INTRODUCTION

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The high-affinity immunoglobulin E (IgE) receptor (FcRI)-mediated activation of mast cells and basophils triggers a cascade of intracellular biochemical events that ultimately lead to the secretion of preformed pharmacological agents and the transcription of cytokine genes. This process is initiated by aggregation of the receptor by means of multivalent antigen (Ag)-IgE complexes, followed by tyrosine phosphorylation of the receptor subunits by Src family protein tyrosine kinases (14, 31).

FcRI has a tetrameric structure comprised of an IgE-binding  subunit, a  subunit, and a disulfide-bonded  dimer (32). The  and  subunits possess immunoreceptor tyrosine-based activation motifs (ITAMs), which are rapidly phosphorylated by protein tyrosine kinase Lyn. Tyrosine-phosphorylated ITAMs of the the  subunits serve as novel binding sites for Src homology 2 (SH2) domains of Syk kinase (6, 22, 28), leading to phosphorylation and activation of Syk. Thereafter, a number of other signaling and adaptor molecules become phosphorylated and recruited into the regions of activated FcRI/Syk complexes. These include PLC1 (26), the proto-oncogene product Vav (41), PKC- (18), and the linker for activation of T cells (LAT) (37, 52).

Detailed molecular mechanisms of the initial engagement of the Lyn kinase and FcRI are not completely understood, but two different models have been proposed. One model, based on protein-protein interactions, postulates that a small fraction of Lyn is constitutively associated with the  subunit of the FcRI prior to activation. FcRI aggregation effected by multivalent antigen-IgE complexes or by other means facilitates the transphosphorylation of one FcRI by Lyn bound to a juxtaposed receptor (33). The tyrosine phosphorylation of  and  subunits of FcRI supports the recruitment of additional Lyn to  subunit and of the Syk kinase to  subunits, promoting further propagation of the activation signal.

The alternative model postulates that the initial coupling of Lyn with aggregated FcRI is mediated by protein-lipid interactions. According to this model, Lyn is anchored to the inner leaflet of the plasma membrane via myristate and palmitate chains that localize it in lipid rafts enriched in glycosphingolipids, cholesterol, and glycosylphosphatidylinositol-anchored proteins. These domains, which are also referred to as detergent-insoluble glycosphingolipid domains, have been found in numerous cell types (9, 40). Upon aggregation, the FcRI rapidly translocates into lipid rafts, where it is phosphorylated by Lyn kinase (15, 39).

Although the model based on the sequestration of signal transduction molecules into lipid rafts is very attractive and was recently supported by analyses of activation via the T-cell receptor (51, 23, 29) and B-cell receptor (10), it is not completely clear how the receptor aggregation leads to its inclusion in lipid rafts and whether this is important for early activation events. For example, aggregation of FcRI on rat basophilic leukemia (RBL) cells (a mast cell tumor analog) by divalent monoclonal antibodies (MAbs), which induce little visible receptor aggregation, as determined by light and scanning electron microscopy (30), results in a cellular response similar to that caused by aggregation induced by a multivalent Ag that causes extensive FcRI aggregation (42).

To investigate the localization of Lyn kinase and its functional consequences, we tagged Lyn with green fluorescense protein (GFP) and followed its distribution in RBL cells, mouse bone marrow-derived mast cells (BMMC), and BMMC from a mouse with a genetically disrupted Lyn gene (BMMC-Lyn^/) (19) before and after FcRI engagement. Various Lyn-GFP constructs with defects in palmitoylation, myristoylation, or both acylation sites, as well as mutations in the SH1 and SH2/SH3 domains, and constitutively tyrosine-phosphorylated FcRI were employed to determine the factors affecting the early stages of FcRI-mediated activation. These experiments suggest that FcRI phosphorylation and early activation events can be initiated outside of lipid rafts.

