Phosphorylation of Fc{gamma}RIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts

Katarzyna Kwiatkowska1, Jürgen Frey2 and Andrzej Sobota1,*

1 Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland
2 Universität Bielefeld, Fakultät für Chemie, Biochemie II, 33615 Bielefeld, Germany

* Author for correspondence (e-mail: asobota{at}nencki.gov.pl)

Accepted 21 October 2002


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Activation of Fc{gamma} receptor II (Fc{gamma}RII) induces rearrangement of the actin-based cytoskeleton that serves as a driving force for Fc{gamma}RII-mediated phagocytosis and Fc{gamma}RII capping. To get insight into the signaling events that lead to the actin reorganization we investigated the role of raft-associated Src family tyrosine kinases in capping of Fc{gamma}RII in U937 cells. After crosslinking, Fc{gamma}RII was found to be recruited to detergent-resistant membrane domains (DRMs), rafts, where it coexisted with Lyn kinase and underwent tyrosine phosphorylation. Lyn was displaced from DRMs under the influence of DL-{alpha}-hydroxymyristic acid and 2-bromopalmitic acid, agents blocking N-terminal myristoylation and palmitoylation of proteins, respectively, and after disruption of DRM integrity by depletion of plasma membrane cholesterol with ß-cyclodextrin. Under these conditions, phosphorylation of the crosslinked Fc{gamma}RII was diminished and assembly of Fc{gamma}RII caps was blocked. The similar reduction of Fc{gamma}RII cap formation correlated with inhibition of receptor phosphorylation was achieved with the use of PP1 and herbimycin A, specific inhibitors of Src family tyrosine kinases. Phosphorylation of Fc{gamma}RIIA expressed in BHK cells, lacking endogenous Fc{gamma}Rs, was abolished by substitution of tyrosine 298 by phenylalanine in the ITAM of the receptor. The mutant receptor did not undergo translocation towards cap-like structures and failed to promote the receptor-mediated spreading of the cells, as compared to BHK cells transfected with the wild-type Fc{gamma}RIIA. On the basis of these data, we suggest that tyrosine phosphorylation of activated Fc{gamma}RIIA by raft-residing tyrosine kinases of the Src family triggers signaling pathways that control the rearrangement of the actin cytoskeleton required for Fc{gamma}RII-mediated motility.

Key words: Fc{gamma} receptor II, Membrane rafts, Lyn, Capping, Actin cytoskeleton


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Out of three classes of Fc{gamma} receptors (Fc{gamma}Rs), only the receptors of class II are single chain, 40 kDa proteins (Ravetch, 1994Go; Daëron, 1997Go). All isoforms of Fc{gamma}RII (A, B and C) bind the Fc domain of IgG via a highly conserved extracellular domain and rely on phosphorylation of their conserved, cytoplasmic tyrosine residues for signal generation. However, although Fc{gamma}RIIB, containing an immunoreceptor tyrosine-based inhibition motif (ITIM) and expressed exclusively in B cells, possesses a single YxxL sequence, which also has been shown to be essential for receptor-mediated endocytosis in B cells (Budde et al., 1994Go), two conserved tyrosine residues of Fc{gamma}RIIA/C are arranged in a so-called immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of tyrosine residues in the ITAM during activation of Fc{gamma}RIIA triggers signaling pathways, including tyrosine phosphorylation of downstream proteins, that eventually lead to reorganization of actin filaments (Greenberg, 1999Go). The actin-based cytoskeleton can in turn drive Fc{gamma}RIIA-mediated uptake of particles as demonstrated in Fc{gamma}RIIA-transfected cells (Hunter et al., 1994Go; Mitchell et al., 1994Go). This particle internalization seems to reflect the physiological ability of Fc{gamma}RIIA of monocytes, macrophages and neutrophils to promote remodeling of the actin cytoskeleton required for phagocytosis of pathogens coated by the IgG antibody (Kwiatkowska and Sobota, 1999aGo). On the other hand, the actin cytoskeleton in monocytic cells can also serve as a driving force for translocation of Fc{gamma}RII clusters (patches) in the plane of the plasma membrane and subsequent assembly of receptor caps (Kwiatkowska and Sobota, 1999bGo). Fc{gamma}RII patching is accompanied by robust protein tyrosine phosphorylation, which is a prerequisite for the assembly of the receptor caps (Kwiatkowska and Sobota, 1999cGo). It is assumed that patching can correspond to clustering of Fc{gamma}RIIA, which takes place upon binding of IgG-coated particles during Fc{gamma}RIIA-mediated phagocytosis (Kwiatkowska and Sobota, 1999aGo). However, it is not known how patched Fc{gamma}RII induces the signaling cascade(s) that control actin-driven capping of the receptor.

Tyrosine residues of the ITAM in Fc{gamma}RIIA are phosphorylated by several protein tyrosine kinases (PTKs) of the Src family among which Lyn is most likely to phosphorylate the clustered receptor in vivo (Bewarder et al., 1996Go; Ibarrola et al., 1997Go). Several data demonstrate that Src family PTKs phosphorylate ITAMs, thus triggering signaling pathways of other immunoreceptors, including T-and B-cell antigen receptors (TCR and BCR, respectively), IgE receptor (Fc{epsilon}RI) and IgA receptor (Fc{alpha}R) (Jouvin et al., 1994Go; Saouaf et al., 1994Go; van Oers et al., 1996Go; Lang et al., 1999Go). Taking into account the importance of Src family PTKs for immunoreceptor signaling it is of special interest that the kinases are sequestrated in sphingolipid/cholesterol-rich domains of the plasma membrane (rafts). Owing to the association of long, saturated fatty acyl chains of the sphingolipids with intercalating cholesterol molecules, these lipid microdomains acquire a liquid-ordered phase (London and Brown, 2000Go). Such a lipid organization allows the separation of the sphingolipid/cholesterol domains from the more liquid, glycerophospholipid-based environment of the plasma membrane, thus rendering them insoluble in non-ionic detergents, a property widely used for raft isolation (detergent-resistant domains, DRMs). In the inner leaflet of DRMs, PTKs of the Src family are anchored through double acylation of the N-terminal region with the saturated fatty acyl chains of myristate and palmitate (Shenoy-Scaria et al., 1993Go; Rodgers et al., 1994Go; Kabouridis et al., 1997Go; van't Hof and Resh, 1997Go). In the outer leaflet of the domains glycosylphosphatidylinositol (GPI)-linked proteins are docked (Friedrichson and Kurzchalia, 1998Go). Immunoreceptors, including TCR, BCR, Fc{epsilon}RI and Fc{alpha}R, were shown to be recruited to rafts upon crosslinking and undergo phosphorylation catalyzed by Src family PTKs residing in the rafts and/or relocated to these sites beside the receptors (Field et al., 1997Go; Montixi et al., 1998Go; Xavier et al., 1998Go; Lang et al., 1999Go; Cheng et al., 2001Go). Recently, merging of BCR- and Fc{gamma}RIIB1-bearing rafts was proposed to facilitate negative regulation of BCR signaling by Fc{gamma}RIIB (Aman et al., 2001Go). It should be noted, however, that there are data which do not support an involvement of membrane rafts in tyrosine phosphorylation of Fc{epsilon}RI but suggest a weak, constitutive Fc{epsilon}RI-Lyn interaction enabling the initial phosphorylation of activated Fc{epsilon}RI (Pribluda et al., 1994Go; Kovarova et al., 2001Go).

