Gilfillan,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMast cell degranulation following FcϵRI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)γ activation. Here we report evidence that the PLCγ1-dependent pathway of FcϵRI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, FcϵRI aggregation induced a rapid translocation and phosphorylation of PLCγ1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of FcϵRI-mediated PLCγ activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLCγ activation. Wortmannin, however, did produce a partial (∼50%) concentration-dependent inhibition of FcϵRI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85α and p85β subunits of PI 3-kinase, also revealed no defects in FcϵRI-mediated PLCγ1 activation. These data are consistent with the conclusion that the PLCγ-dependent component of FcϵRI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of FcϵRI-mediated degranulation in human mast cells. Mast cell degranulation following FcϵRI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)γ activation. Here we report evidence that the PLCγ1-dependent pathway of FcϵRI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, FcϵRI aggregation induced a rapid translocation and phosphorylation of PLCγ1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of FcϵRI-mediated PLCγ activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLCγ activation. Wortmannin, however, did produce a partial (∼50%) concentration-dependent inhibition of FcϵRI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85α and p85β subunits of PI 3-kinase, also revealed no defects in FcϵRI-mediated PLCγ1 activation. These data are consistent with the conclusion that the PLCγ-dependent component of FcϵRI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of FcϵRI-mediated degranulation in human mast cells. Aggregation of the high affinity receptor for IgE (FcϵRI) on the surface of mast cells initiates a cascade of intracellular signaling events leading to degranulation and the release of proinflammatory mediators. Activation of PLC 1The abbreviations used are: PLCphospholipase CBMMCsbone marrow-derived mast cellsFcϵRIhigh affinity receptor for IgEHuMCshuman mast cellsKOknock-outNP-IgEchimeric human Fc-specific anti-4-hydroxy-3-nitrophenylacetyl IgENP-BSA4-hydroxy-3-nitrophenylacetyl-bovine serum albumin conjugatePI 3-kinasephosphatidylinositide 3-kinaseIP3inositol trisphosphateILinterleukinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diolHSPheat shock proteinGSTglutathione S-transferasePIPPIP2, PIP3, phosphatidylinositol monophosphate, bisphosphate, and trisphosphate, respectively. is an important step in the signaling pathway regulating FcϵRI-dependent degranulation (1Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Abstract Full Text PDF PubMed Scopus (368) Google Scholar). PLC catalyzes the hydrolysis of membrane-associated PIP2 thereby generating diacylglycerol and inositol trisphosphate; essential factors, respectively, for the activation of protein kinase C and intracellular calcium mobilization (1Beaven M.A. Metzger H. Immunol. Today. 1993; 14: 222-226Abstract Full Text PDF PubMed Scopus (368) Google Scholar), both of which are necessary signals for degranulation (2Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Munshinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar). The activities of the PLCγ1 and PLCγ2 isoforms are regulated by tyrosine phosphorylation by association with receptors that possess inherent tyrosine kinase activity, such as Kit (3Rottapel R. Reedjik M. Williams D.E. Lyman S.D. Anderson D.M. Pawson T. Berstein A. Mol. Cell. Biol. 1991; 11: 3043-3051Crossref PubMed Scopus (201) Google Scholar), or recruitment of cytosolic tyrosine kinases as is the case with members of the immunoglobulin superfamily. Generally, PLCγ1 and PLCγ2 isoforms are independently expressed in specific cells types. For example, T cells express only PLCγ1 (4Weiss A. Koretsky G. Schatzman R.C. Kaldecek T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5484-5488Crossref PubMed Scopus (279) Google Scholar), whereas B cells express only PLCγ2 (5Coggeshall K.M. McHugh J.C. Altman A. Proc. Natl. Acad. sci. U. S. A. 1992; 89: 5660-5664Crossref PubMed Scopus (114) Google Scholar). Mast cells are one of the few cell types that express both PLCγ1 and PLCγ2 isoforms (6Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 6: 1145-1158Crossref Scopus (124) Google Scholar). phospholipase C bone marrow-derived mast cells high affinity receptor for IgE human mast cells knock-out chimeric human Fc-specific anti-4-hydroxy-3-nitrophenylacetyl IgE 4-hydroxy-3-nitrophenylacetyl-bovine serum albumin conjugate phosphatidylinositide 3-kinase inositol trisphosphate interleukin 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol heat shock protein glutathione S-transferase PIP2, PIP3, phosphatidylinositol monophosphate, bisphosphate, and trisphosphate, respectively. Two alternative models have been proposed for receptor-mediated regulation of the catalytic activities of PLCγ1 and PLCγ2 (7Wilde J.I. Watson S.P. Cell. Signal. 2001; 13: 691-701Crossref PubMed Scopus (125) Google Scholar). In B cells, PLCγ2 appears to be regulated by a PI 3-kinase dependent mechanism preceded by activation of the src kinases, p56lyn and fyn, and p72syk (8Okada T. Maeda A. Iwamatsu A. Gotoh K. Kurosaki T. Immunity. 2000; 13: 817-827Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). In T cells, PLCγ1 appears to be activated following recruitment to the cell membrane via the adaptor molecules SLP76 and LAT, mediated by the src kinase, lck, and ZAP70 (9Samelson L.E. Annu. Rev. Immunol. 2002; 20: 371-394Crossref PubMed Scopus (470) Google Scholar) in a PI 3-kinase independent manner (10Reynolds L.F. Smyth L.A. Norton T. Freshney N. Downward J. Kioussis D. Tybulewick V.L. J. Exp. Med. 2002; 195: 1103-1114Crossref PubMed Scopus (179) Google Scholar, 11Fruman D.A. Snapper S.B. Yballe C.M. Davidson L. Yu J.Y. Alt F.W. Cantley L.C. Science. 1999; 283: 393-397Crossref PubMed Scopus (572) Google Scholar). Based on studies conducted primarily in the rodent RBL 2H3 mast cell line, the current view is that PLCγ activation in mast cells, as in B cells, is dependent on PI 3-kinase (6Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 6: 1145-1158Crossref Scopus (124) Google Scholar, 7Wilde J.I. Watson S.P. Cell. Signal. 2001; 13: 691-701Crossref PubMed Scopus (125) Google Scholar), however, our data with human mast cells indicated that this is not the case. This finding prompted us to investigate the pathways of activation of PLCγ1 and PLCγ2 and the role of PI 3-kinase in the intracellular events leading to degranulation in CD34+ peripheral blood progenitor-derived human mast cells (HuMCs) after FcϵRI aggregation. To further investigate the role of PI 3-kinase in FcϵRI-dependent PLCγ1-mediated mast activation, we examined these responses in BMMCs deficient in the p85α and p85β subunits of PI 3-kinase. As will be shown, the immediate PLCγ1-mediated component of mast cell degranulation following FcϵRI aggregation is independent of PI 3-kinase, however, PI 3-kinase may contribute to a later phase of the degranulation response. Cell Isolation and Culture—HuMCs were developed from CD34+ cells in StemPro-34 culture media (Invitrogen) containing l-glutamine (2 mm) (Biofluids, Rockville, MD), penicillin (100 units/ml) (Biofluids), streptomycin (100 μg/ml) (Biofluids), IL-6 (100 ng/ml) (PeproTech), and stem cell factor (100 ng/ml) (PeproTech), as described (12Kirshenbaum A.S. Goff J.P. Semere T. Foster B. Scott L.M. Metcalfe D.D. Blood. 1999; 94: 2333-2342Crossref PubMed Google Scholar). IL-3 (30 ng/ml) was included for the first week of culture. Experiments were conducted on these cells 8–10 weeks after the initiation of culture, at which point, the population was greater than 99% mast cells. For studies using mouse BMMCs the following KO mice were obtained: p85α-/-:p85β+/+ (BALB/c) (13Terauchi Y. Tsuji Y. Satoh S. Minoura H. Murakami K. Okuno A. Inukai K. Asano T. Kaburagi Y. Ueki K. Nakajima H. Hanafusa T. Matsuzawa Y. Sekihara H. Yin Y. Barrett J.C. Oda H. Ishikawa T. Akanuma Y. Komuro I. Suzuki M. Yamamura K. Kodama T. Suzuki H. Kadowaki T. Nat. Genet. 