	MATERIALS AND METHODS

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Antibodies, reagents, and cell cultures. Mouse MAbs specific for Lyn, Syk,  subunit of FcRI, 2,4,6-trinitrophenyl (TNP)-specific IgE (IGEL b4 1), and 2,4-dinitrophenyl (DNP)-specific IgE have been described previously (13, 35, 36, 45, 46). Rabbit polyclonal Ab specific for Syk, Lyn, LAT, GFP, and IgE were prepared by immunization with the recombinant fragments of Syk (2), Lyn (13), LAT, and GFP (unpublished) or IGEL b4 1 MAb, respectively. A chicken antibody to FcR has been described previously (45). Horseradish peroxidase (HRP)-conjugated mouse antiphosphotyrosine MAb PY-20, goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Transduction Laboratories. Antiphosphotyrosine MAb (4G10) and anti-Shc were obtained from Upstate Biotechnology, and anti-c-Src MAb (B 12) was from Santa Cruz Biotechnology. RBL-2H3 cells and their culture conditions have been described (11). RBL-2H3 cells defective in FcRI and transfected with wild-type FcRI (wt) or with mutated FcRI chain in which a threonine at position 52 was substituted for alanine (T52A) have been described (45). BMMC and BMMC-Lyn^/ (19) were kindly provided by M. Hibbs (Ludwig Institute for Cancer Research, Melbourne, Australia).

DNA constructs, recombinant SFV, and infection. The Semliki Forest virus (SFV) gene expression system, pSFV1 and helper pSFV2, was purchased from Life Technologies. pSFV1 was further modified to contain a multiple cloning site and a GFP expression cassette as previously described (4). Lyn mutants were generated by PCR from cDNA encoding wild-type rat LynA and LynB (48).

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View larger version (26K): [in this window] [in a new window]   FIG. 1.   Schematic representation of Lyn constructs. All constructs were tagged at the C terminus with enhanced GFP (solid circle). LynA-WT and LynB-WT are constructs with two forms of wild-type Lyn kinase that differ in a 20-amino-acid-long insert in the unique domain (shaded box). LynB-CA, LynB-GA, and LynB-GCA are constructs mutated in the palmitoylation, myristoylation, or both acylation sites, respectively. LynA-UNI and LynB-UNI contain only the N-terminal unique domains of LynA and LynB, respectively. LynB-CI is catalytically inactive Lyn B (mutation of Lys279 to Arg). LynA-SH2 is LynA without the catalytic domain. Arrows mark sites of introduced point mutations. The kinase activity of all Lyn constructs is indicated as full (+) and null () kinase activity.

Confocal microscopy. The cells were sensitized with biotinylated IgE (1.5 µg/ml) immediately after infection. After incubation for 3 h at 37°C, the cells were washed twice in buffered saline solution (BSS) containing 20 mM HEPES (pH 7.4), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 1 mg of bovine serum albumin (BSA) per ml and suspended at a concentration of 10⁶ cells/ml in indocarbocyanine (Cy3)-conjugated streptavidin (Pierce; 2 µg/ml) in BSS/BSA. After 2 min at 37°C, the cells were washed in phosphate-buffered saline (PBS), transferred onto cover slips, pretreated with poly-L-lysine in PBS (Sigma; 100 µg/ml) in a 24-well plate, and centrifuged for 1 min at 200 × g. Attached cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed twice in PBS, dried in air, and mounted in ProLong antifade reagent (Molecular Probes). Control cells were fixed with paraformaldehyde before exposure to Cy3-streptavidin.