In previous work concerning Fc{gamma}RII of U937 monocytic cells, which express Fc{gamma}RIIA/C isoforms, we established that upon crosslinking the receptor associated with high molecular mass complexes containing Lyn kinase. This association of Fc{gamma}RII with Lyn, as well as the accompanying tyrosine phosphorylation of Fc{gamma}RII, was regulated by plasma membrane cholesterol level (Kwiatkowska and Sobota, 2001Go). These data suggested an involvement of membrane rafts and Lyn in Fc{gamma}RII phosphorylation and were further underscored by studies of Fc{gamma}RII in HL-60 and K562 cells (Katsumata et al., 2001Go). In the present study we examined an engagement of membrane rafts in remodeling of the actin cytoskeleton after activation of Fc{gamma}RIIA. It was found that tyrosine phosphorylation of Fc{gamma}RIIA, catalyzed by raft-associated PTKs controlled rearrangement of the actin cytoskeleton and assembly of receptor caps. The C-terminal tyrosine residue in the ITAM of Fc{gamma}RIIA (Y298) was critical for triggering of this signaling pathway.


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Cell culture
U937 human monocytic cell line (ATTC) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco BRL) as described earlier (Kwiatkowska and Sobota, 1999cGo). The baby hamster kidney BHK-21 cells stably transfected with either wild-type Fc{gamma}RIIA or Y298F mutant of Fc{gamma}RIIA (C-terminal tyrosine 298 of ITAM was replaced by phenylalanine) were generated and cloned in the eukaryotic expression vector pBEH pac18 containing the puromycin resistance gene as described previously (Engelhardt et al., 1991Go; Bewarder et al., 1996Go). BHK cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml puromycin, 1 mM sodium pyruvate and 2 mM glutamine (all Gibco BRL).

Capping of Fc{gamma}RII and cell spreading
U937 cells (1.2x106 cells/ml) were washed and maintained in HEPES-buffered saline (HBS) containing 125 mM NaCl, 4 mM KCl, 10 mM NaHCO3, 1 mM KH2PO4, 10 mM glucose, 0.2% bovine serum albumin (BSA) and 20 mM Hepes, pH 7.4. BHK cells were plated with a density 5x104/ml and 20 hours later were used for experiments conducted in HBS buffer supplemented with 1 mM CaCl2 and 1 mM MgCl2. To induce crosslinking of Fc{gamma}RII (patching), the cells were exposed for 30 minutes at 0°C to unlabeled or biotin-conjugated anti-Fc{gamma}RII IgG clone IV.3 followed by unlabeled or FITC-conjugated goat anti-mouse IgG (Calbiochem). The IV.3 antibody was purified from hybridoma (ATCC) supernatants on a Protein A-agarose column (Pierce). Subsequent warming of the cells for 10 minutes at 20°C induced formation of Fc{gamma}RII caps. The cells were fixed with 3% formaldehyde in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgCl2, pH 6.9) for 10 minutes at 0°C and 20 minutes at 20°C and mounted in mowiol containing 2.5% DABCO (Sigma). The capping efficiency was quantified in 100-150 cells per sample using a Nikon fluorescence microscope equipped with a 63x oil immersion objective. In U937 cells the cap was assumed to be formed if the crosslinked Fc{gamma}RII was accumulated on less than half of the cell surface (Kwiatkowska and Sobota, 1999bGo; Kwiatkowska and Sobota, 1999cGo). BHK cells with distinct, large conglomerates of crosslinked Fc{gamma}RII formed at the cell margins were scored as `cap positive'.

To induce BHK cell spreading, suspensions of 1x105 cells in 300 µl of DMEM/10% fetal bovine serum were plated onto precoated coverslips (18 mm in diameter) and incubated at 37°C in a 5% CO2 humidified atmosphere for up to 5 hours. The coverslips were precoated with either 20 µg/ml IV.3 antibody or with 20 µg/ml fibronectin (Sigma) for 25 minutes, washed in distilled water, air-dried and incubated with 30 mg/ml BSA for 15 minutes to block unspecific binding sites. Before use, the coverslips were washed and dried as above. In control samples, coverslips were exposed to 30 mg/ml BSA only. At various stages of spreading the cells were fixed with 3% formaldehyde, mounted in mowiol/DABCO and analyzed using a Nikon microscope and 20x objective. To quantify spreading efficiency, cells with irregular outlines and at least one prominent protrusion were scored per 100-150 cell population.

Drug treatment
Cells were pretreated for 30 minutes at 37°C in HBS medium with kinase inhibitors: piceatannol (Alexis), wortmannin (Sigma) and for 10 minutes with PP1; herbimycin A was applied overnight in serum-free growth medium supplemented with 20 mM HEPES, pH 7.4. All drugs were also present during Fc{gamma}RII crosslinking and capping or cell spreading. Control samples contained up to 0.1% DMSO as the drug carrier. The content of unesterified cholesterol in cells was measured by a fluorimetric method (Drzewiecka et al., 1999Go). In U937 cells the level of plasma membrane cholesterol was depleted for 1 hour at 37°C using ß-cyclodextrin (CDX), and cholesterol was reincorporated as described previously (Kwiatkowska and Sobota, 2001Go). Since cholesterol concentration in BHK transfectants was nine times higher than in U937 cells, BHK cells were treated for 1 hour at 37°C with methyl-ß-cyclodextrin (MCDX) owing to higher efficiency of MCDX for cholesterol release (Kilsdonk et al., 1995Go). CDX and MCDX were from Sigma. Cells were pretreated with cytochalasin B (Sigma) for 45 minutes at 37°C in HBS medium. DL-{alpha}-hydroxymyristic acid (2-hydroxytetradecanoic acid) (HMA) and 2-bromopalmitic acid (2-bromohexadecanoic acid) (BPA), from Sigma and Aldrich, respectively, were introduced to the cells as complexes with BSA (Nadler et al., 1993Go). To this end, 1 mmol of HMA or BPA was mixed with 5 g of Celite (BDH) for 10 minutes. After drying, Celite covered with the fatty acids was stirred with 60 mg/ml delipidated BSA (Sigma) for 1 hour at room temperature. The Celite was removed by centrifugation (2°C, 10 minutes, 15,000 g). In control samples HMA and BPA were absent in the prepared mixture. The obtained fatty-acid-BSA complexes and control solutions were diluted in growth medium supplemented with 2% fetal bovine serum and 20 mM HEPES, pH 7.4. U937 cells were suspended in the media at a concentration of 2x106/ml and incubated overnight at 37°C prior to experiments. BHK cells (1x105/ml) were plated and cultured for 12 hours before exposure to the drugs. The viability of the drug-treated cells was over 90% as estimated by Trypan Blue exclusion.

