Article |
Address correspondence to Bridget S. Wilson, Department of Pathology, University of New Mexico Health Sciences Center, CRF 205, 2325 Camino de Salud, Albuquerque, NM 87131. Tel.: (505) 272-8852. Fax: (505) 272-1435. E-mail bwilson{at}salud.unm.edu
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Abstract |
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Key Words: microdomains; PLC; phosphatidylinositol 3-kinase; LAT; Gab2
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Introduction |
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Current interest in organized regions of membrane, variously called microdomains, rafts, detergent-resistant membranes, and glycosylphosphatidylinositol-enriched membranes, centers around their potential roles in signal propagation and membrane trafficking (Edidin, 1997; Simons and Ikonen, 1997; Anderson, 1998; Brown and London, 1998; Jacobson and Dietrich, 1999). Typically, these membranes are isolated by detergent extraction and sucrose gradient centifugation, yielding a light fraction that accumulates acylated Src-family kinase members and is also enriched for glycerophosphatidylinositol-linked proteins, glycosphingolipids, gangliosides, and cholesterol. Detergent-resistant microdomains on leukocyte surfaces are particularly implicated in signaling via multi-chain immune recognition receptors, including the TCR, BCR, several Fc receptors, and the high-affinity IgE receptor (Fc
RI)* of mast cells and basophils (for reviews see Horejsi et al., 1999; Langlet et al., 2000; Dráber et al., 2001).
The high-affinity IgE receptor of mast cells and basophils is an ß
2 tetramer with immunoreceptor tyrosine-based activation motifs (ITAMs) in both the ß and
subunit cytoplasmic tails. Cross-linking this receptor activates the Src-family kinase, Lyn, initiating a cascade of events that include the activation of Syk, PLC
and phosphatidylinositol 3-kinase (PI3-kinase), the mobilization of Ca2+, the secretion of inflammatory mediators from granules, the production of Th2 cytokines, and other events including membrane ruffling, spreading and increased adhesive activity. From detergent extraction and sucrose gradient centrifugation studies, Field et al. (1995)(1997, 1999) suggested that cross-linked Fc
RI moves into detergent-resistant microdomains to encounter Lyn. Stauffer and Meyer (1997) localized a fluorescent Syk-SH2 domain with aggregated Fc
RI in ganglioside-enriched membrane patches. These and other recent experiments using fluorescent reporters (Teruel and Meyer, 2000) have provided new insights into dynamics of signal transduction both temporally and spatially but are limited by the resolution of the light microscope.
Recently, we used immunogold labeling of mast cell membrane sheets and analysis by transmission electron microscopy to document the locations of signaling proteins at higher resolution during FcRI signaling in rat basophilic leukemia cell line 2H3 (RBL-2H3) mast cells. We showed that the sequential association of Fc
RI with Lyn and Syk occurs in topographically distinct microdomains (Wilson et al., 2000). In resting cells, Fc
RI are distributed as dispersed small aggregates that are often loosely colocalized with small Lyn aggregates. After cross-linking, Fc
RI redistribute to membrane domains that stain more intensely than bulk membrane with osmium. These osmiophilic membrane patches exclude Lyn and recruit Syk. Receptors are ultimately internalized through coated pits that bud from the periphery of the patches.
Here, plasma membrane sheets from RBL-2H3 mast cells are used to map the distribution of FcRI ß in relation to a further subset of proteins in the signaling cascade, including PLC
1, PLC
2, and PI3-kinase implicated in the remodeling of membrane inositol phospholipids, as well as the scaffolding/adaptor proteins, linker for activation of T cells (LAT) and Grb2-binding protein 2 (Gab2). Our results suggest that activated mast cells may propagate signals from primary signaling domains organized around Fc
RI ß and from secondary signaling domains, including one organized around the transmembrane protein, LAT.
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Results |
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These results are confirmed and extended here. As shown in Fig. 1 A, PLC2 interacts strongly with the membrane of resting cells. Nevertheless, very little of this enzyme associates with Fc
RI ß (Fig. 1 A, Table I). Instead, PLC
2 occurs in resting cells either in small dispersed clusters or along submembranous cables (Fig. 1 A). The cables can be labeled with gold conjugates of antimyosin antibodies (Fig. 2, A and B)
and phalloidin (Fig. 2, B and C), identifying them as components of the cortical actomyosin cytoskeleton.