Isolation of plasma membranes and lipid rafts. Cell membranes were isolated 4 h after infection. The cells were harvested and washed twice with ice-cold PBS, and 5 × 10⁶ cells were resuspended in 500 µl of ice-cold homogenizing buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM MgCl₂) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF] plus 0.5 U of aprotinin and 0.5 U of leupeptin per ml). After 10 min of incubation on ice, the cells were homogenized by passing them 10 times through a 27-gauge needle, followed by an addition of 150 µl of tonicity restoration buffer (10 mM Tris-HCl [pH 8.0] 0.5 mM MgCl₂, 0.6 M NaCl). Insoluble material was removed by centrifugation, and the postnuclear supernatant was supplemented with 6.5 µl of 0.5 M EDTA and then centrifuged at 100,000 × g for 45 min. The membrane pellet was resuspended in ice-cold 1% Triton X-100 lysis buffer (20 mM Tris-HCl [pH 8.0], 140 mM NaCl, 2 mM EDTA, 1 mM Na₃VO₄, 1 mM PMSF, and 0.5 U of aprotinin and 0.5 U of leupeptin per ml). After 20 min on ice, the insoluble material was removed by centrifugation for 10 min at 4°C at 10,000 × g, and the supernatant was used for further analysis.

Cell activation, immunoprecipitation, immunoblotting, and kinase assay. Transfected cells were sensitized with TNP-specific IgE in suspension. After incubation for 30 min at 37°C, the cells were washed twice in BSS/BSA and activated for 5 min with TNP-BSA at a final concentration of 0.5 µg/ml. Towards the end of the activation period, the cells were briefly centrifuged and the pellet was resuspended in ice-cold 0.5 % Triton X-100 lysis buffer. After 20 min on ice, the lysate was centrifuged at 12,000 × g for 10 min to remove nuclei and insoluble remnants. IgE-FcRI complexes, Syk, or LAT was immunoprecipitated from samples equivalent to 10⁷ cells with rabbit anti-IgE, anti-Syk, or anti-LAT bound to UltraLink-immobilized protein A (Pierce), and immunoblotting with the corresponding antibodies was performed as described (2). Kinase activity of all Lyn-GFP constructs was determined by in vitro kinase assay as described (3).

Intracellular calcium measurements. Changes in the concentration of free intracellular Ca²⁺ ([Ca²⁺]_i) in transfected cells expressing GFP or GFP-containing constructs were monitored using Fura Red AM probe (Molecular Probes). GFP and Fura Red have similar excitation but different emission maxima, allowing determination of [Ca²⁺]_i only in the transfected GFP-expressing cells. The transfected cells were sensitised with IgE and at 3 h postinfection were washed and loaded with Fura Red AM (4 µg/2 × 10⁶ cells). After incubation for 1 h at 37°C, the cells were washed twice in PBS and transferred to tubes for flow cytometry. TNP-BSA at a final concentration of 0.5 µg/ml was added 60 s after the start of fluorescence-activated cell sorting (FACS) analysis, and thapsigargin (Sigma; 1 µM final concentration) was added at 180 s. The measurements were performed with a FACSscan flow cytometer (Becton Dickinson) equipped with a single 488-nm argon laser used as the excitation source. GFP fluorescence was collected at 515 to 535 nm, and Fura Red emission at 665 to 685 nm using linear amplification. GFP-negative cells were gated out, and Fura Red fluorescence was collected at the indicated time intervals.

	RESULTS

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N-terminal myristoylation but not palmitoylation is required for Lyn kinase anchoring to the plasma membrane. The unique domain of Lyn kinase contains specific sequences for myristoylation and palmitoylation at positions Gly2 and Cys3, respectively. It has been shown that a mutation in the acylation sites of other members of the Src kinase family, Lck and Fyn, leads to their inability to associate with the plasma membrane (7, 39). To analyze the significance of N-terminal acylation and SH1 and SH2/SH3 domains for the anchoring of Lyn to the plasma membrane and partitioning in lipid rafts and the in vivo functional consequences thereof, we prepared various Lyn constructs with defects in one or both acylation sites as well as in SH domains. In all constructs GFP was added at the C terminus to allow fluorescence visualization of the constructs and to distinguish them from endogenous Lyn. The kinase activities of all constructs are shown in Fig. 1. The constructs were transfected into RBL cells using an SFV expression system, and the subcellular distribution of the constructs was analyzed at 4 h postinfection.