Immunofluorescence microscopy
To study the colocalization of Fc{gamma}RII with cell-surface proteins, serum-starved U937 cells were incubated with IV.3 mouse anti-Fc{gamma}RII IgG followed by goat anti-mouse IgG-lissamine rhodamine (Jackson ImmunoResearch) supplemented with either mouse anti-CD55 IgM (clone MEM-118, provided by Vaclav Horejsi) or human transferrin (10 µg/ml, Sigma) (30 minutes at 0°C of each incubation). At this stage (Fc{gamma}RII patching) the cells were either fixed with 3% formaldehyde or warmed for 10 minutes at 20°C (Fc{gamma}RII capping) and then fixed. Goat anti-mouse IgM-FITC (Sigma) or rabbit anti-transferrin IgG (Boehringer) followed by goat anti-rabbit IgG-FITC (Sigma) were applied after cell fixation to avoid patching of CD55 and transferrin receptor (TfR) induced by the secondary antibodies. Samples were mounted in mowiol/DABCO. Images were collected using an Olympus Fluoview confocal laser microscope in the mode of sequential excitation of FITC and rhodamine dyes to exclude crossover of their fluorescence. To quantify the degree of protein colocalization, confocal images of cells were analyzed with Quantity One software (Bio-Rad). In each cell, seven parallel lines of 1 µm in width were drawn across a pair of the confocal cell sections generated for crosslinked Fc{gamma}RII and CD55 or crosslinked Fc{gamma}RII and TfR. The distance between the lines was calculated for each cell to divide the cell diameter into constant sections. Two-colored fluorescence intensity profiles obtained for a pair of lines were superimposed. Peaks of intensity of Fc{gamma}RII labeling that corresponded to the receptor patches and overlapped with peaks of fluorescence of CD55 or TfR were scored and expressed as a percentage of total number of Fc{gamma}RII peaks found in the seven analyzed profiles per cell. At least 10 cells were analyzed in each variant.

In BHK cells, studies of colocalization of Fc{gamma}RII and tyrosine-phosphorylated proteins or actin filaments were performed after labeling of the receptor with IV.3 mouse anti-Fc{gamma}RII IgG and donkey anti-mouse IgG-Texas Red (Jackson ImmunoResearch) for 30 minutes at 0°C each. The cells were fixed with 3% formaldehyde in PHEM buffer either after binding of IV.3 anti-Fc{gamma}RII or after Fc{gamma}RII crosslinking or after subsequent cell warming for 10 minutes at 20°C. The fixed cells were permeabilized with 0.1% Triton X-100 in Tris-buffered saline (TBS) (5 minutes, 0°C), blocked in 3% BSA for 30 minutes at room temperature and exposed for 1 hour at room temperature either to rabbit anti-phosphotyrosine IgG (Transduction Laboratories) followed by anti-rabbit IgG-FITC (Sigma) or phalloidin-FITC (10 ng/ml, Sigma). In the case of labeling of tyrosine-phosphorylated proteins, TBS was supplemented with a cocktail of tyrosine phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, 50 M µM phenylarsine oxide). To visualize actin filaments in spreading BHK cells, the cells were fixed, permeabilized and incubated with phalloidin-TRITC (2 ng/ml, Sigma) as described above. Samples mounted in mowiol/DABCO were analyzed using a Nikon fluorescence microscope as described above.

Gradient ultracentrifugation
U937 cells, 1.2x107 per sample, were lysed for 30 minutes at 0°C in 220 µl of Triton X-100 (TX-100) lysis buffer composed of 0.2% TX-100, 100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 30 mM Hepes, pH 7.5, phosphatase inhibitors: 1 mM Na3VO4, 50 µM phenylarsine oxide, 30 mM p-nitrophenylphosphate and a cocktail of protease inhibitors (Boehringer). Cells were further sheared by passing them five times through a 25-G needle. After clarification (1.5 minutes, 480 g, 4°C), 200 µl of the cell lysate was adjusted to 40% OptiPrep (Sigma) and 10% sucrose. 600 µl of the mixture was transferred to RP55-S centrifuge tubes (Sorvall) and overlaid with 400 µl of ice-cold solutions of 30%, 25%, 20% and 300 µl of 0% OptiPrep in 0.2% TX-100 lysis buffer supplemented with 10% sucrose. In experiments where phosphorylation of Fc{gamma}RII was analyzed, 4x107 cells were lysed in 600 µl of 0.2% TX-100 lysis buffer containing 40% OptiPrep and 10% sucrose, clarified, placed on the bottom of the ultracentrifuge tube and overlaid with an OptiPrep gradient as above. Gradients were spun for 3 hours at 170,000 g, 4°C (RCM 100 ultracentrifuge, Sorvall). Seven fractions of 300 µl were collected from the top of the gradient.

Immunoprecipitation and in vitro kinase assay
Fc{gamma}RII was immunoprecipitated from whole U937 cell lysates and from OptiPrep gradient fractions. For immunoprecipitation, cells were either exposed only to IV.3 mouse anti-Fc{gamma}RII (30 minutes, 0°C) or incubated with anti-Fc{gamma}RII followed by goat anti-mouse IgG (30 minutes, 0°C) or left untreated. Subsequently, the cells (1x107 per sample) were solubilized for 30 minutes at 0°C in 3 ml of TX-100/Nonidet P-40 lysis buffer (1% TX-100, 0.5% Nonidet P-40, 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 30 mM Hepes, pH 7.4, protease and phosphatase inhibitors as described above). The lysates were clarified by centrifugation (1.5 minutes, 480 g, 4°C) and supplemented with 50 µl of 10% protein G-bearing Omnisorb (Calbiochem). When cells were treated only with IV.3 antibody, Omnisorb preadsorbed with 1.5 µg of goat anti-mouse IgG was added to the cell lysates to facilitate precipitation of non-crosslinked Fc{gamma}RII. After incubation for 2.5 hours at 4°C and an additional 30 minutes at 20°C, the Omnisorb beads were collected by centrifugation and washed five to seven times in lysis buffer containing 0.5% TX-100 and finally in TBS. The precipitates were boiled in 30 µl of 2xSDS-sample buffer and subjected to SDS-PAGE. To precipitate Fc{gamma}RII from OptiPrep gradient fractions, 200 µl of each fraction were diluted twice with 0.2% TX-100 lysis buffer and supplemented with 30 µl of 10% Omnisorb. Immmunoprecipitation was conducted overnight at 4°C. The precipitates were washed seven times with 0.2% TX-100 lysis buffer and prepared for SDS-PAGE as described above.