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In contrast to PLC2, very little PLC
1, the minor isoform of PLC
in RBL-2H3 cells, was bound to the membrane of unstimulated mast cells (Fig. 3
A; Table I).
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The distribution of PLC1 on membrane sheets from activated cells is shown in Fig. 3 B and quantified in Table I. Fc
RI cross-linking doubles the number of PLC
1-gold particles on membrane sheets. These gold particles occur as singlets and small clusters that are only occasionally seen near Fc
RI ß or in osmiophilic patches (Fig. 3 B). Wortmannin treatment, shown previously to inhibit PLC
1 translocation and tyrosine phosphorylation (Barker et al., 1998), reduces the Fc
RI-mediated recruitment of PLC
1 to membrane sheets (Table I).
Distribution of PI3-kinase in activated cells
Maximal PLC activation after Fc
RI cross-linking is dependent on both tyrosine phosphorylation and on the activation of PI3-kinase to form phosphatidylinositol-3,4,5-P3 (Ptd-Ins[3,4,5]P3), implicating D-3 phosphoinositides in determining both the location and catalytic activity of tyrosine phosphorylated PLC
(Barker, et al., 1999; Smith et al., 2001). In Fig. 4
, polyclonal antibodies to the noncatalytic p85 subunit were used to localize the heterodimeric class IA PI3-kinases on membrane sheets. The sheets were colabeled with monoclonal antibodies to Fc
RI ß or to PLC
isoforms. Fig. 4 A shows the scattered distribution of p85 label on membrane sheets prepared from resting cells. After 2 min of antigen stimulation, a substantial portion of p85 is strongly colocalized with Fc
RI in osmiophilic patches (Fig. 4 B, boxed regions).
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Distinct localizations for two adaptor proteins
Tyrosine kinase cascades typically involve the phosphorylation and/or translocation of a series of tyrosine-phosphorylated adaptor proteins that serve as platforms for macromolecular assembly of downstream components. We hypoth-esized that PLC1 may be selectively segregated away from osmiophilic patches on membrane sheets from activated cells by its interaction with a scaffolding or adaptor protein. We selected Gab2 and LAT as candidates for analysis. Both of these proteins have been implicated in the coupling of receptor activation to changes in inositol phospholipid metabolism.
Grb2-associated binder 2 (Gab2) is a 100-kd cytoplasmic protein with a documented role in growth factor receptor coupling to PI3-kinase (Nishida et al., 1999). Results in Fig. 6
A show that Gab2 gold particles are relatively sparse on membrane sheets from resting cells. Nevertheless, a fraction of these gold particles (18%) are in close proximity to Fc
RI ß (Table II). Gab2 gold label is increased 2.6-fold after Fc
RI cross-linking. Furthermore, >50% of Gab2 is found in the Fc
RI-, p85- and PLC
2-containing osmiophilic patches that are induced by Fc
RI cross-linking (Fig. 6 B; Table II).
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LAT is an integral membrane protein whose cytoplasmic tail is palmitoylated and contains multiple tyrosine phosphorylation sites. LAT has been proposed to act as a scaffold for downstream signaling components and was recently shown to be a critical element upstream of PLC activation and calcium responses in antigen-stimulated mast cells (Saitoh et al., 2000; Kimura et al., 2001).
Counts of >10,000 LAT gold particles showed no differences in the density of LAT between membrane sheets from resting and cells that have stimulated for 2 min with antigen (data not shown). However, the organization of LAT is dramatically altered after short periods of FcRI cross-linking. In Fig. 7
A, gold labeling for LAT identifies some singlets and numerous small clusters across membrane sheets prepared from unstimulated RBL-2H3 cells. In the majority of membrane sheets from resting cells, the number of gold particles per cluster is <20 (Fig. 8, A and B) . Very few (<5%) of the 5-nm gold particles labeling LAT colocalize with 10-nm gold particles marking Fc
RI ß in unstimulated cells. Within 30 s of Fc
RI cross-linking, very large elongated LAT clusters are found on membrane sheets (Fig. 7, BE). Multiple groups of 50150 gold particles were documented in cells activated for 30 s or 1 or 2 min (Fig. 8, BE). LAT aggregates often transect the osmiophilic signaling patches that accumulate Fc
RI ß. However, rather than uniform mixing of the two sizes of gold label, they remain separate from each other. This is particularly evident in the large osmiophilic membrane patch seen in the upper left half of the micrograph in Fig. 7 B. Numerous LAT clusters are also found in apparently unspecialized membrane remote from aggregated Fc
RI (Fig. 7, B and C).