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View larger version (81K): [in this window] [in a new window]   FIG. 2.   Subcellular localization of different Lyn constructs. The indicated Lyn constructs were introduced into RBL cells. (A) Confocal images. Cells expressing GFP alone, wild-type LynB (LynB-WT), the Lyn palmitoylation site mutant (LynB-CA) or the Lyn myristoylation and palmitoylation site mutant (LynB-GCA) were fixed and stained for surface FcRI using biotinylated IgE and Cy3-streptavidin. The left column shows the green fluorescence of Lyn-GFP constructs or GFP alone, the middle column shows the red fluorescence indicating FcRI, and the right column shows the fluorescence overlap of merged images (in yellow). (B) Association of Lyn constructs with isolated membrane fractions. Membranes were isolated from RBL cells transfected with the indicated Lyn constructs, solubilized in 1% Triton X-100 lysis buffer, and size fractionated by SDS-PAGE. Lyn constructs were detected by immunoblotting with anti-Lyn (left) or anti-GFP (right) Abs. The left arrow indicates the position of endogenous p53/p56 Lyn. The positions of size markers (in kilodaltons) are shown on the right. (C) Association of Lyn constructs with lipid rafts. The cells were lysed in 1% Triton X-100 lysis buffer, and the whole lysates were fractionated by sucrose density gradient ultracentrifugation. Individual fractions, as indicated at the bottom, were analyzed for the presence of the indicated Lyn constructs or endogenous Lyn by immunoblotting (IB) as in panel B. Lipid rafts are present in fractions 2 to 4.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Detergent-sensitive association of aggregated and unaggregated FcRI with lipid rafts as detected by sucrose density gradient analysis. RBL-2H3 cells were sensitized with [125I]IgE (1 µg/ml), and [125I]IgE-FcRI complexes were aggregated (open symbols) or not (solid symbols) with rabbit anti-IgE antibody (10 µg/ml) for 5 min. The cells were solubilized on ice in a lysis buffer containing 0.06% (circles), 0.1% (squares), or 0.2% (diamonds) Triton X-100. Total cell lysates were loaded within 40% sucrose fractions of the sucrose step gradient and fractionated by ultracentrifugation for 4 h. Points show the percentage of total cpm present in individual fractions (left axis). The percentage of the FcRI found in the lipid raft fractions was 65.6% ± 2.4%, 52.9% ± 2.5%, and 15.2% ± 1.3% for detergent concentrations of 0.06, 0.1, and 0.2%, respectively. Sucrose concentrations are also indicated (, right axis).

Co-redistribution of Lyn kinase with aggregated FcRI is dependent on Lyn N-terminal acylation, kinase activity, and conformation. FcRI and fully acylated Lyn are uniformly distributed in the plasma membrane (Fig. 2A). If the FcRI is aggregated with IgE and multivalent Ag or by other means, the complexes redistribute into small patches. As can be seen in the representative images in Fig. 4A, the aggregation of surface FcRI in RBL cells results in co-redistribution of wild-type Lyn kinase (LynB-WT), as reflected by the formation of yellow patches from the overlay of green and red fluorescence profiles. An even more extensive co-redistribution was observed when Lyn with a deleted SH1 domain (LynA-SH2) was tested. The palmitoylation-defective Lyn construct (LynB-CA) showed decreased co-redistribution, indicating that a firm anchor into lipid rafts contributes to this process. GFP alone did not show any co-redistribution.

View larger version (65K): [in this window] [in a new window]   FIG. 4.   Co-redistribution of Lyn constructs with aggregated FcRI. The indicated Lyn constructs or GFP alone was introduced into RBL cells, BMMC, or BMMC-Lyn/. Cells were sensitized with biotinylated IgE, and surface FcRI-IgE complexes were aggregated with Cy3-conjugated streptavidin for 2 min. The cells were fixed and analyzed by confocal microscopy. (A) Confocal images of RBL cells. Images are arranged as in Fig. 2A. (B) Quantitative analysis of co-redistribution of the indicated Lyn constructs with aggregated FcRI in RBL cells, BMMC, and BMMC-Lyn/. Means ± SD of correlation coefficients were calculated from at least two independent experiments, with 12 to 16 cells analyzed in each experiment.