To estimate the activity of Lyn and Syk, the kinases were immunoprecipitated from U937 cells (5x106 per sample) pretreated with PP1 or piceatannol or DMSO and subsequently incubated in the presence of the drugs for 30 minutes at 0°C with IV.3 mouse anti-Fc{gamma}RII and goat F(ab)2 anti-mouse IgG and warmed for 30 seconds at 20°C. Cells were lysed in 750 µl of lysis buffer as described for Fc{gamma}RII immunoprecipitation. However, Nonidet P-40 was exchanged in the lysis buffer for 20 mM N-octylglucoside to release Lyn from TX-100-insoluble membrane fragments. After clarification, lysates (4°C) were supplemented with 3 µg of rabbit anti-Lyn IgG or rabbit anti-Syk IgG (N-19 and C-20, 1.5 µg each) (Santa Cruz Biotechnology) and 3 hours later 50 µl of 10% protein A-bearing Pansorbin (Calbiochem) was added for a additional 2 hours. The beads were washed four times in lysis buffer containing 0.5% TX-100, once in TBS and once in kinase buffer (50 mM NaCl, 15 mM MnCl2, 2 mM DTT, 30 mM imidazole, pH 7.4) and suspended in 200 µl of the kinase buffer containing 2 mM ATP. The kinase assay was started by transferring the samples to 37°C. After 30 minutes, the beads were pelleted and boiled in 2xSDS-PAGE buffer.

Immunoblotting
Immunoprecipitates, OptiPrep gradient fractions of U937 cells and lysates of BHK cells were separated by to 10% SDS-PAGE. For lysis, BHK cells (3.4x106 per sample) were treated with 100 µl of the TX-100/Nonidet P-40 lysis buffer containing the protease and phosphatase inhibitors mentioned above (0°C), and 10 minutes later, 30 µl of 4xSDS-sample buffer were added. After electrophoresis, proteins were transferred to nitrocellulose membranes (Kwiatkowska and Sobota, 2001Go). The membranes were incubated with antibodies: mouse or rabbit anti-Lyn IgG, rabbit anti-Syk IgG, rabbit anti-TfR IgG, mouse anti-phosphotyrosine IgG, clone PY99 (all from Santa Cruz Biotechnology), mouse anti-actin (Boehringer) followed by goat anti-mouse IgG or anti-rabbit IgG labeled with peroxidase (Santa Cruz Biotechnology). Mouse anti-phosphotyrosine, clone PY-20, conjugated with peroxidase (Transduction Lab) was used to reveal autophosphorylation of immunoprecipitated Lyn and Syk. Distribution of Fc{gamma}RII in the OptiPrep gradient fractions was analyzed on immunoblots by detection of biotin-labeled IV.3 anti-Fc{gamma}RII IV.3 antibody with the use of goat anti-biotin peroxidase-conjugated IgG (Sigma). As non-labeled IV.3 was applied to immunoprecipitate Fc{gamma}RII from whole cell lysates, rabbit anti-Fc{gamma}RII serum, kindly provided by J.-L. Teillaud, was applied to detect the receptor in a corresponding series of blots. CD55 was detected in the OptiPrep gradient fractions on slot-blots with the use of mouse anti-CD55 IgG (clone IA10, kindly provided by V. Horejsi). These studies were performed on fractions obtained from cells untreated with IV.3 mouse anti-Fc{gamma}RII to avoid crossreactivity of anti-mouse IgG-peroxidase applied for detection of mouse anti-CD55 IgG. Immunoreactive bands were visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce) in a Fluor-S MultiImager and quantified densitometrically using Quantity One software (Bio-Rad). Data shown are the mean±s.e.m. from n number of experiments. Prestained molecular mass standards were from Bio-Rad.


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Capping of Fc{gamma}RII depends on DRM integrity and activity of Src family kinases
In U937 cells exposed to mouse anti-Fc{gamma}RII IgG, clone IV.3, the receptor was dispersed in the plane of the plasma membrane; however, after subsequent crosslinking with antimouse antibody (0°C), Fc{gamma}RII was concentrated into small, distinct patches (Fig. 1A,B). Warming of the cells for 10 minutes at 20°C induced the accumulation of the receptor clusters into polar caps in 76.4±5.3% of the cells (n=5) (Fig. 1C).



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Fig. 1. Cholesterol and Src family kinases control the assembly of Fc{gamma}RII caps and tyrosine phosphorylation of the receptor in U937 cells. (A-F) Localization of Fc{gamma}RII on the cell surface. (A) Cells exposed to IV.3 mouse anti-Fc{gamma}RII, fixed and postlabeled with anti-mouse FITC-IgG. (B) Cells treated at 0°C with IV.3 mouse anti-Fc{gamma}RII and anti-mouse FITC-IgG (crosslinking). (C) Capping of crosslinked Fc{gamma}RII after 10 minutes of cell warming at 20°C. (D) Cells pretreated with 4 mM CDX (1 hour, 37°C) followed by crosslinking of Fc{gamma}RII at 0°C and (E) cells after subsequent incubation for 10 minutes at 20°C. (F) Reconstitution of Fc{gamma}RII cap assembly after reincorporation of cholesterol (30 minutes, 37°C) into cells pretreated with 4 mM CDX. Bar, 10 µm. (G) Quantification of Fc{gamma}RII cap formation as a function of cholesterol content. Bars on the left side depict a dose-dependent inhibition of Fc{gamma}RII capping by CDX treatment (1 hour, 37°C). Bars on the right side show the assembly of Fc{gamma}RII caps in cholesterol-depleted cells after 30 minutes of cholesterol reincorporation (+) or without the reincorporation (-). (-•-) Cholesterol content. (H) Tyrosine phosphorylation (PY) of Fc{gamma}RII immunoprecipitated from whole lysates of U937 cells (upper panel). Cells were either untreated with antibodies or exposed at 0°C to IV.3 anti-Fc{gamma}RII alone (non-crosslinked) or incubated at 0°C with mouse anti-Fc{gamma}RII and goat anti-mouse IgG (crosslinking). Prior to Fc{gamma}RII crosslinking, cells were preincubated with 8 µM herbimycin A (Herb), 5 mM CDX and 1 mM HMA or without inhibitors (control, Ctrl). A subset of the control cells was warmed at 20°C for 10 and 20 minutes to induce the formation of Fc{gamma}RII caps (capping). 52 is a molecular mass standard in kDa. Arrowhead indicates tyrosine phosphorylated Fc{gamma}RII. A corresponding part of the membrane was reprobed with rabbit anti-Fc{gamma}RII to reveal amounts of the precipitated receptor (lower panel). (I) Influence of herbimycin A (Herb), PP1, HMA, BPA, piceatannol (PCT) and wortmannin (Wort) on Fc{gamma}RII cap assembly. Results are the mean±s.e.m. of three to five experiments. (J) In vivo treatment of cells with PP1 and piceatannol led to the inhibition of Lyn and Syk activity. Cells were treated with 15 µM PP1 and 25-100 µM piceatannol (PCT) before (30 minutes) and during Fc{gamma}RII crosslinking. Syk and Lyn kinases were immunoprecipitated from lysates of the cells and the kinase assay was performed on the obtained immunocomplexes. The samples were blotted with anti-PY to reveal autophosphorylation of Syk and Lyn (arrowheads) and later reprobed with rabbit anti-Syk and mouse anti-Lyn IgG to indicate the amounts of the precipitated kinases. Lanes `non-crosslinked' show the autophosphorylation of the kinases in cells exposed to IV.3 anti-Fc{gamma}RII only. The level of Lyn and Syk autophosphorylation was estimated densitometrically in relation to the kinase content and is shown over corresponding lanes (the mean from two experiments). Lane "-", control of immunoprecipitation - cell lysates supplemented with Pansorbin only.