1999; 21: 230-235Crossref PubMed Scopus (348) Google Scholar) from Taconic Labs, Germantown, NY, and p85α+/+:p85β-/- (129/Sv X C57BL/6) as described (14Ueki K. Yballe C.M. Brachmann S.M. Vicent D. Watt J.M. Kahn C.R. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 419-424Crossref PubMed Scopus (199) Google Scholar). The absence or presence of p85α and p85β in these mice was confirmed by PCR and, in the case of p85α, by immunoblot analysis (data not shown). BMMCs were obtained by flushing bone marrow cells from the femurs of either these KO mice or wild type mice, then culturing for 4–6 weeks in RPMI 1640 supplemented with 10% fetal calf serum, glutamine (4 mm), sodium pyruvate (1 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), nonessential amino acids (1 mm), HEPES (25 mm), β-mercaptoethanol (50 mm), and mouse recombinant IL-3 (30 ng/ml) (Peprotech). At this point, the murine mast cell population was greater than 99% pure. RBL 2H3 cells and the U937 human monocytic cell line were cultured in Iscove's media, supplemented with fetal calf serum (10%) (Biofluids), l-glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cultures were maintained at 37 °C in a humidified incubator of 95% air, 5% CO2. Cell Activation—HuMCs were sensitized overnight in culture media containing chimeric human Fc anti-4-hydroxy-3-nitrophenylacetyl (NP)-specific IgE (NP-IgE) (Serotec, Raleigh, NC) (1 μg/ml) and then triggered by the addition of 10 μl of 10× NP-BSA (30:1) conjugate (Biosearch Technologies Inc. Novoto, CA) (final concentration 100 ng/ml), as described (15Tkaczyk C. Metcalfe D.D. Gilfillan A.M. J. Immunol. Methods. 2002; 268: 239-243Crossref PubMed Scopus (46) Google Scholar). When the effects of inhibitors were examined, these, or controls, were added 10 min prior to the addition of NP-BSA. BMMCs were similarly treated, however, these cells were sensitized using mouse monoclonal anti-dinitrophenyl IgE and then triggered by the addition on dinitrophenyl-human serum albumin. Subcellular Fractionation—To separate the membrane and cytosolic fractions, sensitized HuMCs or BMMCs were re-suspended in 200 μl of HEPES/BSA (0.04%) and activated as above. At predetermined times, the tubes were transferred into ice and equal volumes of ice-cold lysis buffer (Tris-HCl, 20 mm; dithiothreitol, 2 mm; EGTA, 1 mm; EDTA, 2 mm; pH 7.5), containing protease and phosphatase inhibitors were added (15Tkaczyk C. Metcalfe D.D. Gilfillan A.M. J. Immunol. Methods. 2002; 268: 239-243Crossref PubMed Scopus (46) Google Scholar). The cells were disrupted by sonication twice on wet ice for 10 s and then the tubes were spun at 20,800 × g for 15 min at 4 °C and the supernatants (cytosolic fractions) and the pellets (membrane fractions) were re-suspended in sample buffer. The samples were then boiled for 3 min and centrifuged at 20,800 × g for 5 min prior to loading the samples onto gels. For inhibitor studies, the cells were pretreated with PP2, piceatannol, or wortmannin (Calbiochem, Carlsbad, CA) for 10 min prior to the addition of antigen. Gel Electrophoresis—All gel electrophoresis supplies were obtained from Invitrogen. Proteins were separated on 4–12% NuPage BisTris gels and probed with the following primary antibodies: anti-phosphotyrosine (4G10) conjugated to biotin, anti-human p72syk (UBI, Lake Placid, NY), anti-rodent p72syk, anti-PI 3-kinase p85α and p110α, -β, and -δ, anti-PLCγ1, anti-PLCγ2, (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-PLCγ1 (pY783), (BIOSOURCE, Camrillo, CA), anti-phospho-Src (pY416), anti-phospho-AKT (polyclonal pS473 and monoclonal pS473), anti-phospho-p70 S6 kinase (pT389) (Cell Signaling Technology, Beverly, MA), and anti-PI 3-kinase p85β (Acris, Bad Nauheim, Germany). Following rinsing and incubation with the appropriate horseradish peroxidase-conjugated secondary antibody or streptavidin, the immunoreactive bands were visualized utilizing a Renaissance Western blot chemiluminescence kit (PerkinElmer Life Sciences, Boston, MA). To confirm equal protein loading onto gels, membranes were stripped and re-probed as described (12Kirshenbaum A.S. Goff J.P. Semere T. Foster B. Scott L.M. Metcalfe D.D. Blood. 