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Importantly, PLC1 colocalizes with LAT after 2 min of Fc
RI cross-linking. Gold particles marking PLC
1 are frequently found at the edge of large LAT clusters (Fig. 9, B and C, arrowheads) and mixed with LAT in smaller clusters (Fig. 9, BC, triangle regions). The inducible association of PLC
1 with LAT inferred from microscopy of native membranes could also be demonstrated biochemically. Fig. 10 A shows a dramatic increase in PLC
1 coprecipitating with anti-LAT immunoprecipitates from activated cells. Conversely, anti-PLC
1 immunoprecipitates from activated but not resting cells contain coprecipitating LAT. Coprecipitation studies showed additionally that a portion of PLC
2 also associates with LAT. However, the extent of PLC
2-LAT coprecipitation was similar from both resting and activated cells, suggesting that PLC
2 associates constitutively with LAT (Fig. 10 A). (TEM confirmation of the inherent association of PLC
2 with LAT was not obtained because the available antibodies to both molecules are rabbit polyclonals and thus unsuitable for double label studies.)
Biochemical analyses of isolated lipid rafts do not reproduce the protein interactions observed directly on native membrane sheets
Previous investigators have inferred properties of mast cell FcRI signaling complexes from the composition of detergent-resistant membranes isolated by detergent extraction and sucrose density gradient centrifugation analysis of activated mast cells (Field et al., 1995, 1997, 1999; Surviladze, 1998). The results in Fig. 10 B reproduce published experiments showing that Lyn and LAT are both in the light fractions containing detergent-resistant membrane. A portion of Fc
RI ß is also recovered in these light fractions, with a modest increase seen in activated cells. This experiment is popularly interpreted as showing the activation-induced recruitment of cross-linked Fc
RI to Lyn microdomains at the onset of signaling. However, it is difficult to reconcile with studies in native membranes showing that cross-linked Fc
RI in fact segregate rapidly away from Lyn on native membranes (Wilson et al., 2000) and that receptor and LAT show little colocalization in resting cells and only transient colocalization in activated cells (this study).
The discrepancies are compounded when other signaling molecules are included in the analysis. A portion of PLC1 is redistributed to the detergent-resistant membrane, as might be expected if this membrane represents regions of active signaling. However, Syk is found separated from receptor in the heavy fractions of the sucrose gradient (Fig. 10 B). In contrast, Syk accumulates with cross-linked Fc
RI in the osmiophilic patches of native membranes (Wilson et al., 2000). Similarly, Gab2, PLC
isoforms, and PI3-kinase are all separated from both receptor and LAT in the heavy fractions of the gradient (Fig. 10 B), even though they can be found in close proximity to receptors and LAT in native membranes. In short, the results of sucrose gradient centrifugation analysis of detergent-extracted mast cells give a strikingly different impression of proteinprotein interactions involved in Fc
RI signaling than the impression obtained by direct microscopy on native membranes.
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Discussion |
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Previously, we demonstrated that cross-linked FcRI encounter Syk in characteristic regions of the mast cell membrane that are easily identified by their dark staining with osmium and by the frequent formation of clathrin-coated pits at the periphery of the patch. Here, we show that the osmiophilic patches also accumulate PLC
2, apparently reflecting a redistribution from its intrinsic associations in part with bulk membrane and in part with components of the cortical cytoskeleton. Osmiophilic patches also attract a substantial portion of class IA PI3-kinase heterodimers, marked here by antibodies to the p85 regulatory subunit, that are translocated from the cytosol during mast cell activation. Additionally, these membrane patches recruit Gab2 that couples to Shc, Grb2, and SHP-2, as well as PI3-kinase (unpublished data). The accumulation of these signaling molecules further establishes the identity of the osmiophilic patches as sites of signaling from cross-linked Fc
RI to downstream responses.