 

To assess the role of DRMs in Fc{gamma}RII capping, the cells were exposed to CDX. The drug removed cholesterol and reduced the formation of Fc{gamma}RII caps in a dose-dependent manner, arresting the receptor in patches (Fig. 1D,E,G). After reincorporation of cholesterol, reconstitution of Fc{gamma}RII capping was observed (Fig. 1F,G). The level of Fc{gamma}RII capping in the cholesterol-reloaded cells reached, but did not exceed, that in CDX-untreated cells, despite the cells acquiring twofold higher amounts of cholesterol over their initial cholesterol content (4.2 µg/2x106 cells after 30 minutes of cholesterol reincorporation versus 2.6 µg/2x106 control cells). Incubation of the CDX-treated cells without cholesterol-delivering complexes did not restore either Fc{gamma}RII cap assembly or cholesterol level (Fig. 1G).

As DRMs are centers of anchorage for kinases of the Src family, we examined an involvement of these kinases in the phosphorylation and capping of Fc{gamma}RII. Fig. 1H shows that crosslinking (patching) of Fc{gamma}RII induced an intensive phosphorylation of the receptor while progressive dephosphorylation of Fc{gamma}RII correlated with the formation of the receptor caps. Several downstream proteins undergo a similar cycle of tyrosine phosphorylation/dephosphorylation during patching and capping of Fc{gamma}RII, as shown earlier (Kwiatkowska and Sobota, 1999cGo). The phosphorylation of crosslinked Fc{gamma}RII was completely blocked by depletion of the plasma cholesterol (Fig. 1H, lane CDX). Further studies indicated that activity of Src family kinases associated with DRMs was required for both Fc{gamma}RII phosphorylation and receptor cap assembly. Two Src-family-specific PTK inhibitors, PP1 at 15 µM and herbimycin A at 8 µM, were found to significantly reduce the assembly of Fc{gamma}RII caps (Fig. 1I). Herbimycin A was likely to exert its prominent inhibitory effect by degradation of the Src family PTKs (June et al., 1990Go). An overnight treatment of cells with 8 µM of this drug led to a 54.1±3.2% reduction in Lyn content, estimated in relation to the actin level, in whole cell lysates by densitometric analysis of immunoblots. Under these conditions, phosphorylation of crosslinked Fc{gamma}RII was strongly attenuated (Fig. 1H, lane Herb) and assembly of Fc{gamma}RII caps was inhibited by 89.2±1.1% (Fig. 1I). Since the Src family PTKs rely on dual acylation as a signal for DRM location (Shenoy-Scaria et al., 1993Go; Kabouridis et al., 1997Go; van't Hof and Resh, 1997Go), we next altered the protein fatty acylation level in cells with the use of HMA and BPA. HMA is a potent inhibitor of N-myristoyltransferase, blocking co-translational attachment of myristate to proteins including Src family kinases (Nadler et al., 1993Go; van't Hof and Resh, 1999Go). BPA preferentially disturbs protein palmitoylation although a negative influence on Fyn myristoylation was also found (Webb et al., 2000Go). Both agents markedly suppressed the assembly of Fc{gamma}RII caps; nearly 90% inhibition of capping by 1 mM HMA, accompanied by abrogation of phosphorylation of crosslinked Fc{gamma}RII, was prominent (Fig. 1H, lane HMA and 1I). The overall data indicate a sensitivity of Fc{gamma}RII capping to DRM-targeting agents and point to an involvement of Src family PTKs in this event, although the effect exerted by HMA and BPA on capping owing to their influence on lipid synthesis can not be excluded.

It is of interest that the negative influence of different concentrations of piceatannol, a Syk kinase inhibitor, on Fc{gamma}RII capping was less profound, reaching 48% of capping inhibition at 100-200 µM (Fig. 1I); however, 200 µM piceatannol affected cell viability. No reduction of Fc{gamma}RII cap assembly occurred under the influence of 0.001-1 µM wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase) (Fig. 1I). Syk is proposed to control actin rearrangement during phagocytosis mediated by Fc{gamma}Rs and the moderate influence of piceatannol on actin-dependent capping of Fc{gamma}RII was unexpected. Therefore, we examined Syk activity, reflected by kinase autophosphorylation, in cells treated with the drug. In our hands, 25 µM piceatannol blocked Fc{gamma}RII-induced Syk activity by 67%, whereas at 100 µM the drug reduced the kinase activity by 83% (Fig. 1J). The discrepancy between the strong inhibition of Syk activity and weaker effect on Fc{gamma}RII capping exerted by piceatannol indicated that Syk is not a key kinase for cap assembly. In addition, piceatannol at concentrations above 25 µM may affect Fc{gamma}RII capping not only by Syk inhibition but also as non-specific inhibitor of Src family PTKs (Majeed et al., 2001Go). In fact, examination of Lyn activity revealed that 100 µM piceatannol diminished autophosphorylation of this kinase by 61%, whereas PP1 at 15 µM inhibited Lyn activity by 79% (Fig. 1J). This PP1 concentration also inhibited the enhancement of Syk activity induced by Fc{gamma}RII crosslinking, which is in line with the supposition that Src family PTKs act upstream of Syk in the signaling pathway of Fc{gamma}RIIA (Cooney et al., 2001Go).

Phosphorylation of crosslinked Fc{gamma}RII within DRMs controls assembly of Fc{gamma}RII caps
The majority of the total Lyn kinase in U937 cells, 78.2±3.9% (n=3), was accumulated in DRMs as revealed by flotation of Lyn to fractions 1-2 of an OptiPrep density gradient (buoyant density 1.08-1.13 g/ml). These fractions also contained 63.2±2.2% of the total CD55, a GPI-anchored protein (Fig. 2A). Depletion of cholesterol in cells with the use of 5 mM CDX evoked a significant shift of Lyn and CD55 within the density gradient from DRMs to high density fractions 6-7, in which 64.8±0.7% (n=3) of the kinase content and total CD55 were found (Fig. 2A). Fractions 6-7 (buoyant density 1.28-1.34 g/ml) contained detergent-soluble proteins, including TfR. No traces of this receptor were found in DRM fractions 1-2 isolated either from control cells or cells cultured under inhibitory conditions (Fig. 2A, bottom). Pretreatment of cells with 1 mM HMA or 0.3 mM BPA diminished the amount of Lyn associated with DRMs to 35.9±3.1% and 44.9±3.8% of the total (n=4), respectively (Fig. 2A, left panel). The exposure of cells to BPA led to an enrichment of Lyn in the intermediate fractions 4-5 of the gradient (buoyant density 1.21-1.24 g/ml). On the other hand, the HMA treatment caused a displacement of Lyn toward high-density fractions 6-7. Moreover, under the influence of HMA, the cellular level of Lyn estimated in relation to actin content was reduced by 40.8±5.5% (n=5). As myristoylation of the Src family PTKs is required for the palmitoylation to occur (Shenoy-Scaria et al., 1994Go) the non-acylated Lyn formed after HMA treatment is likely to undergo a rapid degradation that leads to the detected reduction of the Lyn content in U937 cells. Such a phenomenon was described for Lck and Lyn kinases in HMA-treated T and B cells (Nadler et al., 1993Go). In striking contrast to Lyn, an association of CD55 with DRMs was not disrupted by preincubation of U937 cells with HMA and BPA (Fig. 2A, right panel). These data indicate that the impairment of protein fatty acylation by HMA and BPA significantly affected PTKs of the Src family that are located in the inner leaflet of DRMs without disturbing the organization of the outer leaflet of the domains where CD55 is anchored. In addition, docking of CD55 in DRMs relies on the GPI moiety, which is expected to be acylated with palmitate or other long-chain fatty acids. As BPA did not diminish association of CD55 with DRMs it seems that the drug affects mainly protein palmitoylation without general reduction of the fatty acyl coenzyme A population in the cells.