1999; 94: 2333-2342Crossref PubMed Google Scholar) with anti-p72syk. Alternatively, identically loaded membranes were probed for normalization. For translocation experiments, protein equivalents were established by probing the membrane extracts with anti-Kit (CD117) (Santa Cruz Biotechnology) and/or heat shock protein (HSP)90 (BD Transduction Laboratories, San Diego, CA). Kit was used as a marker for membrane proteins and HSP90 as a marker for the presence of cytosolic proteins in the membrane fraction. Although, in some experiments, HSP90 was detected in the membrane fraction, this was generally a minor component of total cellular protein and was unaffected by antigen challenge. To quantitate changes in protein phosphorylation, the ECL films were scanned using an ImageQuant 5.0 scanner (Amersham Biosciences). GST Fusion Protein Capture Studies—HuMCs were stimulated and processed as for immunoprecipitation studies. After 2 min of challenge with NP-BSA (100 ng/ml), boiling lysis buffer was added to the cells and processed as above. The supernatants were diluted 10× in Tris-buffered saline containing Triton X-100 (0.1%) and protease and phosphatase inhibitors, and precleared with GST-agarose for 1 h at room temperature under rotation. After centrifugation, the supernatants were removed and incubated with 10 μg of agarose beads-GST-SH2-SH2 domains of PLCγ1 or the GST-SH3 domain of PLCγ1 (Santa Cruz) for 2 h at room temperature under rotation. The beads were washed 3× with Triton X-100 (0.1% in Tris-buffered saline) and the proteins were solubilized and separated by electrophoresis as above. The samples were then probed with anti-Vav, anti-SLP76 (UBI), and anti-LAT (gift from Larry Samelson, NCI, National Institutes of Health) antibodies. Fusion proteins alone were also run on the gels as controls for nonspecific binding of the primary and secondary antibodies. IP3Assay—Cellular IP3 concentrations were determined utilizing a commercially available kit (Amersham Biosciences) according to the manufacturers instructions. Briefly, following cell activation, cells were extracted with trichloroacetic acid on ice for 15 min. IP3 was then extracted from the trichloroacetic acid precipitate using 1,1,2-trichlorofluorethane-trioctylamine. The IP3 containing fractions were then incubated for 1 h at 4 °C with membrane fractions of calf cerebellum containing the IP3 receptor and tracer d-myo-[H3]inositol 1,4,5-trisphosphate. The mixtures were then centrifuged at 3700 × g for 10 min and the tracer remaining bound to the membranes was measured with a β-scintillation counter. The results were expressed as picomoles of IP3 per 106 cells. Calcium Measurements—HuMCs and BMMCs were sensitized as above and then re-suspended in HEPES buffer containing 0.04% BSA, 0.3 mm sulfinapyrazole, and 0.5 μm Fura-2 AM (Molecular Probes, Eugene, OR). Cells were then incubated for 30 min, washed twice in the same buffer (without Fura-2 AME), and plated in black 96-well plates (Packard Biosciences, Meriden CT; CulturPlate™ -96F) at a density of 10,000 cells/100 μl/well. An additional aliquot of cells was treated similarly, except that Fura-2 AM was omitted from the protocol (non-loaded cells) and plated alongside Fura-2-loaded cells. Fluorescence was determined at 510 nm with alternating excitation at 340 and 380 nm in the Wallac Victor2 1420 Multilabel Counter (PerkinElmer Life Sciences). The instrument was set at 37 °C and a top reading mode. After subtraction of background fluorescence of non-loaded cells, data were calculated as the ratio of fluorescence at 340 and 380 nm excitation wavelengths. Degranulation Experiments—Degranulation was monitored by determining the release of β-hexosaminidase as described (16Chaves-Dias C. Hundley T.R. Gilfillan A.M. Kirshenbaum A.S. Cunha-Melo J.R. Metcalfe D.D. Beaven M.A. J. Immunol. 