We show that FcRI cross-linking also causes the formation of large elongated rafts that label with antibodies to the scaffolding protein, LAT. Although the LAT rafts often intersect osmiophilic patches containing Fc
RI, they do not mix extensively with receptors in the patches. We hypothesize that the small LAT rafts characteristic of resting cells redistribute after Fc
RI cross-linking into the osmiophilic patches, where they encounter and are phosphorylated by Syk. Modification of LAT may then promote the formation of larger, mobile LAT rafts. Our micrographs suggest that these rafts, which become prominent features outside of osmiophilic patches, may form separate domains for the docking of other proteins. At least two proteins are candidates for this docking activity: PLC
1, whose association with LAT rafts is induced by Fc
RI cross-linking (Fig. 10 A), and a portion of PI3-kinase that is apparently distinct from that associated with Fc
RI or Gab2, based on coprecipitation studies (Fig. 5 B).
Several studies have implicated LAT, together with the SH2 domaincontaining cytoplasmic adaptors in the SLP-76 family, in the activation of both PLC isoforms (Jevremovic et al., 1999; Pasquet et al., 1999; Ishiai et al., 2000; for reviews see Pivniouk and Geha, 2000; Myung et al., 2000). A direct association of LAT and PLC
proteins is implied by coprecipitation studies (Fukazawa et al., 1995; Finco et al., 1998; Tridandapani et al., 2000; Martelli et al., 2000; Zhang et al., 2000) and by the isolation of LAT on immobilized PLC
SH2(C) domains (Gross et al., 1999). Our results suggest different roles for LAT in the localization, and likely the activation, of the two PLC
isoforms in mast cells. In RBL-2H3 cells, Fc
RI cross-linking promotes the association of PLC
1 with LAT domains, whereas a significant fraction of PLC
2 appears to interact constitutively with LAT. Both results are consistent with previous evidence by immunogold labeling in thin section TEM that PLC
1 is recruited to antigen-stimulated RBL membranes, whereas PLC
2 is inherently at the membrane (Barker et al., 1998). The present results raise the possibility that PLC
2 is inherently at the membrane as a result of its constitutive interaction with LAT and can redistribute to osmiophilic patches after Fc
RI cross-linking. In contrast, PLC
1 is transiently at the membrane when levels of D3-phosphoinositides produced by PI3-kinase in LAT domains are locally high.
We show that the topography of LAT on membrane sheets is markedly different from another class of adaptor, Gab2. Membrane-associated Gab2 was shown here to increase >2.5-fold in response to FcRI cross-linking. Most of this Gab2 is found in the osmiophilic patches. Gab2 translocation is accompanied by a severalfold increase in associated PI3-kinase activity, identifying sites of Gab2 accumulation as potential sites of D-3 phosphoinositide synthesis.
Why would cells form multiple signaling domains? Because cross-linked FcRI are internalized relatively rapidly by endocytosis through coated pits, whereas LAT is not internalized through coated pits, one prediction is that signaling domains organized by LAT may be more stable than signaling domains organized by Fc
RI. In this case, one role for LAT domains could be to sustain and amplify signaling as receptor levels drop. Interestingly, although secretion from RBL-2H3 cells halts within seconds of the addition of monovalent haptens that disrupt Fc
RI aggregates, calcium levels remain high for several minutes (Lee and Oliver, 1995). This could be explained if LAT continues to serve as a scaffold for propagating signals to PI3-kinase and PLC
proteins for some time after the disruption of Fc
RI signaling patches. Another prediction is that distinct arms of the Fc
RI signaling cascade could be propagated in these separate domains. These distinct arms may be complementary or, alternatively, may have independent outcomes.
Importantly, the p85 regulatory subunit of PI3-kinase can be found in both FcRI and LAT domains. D-3 phosphoinositides represent a small and transient fraction of total membrane phosphoinositides (Traynor-Kaplan et al., 1989). Thus, the recruitment of PI3-kinase to specific domains may serve to generate the locally high concentrations of Ptd-Ins(3,4,5)P3 required for full activation of both PLC
isoforms (Barker et al., 1999; Smith et al., 2001) at their distinct membrane locales (Barker et al., 1998). If this is true, then location is likely to be a factor in the activation of other enzymes, such as Akt, that also require PtdIns(3,4,5)P3 or its metabolite PtdIns(3,4)P2 for activation.