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Fig. 2. Distribution of Lyn, CD55, TfR and Fc{gamma}RII in density gradient fractions. U937 cells unexposed to inhibitors (controls, Ctrl) or preincubated with 1 mM HMA, 0.3 mM BPA or 5 mM CDX were lysed and fractionated on a density gradient in the presence of 0.2% TX-100. Fractions were immunoblotted for the presence of Lyn, CD55 and TfR (A) or biotin-labeled IV.3 anti-Fc{gamma}RII reflecting the receptor distribution (B). In B, the distribution of cross-linked (+) and non-crosslinked (-) Fc{gamma}RII is shown; in A, proteins of fractions derived prior to Fc{gamma}RII labeling are demonstrated. Plots display the results of densitomeric analysis of the blots shown above. The estimated amounts of an individual protein in fractions are expressed as a percentage of this total protein content in the whole gradient. Symbols used for visualization of various experimental conditions in plots of (A) and (B) are shown at the bottom of panel (B). The results are representative of three to five separate experiments.

 

Fc{gamma}RII in unstimulated cells, exposed only to biotin-labeled anti-Fc{gamma}RII IgG, was fully released from the plasma membrane with 0.2% TX-100 and was recovered in fractions 5-7 of the density gradient, as revealed by the distribution of the anti-Fc{gamma}RII antibody (Fig. 2B). However, upon crosslinking (patching), 84.1±1.5% (n=3) of total Fc{gamma}RII was redistributed to DRM fractions 1-2, whereas the rest of the receptor population was found in fractions 3-4 of the gradient (Fig. 2B). Depletion of plasma membrane cholesterol with 5 mM CDX rendered the receptor susceptible to TX-100 solubilization again. This was observed as a clear shift in the crosslinked Fc{gamma}RII toward fractions 4-7 of higher density in the gradient (Fig. 2B). By contrast, exposing the cells to 1 mM HMA or 0.3 mM BPA did not impair the appearance of the crosslinked Fc{gamma}RII in the lightest region of the density gradient, indicating that the fatty acylation of proteins, including that of Src family PTKs, is not crucial for the association of crosslinked Fc{gamma}RII with DRMs (Fig. 2B). Taken together with the lack of influence of HMA and BPA on CD55 presence in DRMs, these data suggest that the drugs had no non-specific effects on DRMs lipids and did not impair association of the crosslinked Fc{gamma}RII with DRMs.

Immunofluorescence studies on Fc{gamma}RII and CD55 distribution on the surface of U937 cells confirmed the interaction of crosslinked Fc{gamma}RII with DRMs. Although in unstimulated cells a diffuse distribution of CD55 was seen (data not shown), crosslinking of Fc{gamma}RII was found to evoke concomitant clustering of CD55, and significant colocalization of both proteins was detected (Fig. 3A-C). This pattern closely resembled copatching of Fc{gamma}RII and Lyn kinase as described previously (Kwiatkowska and Sobota, 2001Go). Quantitative analysis of Fc{gamma}RII and CD55 distribution revealed that 76.9±4.8% of the receptor patches colocalized with CD55 clusters (Fig. 4E). Apparent colocalization of CD55 and Fc{gamma}RII was maintained during assembly of the receptor caps, although a fraction of CD55 remained scattered outside the cap region (Fig. 3D). Surprisingly, during crosslinking of Fc{gamma}RII, aggregation of TfR also occurred; however, the clusters of Fc{gamma}RII (red) and TfR (green) were clearly segregated and only 15.7±3.2% of the clusters overlapped (Fig. 3E-H). The clusters of TfR are likely to reflect an accumulation of the receptor in coated pits.



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Fig. 3. Patches and caps of Fc{gamma}RII colocalize with CD55 but not with TfR. Distribution of Fc{gamma}RII (A, red) and CD55 (B, green) during Fc{gamma}RII crosslinking. (C) The merged image of A and B shows colocalization of CD55 and Fc{gamma}RII in receptor patches (yellow). (D) The merged image of Fc{gamma}RII (red) and CD55 (green) reveals the colocalization of the proteins in Fc{gamma}RII cap (yellow). Distribution of Fc{gamma}RII (F, red) and TfR (G, green) during Fc{gamma}RII crosslinking. (H) Merged images of Fc{gamma}RII (red) and TfR (green) show the separation of the proteins during Fc{gamma}RII patching. E shows representative line profiles of intensity of fluorescent labeling of crosslinked Fc{gamma}RII and CD55 (upper plot) and crosslinked Fc{gamma}RII and TfR (lower plot). Seven such profiles were generated for each analyzed cell to estimate the protein colocalization. Bar, 5 µm.

 


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Fig. 4. Crosslinked Fc{gamma}RII in U937 cells associates with Lyn and undergoes tyrosine phosphorylation in DRMs. (A) Tyrosine phosphorylation (PY) of proteins in OptiPrep gradient fractions. Cells were exposed to 1 mM HMA, 100 µM piceatannol, 15 µM PP1 or 5 mM CDX or left untreated (control) before crosslinking of Fc{gamma}RII with biotin-labeled IV.3 mouse anti-Fc{gamma}RII and goat anti-mouse IgG. For the analysis, 12 µl of gradient fractions 1-2 and only 4 µl of fraction 6 were used for a clearer visualization of proteins. Molecular mass standards are shown in kDa. An arrowhead marks the 40 kDa phosphoprotein corresponding to Fc{gamma}RII. Dots indicate proteins whose phosphorylation was significantly reduced by piceatannol. (B) Immunoprecipitation of Fc{gamma}RII from the gradient fractions (200 µl each). Immunoprecipitates were immunoblotted to simultaneously detect biotin-labeled anti-Fc{gamma}RII, reflecting the presence of the receptor (upper panel) and tyrosine-phosphorylated proteins (PY) (middle panel). An arrowhead points to Fc{gamma}RII; small arrows point to a doublet of 53/56 kDa phosphoproteins. The blots from the middle panel were reprobed with rabbit anti-Lyn (lower panel). The data represent one out of three experiments.