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Palmitoylation-defective Lyn kinase is able to initiate early stages of FcRI-mediated activation. To find out whether or not Lyn kinase must be anchored in the plasma membrane and lipid rafts to initiate early FcRI-mediated activation events, we transfected various Lyn constructs into BMMC-Lyn^/ and followed the tyrosine phosphorylation of FcRI and  subunits, Syk kinase, and the LAT adaptor. For these experiments, we used constructs with Lyn kinase activity, namely, LynB-WT, palmitoylation-deficient LynB-CA, and myristoylation- and palmitoylation-deficient Lyn-B-GCA. In all experiments, a construct with GFP alone served as the negative control. The transfected cells were sensitized with IgE and stimulated with TNP-BSA. Five minutes later the cells were lysed, and FcRI, Syk, or LAT was precipitated using the appropriate Abs. The immunoprecipitates were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Antigen-induced tyrosine phosphorylation of FcRI, Syk, and LAT in cells expressing palmitoylation site-mutated Lyn. BMMC-Lyn/ were transfected with LynB-WT, LynB-CA, LynB-GCA, or GFP alone. The transfected and IgE-sensitized cells were activated or not with TNP-BSA for 5 min. The cells were lysed, and FcRI complexes, Syk, or LAT was immunoprecipitated from the postnuclear supernatants using Abs specific for IgE (A), Syk (B), or LAT (C). Proteins were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with antiphosphotyrosine MAb PY-20-HRP. After stripping, MAbs specific for FcRI  subunit, Syk, and LAT were used to estimate the relative amounts of the immunoprecipitated (IP) protein. Arrows indicate the positions of the phosphorylated  and  subunits of the FcRI (A), Syk (B), and LAT (C). (A) Positions of size markers are shown on the right (in kilodaltons). (D) Quantitative analysis of tyrosine phosphorylation of immunoprecipitated FcRI, Syk, and LAT from cells transfected with LynB-WT (WT), LynB-CA (CA), LynB-GCA (GCA), or GFP alone. Tyrosine phosphorylation of FcRI, Syk and LAT from cells transfected with LynB-WT was taken as 100%. Means ± SD were calculated from four independent experiments in each group. Data were normalized for different transfections efficiencies as determined by flow cytofluorometry.

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View larger version (35K): [in this window] [in a new window]   FIG. 6.   Antigen-induced increase in [Ca2+]i in cells expressing palmitoylation site-mutated Lyn. (A) BMMC-Lyn/ were transfected with LynB-WT, LynB-CA, LynB-GCA, or GFP alone. The cells were sensitized with IgE and loaded with Fura Red AM. At 3 h postinfection, the cells were stimulated with TNP-BSA, and [Ca2+]i was determined only in transfected cells by double-color FACS analysis. The inverse of the quenching of Fura Red fluorescence intensity (rise in intracellular calcium) in transfected cells is reported as a function of time. The long arrow and short arrow indicate time of addition of the antigen and thapsigargin, respectively. (B) The extent of activation is expressed as a ratio between the decrease in Fura Red fluorescence in FcRI-activated cells and that observed after addition of thapsigargin, which was taken as 100%. Means ± SD were calculated from six to eight experiments in each group.

Is constitutively phosphorylated FcRI associated with lipid rafts? In the experiments described above, we showed that LynB-CA is able to phosphorylate FcRI and trigger early signaling events. Because LynB-CA seems to be excluded from lipid rafts, at least those defined by resistance to 0.1% Triton X-100, the above data suggested that FcRI could be phosphorylated outside the lipid rafts. To further analyze this possibility, we studied the lipid raft association of a constitutively tyrosine-phosphorylated FcRI that results from the expression of a mutant  chain (Thr52 to Ala) in RBL-2H3- cells (45). This mutation, resulting in constitutive tyrosine phosphorylation of FcRI, could cause a conformational change of FcRI, allowing its tyrosine phosphorylation cis- or trans-molecularly. Alternatively, the T52A mutation could cause the constitutive formation of FcRI aggregates that would then be targeted to lipid rafts, where they would become phosphorylated. In this case, an increased amount of FcRIT52A should be detected in lipid rafts.