Many questions remain regarding the topography and functions of the class IA PI3-kinases. We showed previously that tyrosine phosphorylated proteins coprecipitate with all three p110 PI3-kinase catalytic subunits and that microinjection of blocking antibodies to the p110 and p110ß isoforms, but not p110
, inhibits calcium responses in RBL-2H3 cells (Smith et al., 2001). Here, we have observed that Fc
RI cross-linking leads to increases in PI3-kinase activity in distinct macromolecular complexes organized around LAT, Fc
RI, and Gab (Fig. 5). Together, these data raise the possibility that the different class IA PI3-kinases, composed of unique p85-p110 heterodimers, may distribute to distinct membrane domains to regulate different cellular functions. In particular, the presence of locally high PI3-kinase activity may induce locally high concentrations of D3-phosphoinositides to serve as lipid anchors for the recruitment of signaling molecules like PLC
1. As well as determining the topography of recruited proteins, the local remodeling of membrane inositol phospholipid composition by nonrandomly distributed PI3-kinase family members may help to create or maintain preferred environments for transmembrane proteins like acylated LAT.
Current models of membrane structure envision membranes as dynamic mixtures of more or less ordered lipids in association with distinct proteins. An important example is the newly described immunological synapse that forms between the T cell and an antigen-presenting cell during conjugation of TCRMHC peptide complexes (for review see Dustin and Chan, 2000). When observed by sophisticated fluorescence microscopic techniques, engaged TCR form the center of a bull's eye (referred to as the central supramolecular activation cluster or cSMAC), surrounded by a ring of adhesion receptors (the peripheral supramolecular activation cluster or pSMAC). Signaling molecules such as PKC may associate stably with the synapse (Monks et al., 1998), whereas others such as CD45 may be conditionally or transiently excluded (Sperling et al., 1998; Leupin et al., 2000; Johnson et al., 2000). Thus, the new models developed around data derived in both the TCR and Fc
RI systems readily accommodate the concept that biological membranes may include one or more domains that are compositionally distinct from bulk membrane and can form and disassemble in a highly dynamic fashion. They also accommodate the hypothesis presented here that the segregation may be initiated in part when enzymes remodel the membrane inositol phospholipid composition after activation by receptor-coupled signaling pathways.
The relationship of the rafts isolated biochemically to the signaling domains observed microscopically remains to be determined. The protein associations we observe in native membrane sheets do not always correlate with implied associations based upon analysis of detergent-solubilized sucrose density fractions. For example, using the sucrose density fractionation protocol, we are able to confirm the localization of acylated proteins such as Lyn kinase and LAT to the light fraction. We also find a portion of PLC1 in the light fraction after Fc
RI cross-linking. However, none of Syk, p85, PLC
2, or Gab2 is found in the light fraction, even though their close interaction with receptor and LAT is readily demonstrated by TEM of native membranes.
In contrast, results using light microscopic approaches to elucidate interactions of FcRI with rafts are more compatible with our results on native membrane sheets. Stauffer and Meyer (1997) showed that GFP chimeric proteins integrating the tandem SH2 domains of Syk were recruited to punctate structures at the plasma membrane, consistent with our observations that Syk is recruited to osmiophilic patches with receptors (Wilson et al., 2000). Using membrane sheets, we demonstrated that Lyn segregates from receptors with the first 2 min of cross-linking. The stringed appearance of Lyn as it segregates from osmiophilic patches, and the colocalization of Lyn with the actin-based cytoskeleton, suggests that cytoskeleton plays a role in Lyn's dissociation from receptors (Wilson et al., 2000). Using a complementary fluorescence confocal microscopy approach, Holowka et al. (2000) showed cytochalasin treatment leads to prolonged associations between cross-linked Fc
RI and Lyn. Thus, a combination of light and electron microscopic approaches are likely to ultimately yield the clearest insight into the different proteinprotein and proteinlipid interactions involved in the Fc
RI signaling cascade.
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Materials and methods |
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Cell activation and membrane labeling
RBL-2H3 cells were allowed to settle overnight onto 15-mm round clean glass coverslips in the presence of anti-DNP IgE (1 µg/ml) to prime cell surface FcRI. After washing to remove excess IgE, Fc
RI were cross-linked by incubation for 2 min at 37°C with DNP-BSA (1 µg/ml). Plasma membrane sheets were prepared and labeled with antibody- or phalloidin-conjugated colloidal gold particles as described in Wilson et al. (2000), using a modification of procedures developed by Sanan and Anderson (1991). Samples were examined and photographed using an Hitachi H600 transmission electron microscope.