2001; 166: 6647-6656Crossref PubMed Scopus (29) Google Scholar). Briefly, HuMCs or BMMCs were sensitized overnight as above. Following rinsing with HEPES buffer (17Lin P. Fung S.J. Chen T. Repetto B. Huang K.S. Gilfillan A.M. Biochem. J. 1994; 299: 109-114Crossref PubMed Scopus (22) Google Scholar) containing BSA (0.04%), HuMCs were suspended in the same buffer at 50,000 cells/ml, then triggered by the addition of NP-BSA in a 100-μl total volume. BMMCs were suspended at a concentration of 1 million cells/ml and then triggered in a volume of 200 μl. When the effects of inhibitors were examined, these compounds (or carrier buffer for controls) were added 10 min prior to the addition of NP-BSA. The experiments were terminated by centrifugation at 4 °C and then aliquots of the supernatants were removed for β-hexosaminidase assay. The remaining cells were lysed by the addition of distilled water and freeze-thawing. The β-hexosaminidase content of the supernatants and cell lysates was then determined as described and the release calculated as the % of total β-hexosaminidase (cells + supernatant) found in the supernatant. Phosphatidylinositol 3,4,5-Trisphosphate (PIP3) Production—HuMCs were sensitized as above and then transferred to media without growth factors for 2 h. Following rinsing in HEPES buffer containing 0.04% BSA, the cells were loaded with [32P]orthophosphate (200 μCi/ml) (Amersham Biosciences) for 1 h in the same buffer, rinsed, then 2.5–3 × 106 cells placed in 1.5-ml polyethylene tubes prior to stimulation for the indicated times with NP-BSA (100 ng/ml) in the absence or presence of wortmannin (100 nm). PIP3 production in HuMCs and BMMCs was subsequently measured as described in Ref. 18Manetz T.S. Gonzales-Espinosa C. Arudchandran R. Xirasagar S. Tybulewicz V. Rivera J. Mol. Cell. Biol. 2001; 21: 3763-3774Crossref PubMed Scopus (135) Google Scholar. Expression and Activation of PLCγ —To first examine which isoforms of PLCγ were expressed in HuMCs, cell lysates were probed with anti-PLCγ1 and PLCγ2 antibodies. Extracts of other cell types known to express these isoforms were used as positive controls. As expected, the U937 cell line, mouse BMMCs, and RBL 2H3 cells expressed both PLCγ isoforms (Fig. 1a). Similarly both PLCγ1 and PLCγ2 were found in HuMCs. Having demonstrated that PLCγ1 and PLCγ2 were both expressed in HuMCs, we next determined whether aggregation of FcϵRI resulted in activation of these molecules as assessed by PLCγ translocation and phosphorylation, IP3 production, and intracellular calcium mobilization. HuMCs were sensitized overnight and activated by the addition of NP-BSA (15Tkaczyk C. Metcalfe D.D. Gilfillan A.M. J. Immunol. Methods. 2002; 268: 239-243Crossref PubMed Scopus (46) Google Scholar). This resulted in a rapid translocation of both PLCγ1 and PLCγ2 to the mast cell plasma membrane, which was observed as early as 5 s after cell activation (Fig. 1b). Translocation of PLCγ2, however, was not as marked as the translocation of PLCγ1. We monitored PLCγ phosphorylation in HuMC lysates by probing with an activation state-specific anti-phospho-PLCγ1 antibody (Fig. 1c). There was little constitutive phosphorylation of PLCγ1 in resting HuMCs. However, FcϵRI aggregation induced a marked increase in phosphorylation of the active regulatory tyrosine residue of PLCγ1. This was followed by a noticeable, but incomplete, dephosphorylation by 120 s after FcϵRI aggregation. In these studies, no phosphorylation of PLCγ2 was observed in HuMCs, utilizing conditions (18Manetz T.S. Gonzales-Espinosa C. Arudchandran R. Xirasagar S. Tybulewicz V. Rivera J. Mol. Cell. Biol. 2001; 21: 3763-3774Crossref PubMed Scopus (135) Google Scholar) under which PLCγ2 phosphorylation was observed in mouse BMMCs (data not shown). However, we cannot rule out the possibility that PLCγ2 is activated in HuMCs following FcϵRI aggregation but is under the level of detection. We then confirmed the production of IP3, known to be PLCγ-dependent in HuMCs following FcϵRI aggregation. From Fig. 1d it can be seen that aggregation of FcϵRI resulted in a rapid increase in the production of IP3 that maximized between 10 and 20 s after FcϵRI aggregation. IP3 levels subsequently decreased but still remained elevated for up to 120 s. Finally, as before (19Okayama Y. Tkaczyk C. Metcalfe D.D. Gilfillan A.M. Eur. J. Immunol. 