View larger version (34K): [in this window] [in a new window]   FIG. 7.   Tyrosine phosphorylation of FcRI from wt and T52A cells. The cells (5 × 106) were sensitized with IgE and stimulated (+) or not () with 0.1 µg of DNP-HSA (Ag). After 5 min the cells were lysed in 1% Triton X-100-containing lysis buffer, and tyrosine-phosphorylated proteins were immunoprecipitated (IP) with MAb 4G10. Immunoblots (IB) were probed with chicken antibody to FcRI, followed by stripping and sequential immunoblotting with an antibody recognizing constitutively phosphorylated p46 and p52 isoforms of Shc as a loading control.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The results presented in this study show that Lyn kinase must be anchored in the plasma membrane, but not necessarily firmly in lipid rafts, to be able to initiate early stages of cell activation mediated by FcRI. The first step in this process is aggregation of the FcRI by multivalent ligand and subsequent tyrosine phosphorylation of its  and  subunits by Lyn kinase (14, 31). This is followed by tyrosine phosphorylation of other signaling molecules (see the introduction). Interestingly, most of these molecules have been shown to transiently co-redistribute with the aggregated FcRI (5, 42). As shown in this study, as well as in a previous report (20), Lyn exhibits a homogenous distribution on the plasma membrane before activation, as detected by fluorescence microsocopy, and moves after FcRI aggregation into regions of aggregated FcRI. Quantitative analysis revealed that the co-redistribution of Lyn and aggregated FcRI depends on several structural and functional properties of the Lyn kinase.

First, Lyn must be fatty acylated in the N-terminal end of the molecule. In the absence of both palmitoylation and myristoylation, no anchoring of Lyn into the plasma membrane is observed and there is no co-redistribution of Lyn with aggregated FcRI. The absence of membrane anchoring and co-redistribution with FcRI was found not only in the Lyn-GCA construct, with both acylation sites mutated, but also in the Lyn-GA construct, with a mutation in the myristoylation site. This is consistent with previous data indicating that N-myristoylation is a necessary prerequisite for palmitoylation (7). The absence of palmitoylation in the LynB-CA mutant resulted in its decreased ability to localize into lipid rafts but did not preclude its association with the plasma membrane. The LynB-CA mutant exhibited a decreased co-redistribution with aggregated FcRI. This inhibition was similar in RBL, BMMC, and BMMC-Lyn^/, indicating that it is independent of the presence of the endogenous Lyn. Importantly, the decreased ability to associate with lipid rafts and the decreased co-redistribution with aggregated FcRI did not interfere with the ability of the Lyn-CA mutant to initiate early FcRI phosphorylation and activation of early events (see below).

Second, the inhibition of kinase activity caused by mutating Lys279 to Arg in full-length Lyn B kinase (LynB-CI) had no effect on anchoring of this mutant kinase to the plasma membrane and lipid rafts, but completely suppressed its co-redistribution with aggregated FcRI. This suppression was observed after transfection of LynB-CI into BMMC-Lyn^/ but not into BMMC and RBL cells, which express an endogenous Lyn. These data, together with the comparable co-redistribution of wild-type Lyn with aggregated FcRI in both BMMC and BMMC-Lyn^/, indicated that Lyn kinase activity in cis or trans was necessary for proper Lyn-FcRI interaction.

Third, consistent with these data, the removal of the catalytic domain in the LynA-SH2 construct had no effect on Lyn's incorporation in the plasma membrane and lipid rafts but inhibited co-redistribution of this construct with aggregated FcRI in BMMC-Lyn^/. Compared to LynB-CI, the extent of inhibition was smaller, suggesting that the absence of the kinase domain and/or the regulatory C-terminal tyrosine induced a conformational change improving its co-redistribution. In accord with this interpretation, LynA-SH2, exhibited a higher degree of co-redistribution with FcRI in RBL and BMMC than wild-type Lyn.