Quantifying gold particle distributions
Methods for counting gold particle distributions were established previously (Wilson et al., 2000). Here, micrographs from 24 separate experiments were sorted into groups according to distinct treatment and labeling conditions. For determination of cluster size and codistribution, gold particles were counted for matching sets of micrographs. Gold particles per set range 5003,000 and reflect the relative abundance of label for antigen in the micrographs. For measuring translocation, numbers of gold particles were counted for each experimental condition over equivalent areas of membrane (defined in µm2).
Sucrose gradient centrifugation and analysis of membrane fractions
IgE-primed RBL-2H3 cells (40 x 106 cells per treatment condition) were harvested from culture dishes with 1.5 mM EDTA in Hanks' buffered saline without divalent cations. Washed cells were resuspended in Hanks' buffered saline, divided into two aliquots, and held for 2 min at 37°C with or without DNP-BSA (1 µg/ml). Cells were collected by centrifugation at 4°C, cell pellets were resuspended in 750 µl ice-cold lysis buffer containing low concentrations of detergent (10 mM Tris/HCl, pH 8.0, 0.05% Triton X-100, 50 mM NaCl, 10 mM EDTA, 10 mM glycerophosphate, 1 mM NaV04 and 1x protease inhibitor cocktail from Roche Molecular Chemicals). Lysates were mixed with 750 µl 80% sucrose (prepared in 10 mM Tris-HCl, pH 8.5, 50 mM NaCl, 2 mM EDTA) and overlaid onto 0.5 ml 80% sucrose in polyallomer tubes (13 x 51 mm), followed by 0.5-ml layers of 35, 25, and 20% and 0.6-ml aliquots of 15 and 10%. The gradient was centrifuged in a SW 55 (Beckman) rotor at 200,000 g for 16 h at 4°C. Fractions (0.5 ml) were harvested sequentially from the top of the gradient. For analyses of protein composition, aliquots (35 µl) were mixed with equal volume of 2x SDS sample buffer, boiled for 5 min, and separated by 8 or 10% SDS-PAGE. Alternatively, designated fractions were diluted in 50 mM Tris, 150 mM NaCl, pH 7.4, 1 mM NaV04 to a total volume of 1 ml and rocked for 1 h with beads prebound to primary antibodies, followed by separation of proteins bound to washed beads by SDS-PAGE. Proteins were transferred to nitrocellulose using a semidry blotting system (Labconco). Blots were probed with primary antibodies (1 µg/ml), followed by HRP-conjugated secondary antibodies (antimouse HRP-conjugates were diluted 1:10,000, and antirabbit HRP-conjugates were diluted 1:40,000); immunolabeled proteins were visualized by fluorography (ECL, Pierce Chemical Co.).
PI3-kinase assays
IgE-primed RBL cells were incubated with or without DNP-BSA at 37°C, and reactions were stopped with ice-cold saline. Cells were sedimented by microcentrifugation and lysed in 50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 1 mM NaV04, and protease inhibitor cocktail (Boehringer) containing 1% Brij 96 or 1% Triton X-100, as specified. Lysates were clarified by centrifugation (15,000 g) for 5 min and rocked 2 h at 4°C with 40 µl (per ml of lysate) protein A and GSepharose bead mixture (1:1) (Amersham Pharmacia Biotech) prebound to specified antibodies (1 µg). Washed immune complexes were incubated in reaction buffer (20 mM Hepes, pH 7.4, 5 mM MgCl2, 0.25 mM EGTA), containing 10 µM ATP, 20 µCi [32P]ATP, 500 µg/ml sonicated PtdIns (50 µl total) for 30 min at 37°C. The reactions were stopped by addition of 3N HCl. The lipid fraction was isolated using chloroformmethanol partitioning (Jackson et al., 1992) and resolved by thin-layer chromatography in 1-propyl acetate:2-propanol:ethanol:6% aqueous ammonia (1:3:1:3) (vol/vol) on silica gel 60 plates (Merck) (Hegewald, 1996). Formation of 32P-labeled PtdIns(3)P was imaged on a STORM 860 PhosphorImager (Molecular Dynamics) and quantified by means of Image Quant software. Data shown are duplicates ± SEM and are representative of at least three experiments.
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Footnotes |
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Acknowledgments |
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This work was supported in part by American Cancer Society grant RPG-99-233-01-CIM to B.S. Wilson and by National Institutes of Health grant RO1 GM49814 to J.M. Oliver.
Submitted: 12 April 2001
Revised: 27 June 2001
Accepted: 2 July 2001
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