2003; 33: 1450-1459Crossref PubMed Scopus (50) Google Scholar), we observed a rapid increase in intracellular free Ca2+ levels following FcϵRI aggregation (Fig. 1e). The initial increase closely followed the kinetics of PLCγ1 activation described above, however, levels continued to increase until 120 s after FcϵRI aggregation. This was followed by a slow decrease in levels. Interaction of PLCγ1with Adaptor Molecules—In activated T cells, PLCγ1 is recruited to the cell membrane in a PI 3-kinase-independent manner via the adaptor molecules LAT, SLP76, and Vav (reviewed in Ref. 7Wilde J.I. Watson S.P. Cell. Signal. 2001; 13: 691-701Crossref PubMed Scopus (125) Google Scholar). To examine whether the activation of PLCγ1 in HuMCs following FcϵRI aggregation may also involve similar interactions, we examined the ability of the PLCγ1 SH3 domain and dual SH2 domains to bind to these molecules in extracts of activated HuMCs. Cells were stimulated for 120 s, then proteins were extracted under denaturing conditions. Following capture by the GST-SH3 domain and GST-SH2-SH2 fusion proteins, the samples were probed with antibodies to SLP76, Vav, and LAT. We observed no direct binding of PLCγ to LAT via the SH2 or SH3 domains under these conditions (data not shown) but we did observe an inducible binding of both SLP76 and Vav to the SH2 domains of PLCγ1 (Fig. 2a). Thus, in HuMCs, as in T cells, following cell activation, PLCγ1 is capable of directly associating with SLP76 and Vav. Expression and Activation of PI-3 Kinase—To next investigate the role of PI 3-kinase in the regulation of PLCγ activation leading to degranulation of HuMCs, we first examined the expression of PI 3-kinase in HuMCs. Lysates were probed for the α and β isoforms of the PI 3-kinase p85 subunit, and the α, β, and δ isoforms of the PI 3-kinase p110 subunit utilizing lysates of U937 cells, mouse BMMCs, and RBL 2H3 cells as positive controls (Fig. 3a). In HuMCs, the p85α and p85β and the p110α, -β, and -δ subunits (Fig. 3a) were all expressed. The anti-p85β antibody used in these studies did not recognize rodent p85β. However, expression of p85β in BMMCs has been confirmed utilizing an anti-p85 polyclonal antibody that recognizes both the α and β isoforms (data not shown). To establish that PI 3-kinase was activated in HuMCs following FcϵRI aggregation, HuMCs were sensitized and then loaded with [32P]orthophosphate prior to triggering as above. The formation of PI phosphates was then examined 2 min after FcϵRI aggregation. At this point, we observed a marked increase in PIP, PIP2 (Fig. 3b), and PIP3 (Fig. 3c) formation. As previously observed in mouse BMMCs (18Manetz T.S. Gonzales-Espinosa C. Arudchandran R. Xirasagar S. Tybulewicz V. Rivera J. Mol. Cell. Biol. 2001; 21: 3763-3774Crossref PubMed Scopus (135) Google Scholar), PIP3 was a relatively minor product compared with PIP and PIP2. These responses were blocked by preincubation of the cells with the PI 3-kinase inhibitor wortmannin, thus confirming PI 3-kinase activity following FcϵRI aggregation in HuMCs. To further support this conclusion, control and activated HuMC lysates were probed with antibodies against the phosphorylated forms of AKT and p70 S6 kinase, which have been utilized as markers for PI 3-kinase activity (20Kitamura J. Asai K. Maeda-Yamamoto M. Kikkawa U. Kawakami T. J. Exp. Med. 2000; 192: 729-740Crossref PubMed Scopus (158) Google Scholar, 21Li H.L. Davis W. Pure E. J. Biol. Chem. 1999; 274: 9812-9820Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Following FcϵRI aggregation, as we previously described (19Okayama Y. Tkaczyk C. Metcalfe D.D. Gilfillan A.M. Eur. J. Immunol. 