Fourth, LynA-UNI and LynB-UNI, which lack the SH1, SH2, and SH3 domains but retain both acylation sites, localized properly in the plasma membrane and in lipid rafts but failed to show any co-redistribution with aggregated FcRI. The absence of recruitment of the Lyn unique domains into regions of aggregated FcRI was observed after transfection of the Lyn constructs not only into BMMC-Lyn^/, but also into BMMC and RBL cells, i.e., cells expressing endogenous wild-type Lyn. These data, together with the finding of normal colocalization of LynB-CI in cells expressing endogenous Lyn (see above), suggest that the SH2 and SH3 domains are involved in the recruitment of Lyn to the phosphorylated receptors. Our findings are consistent with in vitro studies (25) demonstrating that the SH2 domain of Lyn interacts with the phosphorylated ITAM of the FcRI subunit; consequently, we assume that this interaction is likely involved in the LynB-CI recruitment.

The relationship between patches of aggregated FcRI, as determined by confocal microscopy, and lipid rafts with aggregated FcRI, as determined by biochemical analysis of detergent-solubilized cells, is not completely clear and will require further study. However, our confocal microscopy findings that Lyn constructs exhibited different potentials to colocalize with patches of aggregated FcRI and that this association corresponded to the activation potential of the constructs indicated that association of Lyn and FcRI in patches has a physiological significance. Furthermore, because most of the aggregated FcRI was located in small patches on the cell surface and at the same time was found associated with lipid rafts, it is very likely that microscopic patches of aggregated FcRI are found in lipid raft fractions of the sucrose gradient, especially when analysis is performed at early stages of receptor activation.

Thus, the collective data favor the co-redistribution of Lyn with FcRI as reflecting an SH2 domain-dependent redistribution of Lyn that follows the initial phosphorylation of the FcRI by a constitutive but weakly associated Lyn kinase. This view is supported by the requirement of Lyn kinase activity for co-redistribution of Lyn-CI in the BMMC-Lyn^/ by the enhanced co-redistribution of a Lyn-SH2 construct, and by our inability to observe the previously described weak interaction of Lyn-UNI with FcRI (48) in the co-redistribution assay. Regardless, the confocal studies demonstrated the unique failure of the SH2 domain-containing and catalytically active Lyn-CA to co-redistribute with the FcRI in the presence or absence of endogenous Lyn. As this Lyn construct is not effectively recruited to lipid rafts (based on our biochemical data), our findings argue the importance of Lyn residence in lipid rafts for the SH2-dependent redistribution of Lyn with FcRI and suggest that this takes place in lipid rafts.

The most important finding of this work is the evidence that LynB-CA, unlike LynB-GCA, was able to initiate early stages of mast cell activation, including tyrosine phosphorylation of FcRI  and  subunits, Syk kinase, and LAT and an increase in [Ca²⁺]_i. These data support the concept that Lyn kinase anchored to the plasma membrane but not necessarily to lipid rafts (defined by resistance to detergents) is important for early stages of mast cell activation. Thus, our data are more consistent with the model suggesting that Lyn kinase in resting cells is somehow weakly associated with a small fraction of FcRI. This association requires Lyn myristoylation and its anchoring into the plasma membrane. The initial phosphorylation of the receptor subunits but not their extensive aggregation promotes a transient association of more Lyn, thereby shifting the balance between phosphorylation and dephosphorylation state (see protein-protein model in the introduction).

Several recent findings support this notion. Using a yeast two-hybrid system, direct interaction was detected between Lyn or its unique domain and the C-terminal cytoplasmic domain of the FcRI  subunit (48). Next, immunoelectron microscopic studies of nonactivated cells indicated that FcRI and Lyn are localized in the plasma membrane in small clusters and that approximately 25% of the FcRI clusters contained Lyn (50). Furthermore, functional studies indicated that catalytically active or inactive Lyn chimeric constructs, which did not localize in lipid rafts, were able to potentiate or inhibit, respectively, aggregation-induced phosphorylation of receptors (49). Finally, our data indicating that constitutively tyrosine-phosphorylated FcRI from resting T52A cells and unphosphorylated FcRI from resting wt cells exhibit the same density on sucrose gradients suggest that FcRI can be tyrosine phosphorylated in the absence of aggregation and increased association with lipid rafts.