 

A distinct subset of strongly tyrosine-phosphorylated proteins was found in the buoyant gradient fractions 1-2 recovered from cells subjected to Fc{gamma}RII crosslinking (Fig. 4A). These included proteins in the range of 53-75 kDa and proteins of 40 kDa (Fig. 4A, arrowhead) and 20 kDa. The 40 kDa phosphoprotein, present in DRM fractions 1-2, was Fc{gamma}RII as demonstrated by its immunoprecipitation with the IV.3 anti-Fc{gamma}RII antibody (Fig. 4B, middle panel, arrowhead). Lyn kinase was found to co-immunoprecipitate with the receptor from low density fractions 1-2 (Fig. 4B, lower panel), most likely as a phosphorylated enzyme judging from the detection of the corresponding 53/56 kDa doublet by antiphosphotyrosine antibody (Fig. 4B, middle panel, small arrows). No phosphorylation of Fc{gamma}RII was visible in the receptor immunoprecipitates obtained from gradient fractions 3-7, and simultaneously barely detectable amounts of Lyn were found in these complexes (Fig. 4B).

An impairment of protein acylation by 1 mM HMA and an inhibition of the activity of Src family PTKs with 15 µM PP1 resulted in an attenuation of phosphorylation of the crosslinked Fc{gamma}RII and the accompanying proteins (Fig. 4A). The amount of Lyn kinase co-immunoprecipitated with Fc{gamma}RII from DRMs in HMA-treated cells was substantially reduced (Fig. 4B). The residual phosphorylation of Fc{gamma}RII detected in PP1-treated cells could either reflect a functional redundancy of multiple endogenous Src family PTKs or could result from the activity of PP1-insensitive Syk kinase. The latter possibility seems unlikely since pretreatment of U937 cells with 100 µM piceatannol enhanced, rather than diminished, the phosphorylation of the cross-linked Fc{gamma}RII and other proteins located in DRMs (Fig. 4A). Accordingly, the co-immunoprecipitation of phosphorylated Lyn kinase with the receptor from DRM fractions was also preserved under these conditions (Fig. 4B). The inhibition of Syk activity reduced the phosphorylation of some soluble proteins in fractions 6-7 (Fig. 4A, dots), which could account for the moderate inhibition of Fc{gamma}RII capping in the piceatannol-treated cells (Fig. 1H). Tyrosine phosphorylation of the crosslinked Fc{gamma}RII and other proteins residing in DRMs, as well as soluble proteins of fractions 6-7, was abolished by depletion of plasma membrane cholesterol with 5 mM CDX (Fig. 4A). No phosphorylated Fc{gamma}RII and no traces of Lyn kinase were seen in receptor immunoprecipitates obtained under these conditions (Fig. 4B).

Taken together, the results show that crosslinked Fc{gamma}RII is recruited to DRMs where it coexists jointly with Lyn kinase and undergoes tyrosine phosphorylation. These events are required for subsequent assembly of Fc{gamma}RII caps.

Tyrosine residue 298 of ITAM in Fc{gamma}RIIA is required for the receptor-triggered actin reorganization
Phosphorylation of crosslinked Fc{gamma}RII is likely to trigger signal transduction cascades targeting the actin cytoskeleton which in turn controls assembly of Fc{gamma}RII caps (Kwiatkowska and Sobota, 1999bGo). To further explore this pathway we examined the actin rearrangement induced by wild-type Fc{gamma}RIIA and Y298F-substituted Fc{gamma}RIIA expressed in BHK cells. By analogy to U937 cells, crosslinking of wild-type Fc{gamma}RIIA in BHK cells with IV.3 mouse anti-Fc{gamma}RII IgG and anti-mouse IgG at 0°C induced an intense phosphorylation of the receptor followed by its progressive dephosphorylation after shifting of the cells to 20°C (Fig. 5A). Furthermore, the receptor phosphorylation was strongly inhibited by 10 µM PP1 and 1 mM HMA, indicating an involvement of Src family tyrosine kinases in this process (Fig. 5B). Substitution of Y298, the C-terminal tyrosine residue of the ITAM of Fc{gamma}RIIA, by phenylalanine completely abrogated phosphorylation of the receptor upon its crosslinking (Fig. 5A). Detection of receptor-bound IV.3-biotin IgG confirmed that cell clones transfected with wild-type and Y298F-mutated Fc{gamma}RIIA expressed comparable amounts of the receptors, estimated in relation to actin content in the cells (Fig. 5A).



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Fig. 5. Tyrosine phosphorylation of Fc{gamma}RIIA expressed in BHK cells is required for translocation of the crosslinked receptor. (A) Comparison of phosphorylation of wild-type (wt) Fc{gamma}RIIA and mutant Y298F Fc{gamma}RIIA in cells after receptor crosslinking at 0°C and after subsequent warming of the cells for 10-20 minutes at 20°C (capping). Distribution of biotin-labeled IV.3 anti-Fc{gamma}RII reflects the amounts of the receptor in the blot, whereas actin labeling demonstrates equal loading of the proteins. On the left, molecular mass standards are shown in kDa. (B) Tyrosine phosphorylation of wild-type Fc{gamma}RIIA, induced by receptor crosslinking (Ctrl), is inhibited by 10 µM PP1 and 1 mM HMA. The blot was reprobed with anti-actin to demonstrate that equal amounts of proteins were loaded on the gel. Arrowheads indicate tyrosine-phosphorylated Fc{gamma}RIIA in A and B. (C) Formation of cap-like structures by crosslinked wild-type Fc{gamma}RIIA and Y298F Fc{gamma}RIIA in BHK transfectants. The results are the means±s.e.m. from four experiments. (D) Colocalization of crosslinked wild-type Fc{gamma}RIIA with tyrosine-phosphorylated proteins and actin filaments during receptor crosslinking (0°C) and formation of cap-like structures (10 minutes at 20°C). Inserts in the `crosslinking' row show a magnified fragment of the cell, which reveals significant colocalization of crosslinked Fc{gamma}RIIA and phosphotyrosine-bearing proteins. (E) Crosslinking of mutant Y298F Fc{gamma}RIIA at 0°C did not induce tyrosine phosphorylation of proteins and no cap-like structures are formed after 10 minutes at 20°C. In D and E, the top panels show cells fixed directly after binding of IV.3 anti-Fc{gamma}RII, which reveals diffuse distribution of the non-crosslinked receptors on the cell surface and low level of phosphotyrosine-bearing proteins inside the cells. Bar, 15 µm.