2003; 33: 1450-1459Crossref PubMed Scopus (50) Google Scholar), there was a marked increase in the phosphorylation of both AKT and p70 S6 kinase (Fig. 3, d and e). From Fig. 3d, it can be seen that wortmannin blocked the increase in phosphorylation of both AKT and p70 S6 kinase over a similar concentration range to that previously reported for the effect of wortmannin on PI 3-kinase-dependent responses (6Barker S.A. Caldwell K.K. Pfeiffer J.R. Wilson B.S. Mol. Biol. Cell. 1998; 6: 1145-1158Crossref Scopus (124) Google Scholar, 19Okayama Y. Tkaczyk C. Metcalfe D.D. Gilfillan A.M. Eur. J. Immunol. 2003; 33: 1450-1459Crossref PubMed Scopus (50) Google Scholar, 22Hiller G. Sundler R. Cell. Signal. 2002; 14: 169-173Crossref PubMed Scopus (19) Google Scholar). Taken together, the above data demonstrate that both PLCγ and PI 3-kinase are activated following FcϵRI aggregation in HuMCs. Relative Kinetics of the Activation of Src Kinases, PLCγ1, and PI 3-Kinase following FcϵRI Aggregation—To determine the sequence of activation events after aggregation, and the relationship between the activation of PI 3-kinase and PLCγ, we next examined the temporal relationships between the FcϵRI-dependent phosphorylation of PLCγ, and phosphorylation of AKT and p70 S6 kinase, and compared these kinetics to those of the activation of src kinases that represents one of the earliest FcϵRI-induced signaling events in mast cells. Following FcϵRI aggregation, the increase in tyrosine phosphorylation of both PLCγ1 and, as expected, src kinase was very rapid, maximizing within 5 to 10 s (Fig. 4a). In contrast, the kinetics of phosphorylation of AKT and p70 S6 kinase, whereas virtually coincidental, was not apparent until ∼30 s after FcϵRI aggregation (Fig. 4b). Thus, PI 3-kinase-dependent responses, as monitored by the phosphorylation of AKT and p70 S6 kinase, were delayed (Fig. 4c), appearing after the activation of PLCγ. These data are consistent with the conclusion that PLCγ activation is coincidental with the activation of src kinases but precedes the activation of PI 3-kinase. The Effect of Src Kinase, p72syk, and PI 3-Kinase Inhibitors on FcϵRI-mediated Protein Phosphorylation and PLCγ Activation—The above data suggest that PLCγ activation is independent of PI 3-kinase but may be regulated by src kinase(s). To verify this conclusion, inhibitors of src kinases, p72syk, and PI 3-kinase, respectively, PP2, piceatannol, and wortmannin, were next employed. We first examined the effect of PP2 on the phosphorylation of PLCγ1 and AKT. These responses were completely blocked in a concentration-dependent manner (Fig. 5a) suggesting that the activation of both PLCγ1 and PI 3-kinase is dependent on src kinases. Src kinase(s) are known to regulate signaling events in activated mast cells in part through p72syk (23Benhamou M. Stephan V. Robbins K.C. Siraganian R.P. J. Biol. Chem. 1992; 267: 7310-7314Abstract Full Text PDF PubMed Google Scholar, 24Yamashita T. Mao S.Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 19. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar). To, therefore, determine whether the regulation of PLCγ activation by src kinases required p72syk activation, we next examined the ability of piceatannol to block FcϵRI-dependent PLCγ activation. The phosphorylation of PLCγ1 and AKT observed following FcϵRI aggregation was again completely inhibited in a concentration-dependent manner by piceatannol (Fig. 5b). These data are consistent with the conclusion that both src kinases and p72syk activation precedes PLCγ phosphorylation. In addition to tyrosine phosphorylation, PLCγ activation also requires membrane translocation. We, thus, next examined the relative abilities of PP2 and piceatannol to prevent the FcϵRI-dependent membrane translocation of PLCγ and PLCγ-dependent IP3 production. Both PP2 and piceatannol blocked the membrane translocation of PLCγ (Fig. 5c) and IP3 production (Fig. 5d) following FcϵRI aggregation, indicating that these responses are also dependent on both src kinases and p72syk activation. Last, and following the data presented in Fig. 3 that shows that PI 3-kinase acti
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