Although our data indicate that Lyn must be anchored in the plasma membrane for its proper signaling, they do not support the model suggesting that Lyn kinase sequestered in lipid rafts is necessary for FcRI-induced phosphorylation (see the introduction). That concept was inferred from observations that the aggregated and tyrosine-phosphorylated FcRI, derived from nonionic-detergent-solubilized activated cells, was found in the buoyant fraction of lipid rafts, together with Lyn kinase and glycosyl phosphatidylinositol (GPI)-anchored proteins (15-17). Furthermore, cholesterol depletion induced by a 60-min incubation of RBL cells with 10 mM methyl--cyclodextrin inhibited the co-redistribution of Lyn with aggregated FcRI, association of aggregated FcRI with lipid rafts, and tyrosine phosphorylation of the FcRI subunits (20, 38). However, newer data indicate that a shorter treatment of RBL cells with 10 mM methyl--cyclodextrin, which removed approximately 60% of cholesterol and led to almost complete solubilization of Lyn kinase in Triton X-100 with only a negligible effect on tyrosine phosphorylation of Syk kinase, could dramatically potentiate the FcRI-mediated secretory response (44).

However, it should be noted that all studies to date, including the present one, do not rule out a role for lipid rafts in FcRI signaling. In fact, we observed an additional increase in phosphorylation of the constitutively tyrosine-phosphorylated FcRIT52A upon antigen aggregation, an event which enhanced its association with lipid rafts. This suggests the possibility that Lyn in lipid rafts may function to sustain the activation state of the FcRI in an SH2 domain-dependent manner. Nevertheless, we also observed the normal phosphorylation of LAT, a lipid raft-resident protein (53), and normal calcium signals independent of Lyn residence in lipid rafts but dependent on Lyn anchoring to the plasma membrane. Thus, because we observed normal calcium signals and LAT is required for this event in mast cells (37), its phosphorylation in lipid rafts can occur independently of Lyn residence in lipid rafts.

Data seemingly contradictory to ours were obtained by Honda and coworkers, who found that only the palmitoylated Src kinases were able to physically interact with FcRI and to mediate signal transduction (21). However, in their experiments the activity of endogenous Src family kinases was first suppressed by introduction of a membrane-anchored C-terminal Src kinase (m-Csk) and then reconstituted with Src family kinases whose C-terminal negative regulatory sequence was replaced with a c-Myc epitope. Thus, as the authors themselves admit, in this system one cannot exclude the possibility that the transfected Src kinases competed for the inhibitory activity of m-Csk and that FcRI signaling was in fact initiated by the endogenous Src kinases instead of the transfected ones. Recent findings that a Csk binding protein, Cbp (24) or PAG (8), is associated with lipid rafts, and therefore only lipid raft-anchored kinases could compete with the substrate, make this scenario plausible.

Taken together, the combined data suggest that the constitutive and functional association between Lyn and FcRI can occur outside the lipid rafts. Nevertheless, membrane lipids seem to be involved in the proper positioning of Lyn in the plasma membrane of both resting and activated cells. The formation of large receptor aggregates in the course of activation by multivalent antigen could lead to changes in lipids surrounding the FcRI, and this could explain previous observations of a redistribution of some lipids in FcRI-activated cells (20, 42). Lipids involved not just in anchoring Lyn in the plasma membrane but also in co-localization of Lyn with FcRI in resting cells could conceivably be responsible for the signaling capacity of FcRI or of chimeras lacking the  subunit (1, 27), by stabilizing or promoting weak interactions with the  cytoplasmic tail (34, 49).