 

Immunofluorescence studies revealed that crosslinking of both wild-type and Y298F-substituted Fc{gamma}RIIA was correlated with clear clustering of the receptors (Fig. 5D,E). Before the receptor clustering, minute amounts of tyrosine-phosphorylated proteins, located mainly at focal contacts, were visible in both types of BHK transfectants (Fig. 5D,E). However, crosslinking of wild-type Fc{gamma}RIIA evoked strong tyrosine phosphorylation of proteins, which colocalized with receptor patches (Fig. 5D). It is of interest that subsequent shifting of the cells from 0°C to 20°C for 10 minutes led to accumulation of the crosslinked receptor as 1-4 large conglomerates located at the edges of rounding cells. The conglomerates of Fc{gamma}RIIA resembled the cap-like structures of crosslinked epidermal growth factor receptor described previously in A431 cells (Kwiatkowska et al., 1991Go). The cap-like structures of Fc{gamma}RIIA were formed in 64.8±2.4% of BHK cells and were enriched in tyrosine-phosphorylated proteins as well as actin filaments (Fig. 5C,D). Disruption of microfilaments by pretreatment of cells with 10 µM cytochalasin B inhibited translocation of the Fc{gamma}RIIA patches into the cap-like aggregates (data not shown). In cells transfected with Y298F Fc{gamma}RIIA, protein tyrosine phosphorylation remained at a low level upon receptor crosslinking and some of the phosphotyrosine-bearing proteins were concentrated at focal contacts (Fig. 5E). Formation of Y298F Fc{gamma}RIIA conglomerates was severely impaired: 8.1±3.9% of the cell population formed small aggregates of crosslinked receptor after 10 minutes at 20°C (Fig. 5C). Tyrosine-phosphorylated proteins remained concentrated in focal contacts, and no accumulation of actin filaments was detected at the crosslinked Y298F Fc{gamma}RIIA (Fig. 5E).

In another approach, we addressed the Fc{gamma}RIIA-actin cytoskeleton relations by studying adhesion and spreading of BHK transfectants on anti-Fc{gamma}RIIA IV.3-coated substratum. When seeded on such substratum, wild-type Fc{gamma}RIIA-expressing cells adhered within 30 minutes and during the next 1.5 hours 71.1±2.9% of the cells spread, forming many thin, finger-like protrusions and flat lamellae (Fig. 6A,B). The adhesion of the cells depended on interaction between Fc{gamma}RIIA and anti-Fc{gamma}RIIA since BSA-coated substratum did not promote the cell attachment (Fig. 6A). Furthermore, spreading of cells onto IV.3-coated substratum required participation of the actin cytoskeleton because it was blocked by 10 µM cytochalasin B (Fig. 6A). Immunofluorescence studies of actin filament organization in spreading cells revealed prominent actin ribs in the protrusions of the cells, abundant dotted staining of actin dispersed through the cell and few, delicate stress-fibers (Fig. 6A). For comparison, cells spreading on fibronectin-coated substratum (81.3±3.4% of the cell population) manifested a more elongated and polarized shape with stronger stress-fibers (Fig. 6A).



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Fig. 6. Wild-type (wt) Fc{gamma}RIIA but not Y298F Fc{gamma}RIIA mediates spreading of BHK transfectants. (A) Cells were plated on coverslips coated with IV.3 anti-Fc{gamma}RII. In control samples, BSA or fibronectin (Fn) were used as a substratum. Where indicated, cells were pretreated with 15 µM cytochalasin B (CB), 10 µM PP1, 1 mM HMA or 8 mM MCDX. Cells after 2 hours (or 5 hours, as indicated) of spreading are shown. Organization of actin filaments visualized with phalloidin-TRITC is demonstrated in the lower panel. Bars, 20 µm. (B) Quantification of spreading of BHK cells transfected with wild-type Fc{gamma}RIIA and Y298F Fc{gamma}RIIA on IV.3-coated substratum 2 hours after plating. Spreading of cells transfected with wild-type receptor was inhibited after pretreatment with 15 µM cytochalasin B, 10 µM PP1, 1 mM HMA or 8 mM MCDX. The results are the means±s.e.m. from two experiments. (C) Tyrosine-phosphorylated Fc{gamma}RIIA is marked by an arrowhead. Phosphorylation of wild-type Fc{gamma}RIIA was induced by spreading of BHK cells on IV.3 but not on fibronectin-coated substratum. No phosphorylation of Y298F Fc{gamma}RIIA was detected during spreading of the receptor-expressing cells on IV.3-coated substratum. Cell spreading was carried out for 0.5, 2 and 3 hours. The blot was reprobed with anti-actin to reveal equal loading of proteins. Molecular mass standards are shown on the left in kDa.

 

To gain insight into signaling events governing the actin rearrangement during Fc{gamma}RIIA-mediated cell spreading, Src tyrosine kinase-targeting agents were applied. It was found that the spreading was strongly diminished by 10 µM PP1 and 1 mM HMA and also affected by depletion of plasma membrane cholesterol with 8 mM MCDX (Fig. 6A,B). Immunoblotting analysis demonstrated that spreading of wild-type Fc{gamma}RIIA-expressing cells on IV.3-coated substratum was correlated with strong tyrosine phosphorylation of the receptor, which remained at the elevated level for at least 3 hours (Fig. 6C). By contrast, fibronectin-promoted spreading of these cells did not induce phosphorylation of the wild-type Fc{gamma}RIIA confirming activation of different receptors and signaling pathways by anti-Fc{gamma}RIIA and fibronectin. Finally, seeding of BHK cells transfected with mutant Y298F Fc{gamma}RIIA on anti-Fc{gamma}RIIA IV.3-coated substratum did not elicit any phosphorylation of the receptor (Fig. 6C). Accordingly, there was no spreading of the cells on the IV.3-coated substratum, although adhesion of the cells was efficient (Fig. 6A,B). The Y298F Fc{gamma}RIIA-expressing cells remained round without any visible major protrusions even 5 hours after plating (Fig. 6A).


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that crosslinked Fc{gamma}RII associates with high molecular mass complexes that share properties with DRMs and can be isolated from TX-100 lysates of U937 cells by gel filtration (Kwiatkowska and Sobota, 2001Go). Here we report that the majority of crosslinked, but not non-crosslinked, Fc{gamma}RII of U937 cells was present in DRMs fractionated by density gradient ultracentrifugation. Plasma membrane cholesterol was crucial for the association of Fc{gamma}RII with DRMs and phosphorylation of the receptor since both these events were blocked in cells pretreated with 5 mM CDX. It should be noted that removal of increasing amounts of the cholesterol by CDX in a range 0.5-5 mM correlated with a progressive reduction of the assembly of Fc{gamma}RII caps and inhibition of protein tyrosine phosphorylation (Fig. 1; data not shown). These data further support the assumption that membrane rafts participate in generation of Fc{gamma}RII signaling pathways. In addition to the biochemical data, the association of crosslinked Fc{gamma}RII with rafts in intact U937 cells was confirmed by immunofluorescence microscopy, which indicated a high degree of colocalization of the receptor clusters with CD55 and concomitant exclusion of TfR from Fc{gamma}RII patches (Fig. 3). Our results are in line with those of Katsumata et al., showing recruitment of Fc{gamma}RII to DRMs of human erythroleukemia K562 cells (Katsumata et al., 2001Go). The association of crosslinked Fc{gamma}RII with DRMs was markedly resistant to TX-100 as the complex was stable in the presence of 0.2%-1% of the detergent (Katsumata et al., 2001Go; Kwiatkowska and Sobota, 2001Go) (this report). This is in clear contrast to detergent solubility of other immunoreceptors, for example, an association of Fc{epsilon}RI with DRMs was disrupted by 0.08-0.2% TX-100 (Field et al., 1999Go; Kovarova et al., 2001Go), which argues against the involvement of membrane rafts in the initial phosphorylation of this receptor (Kovarova et al., 2001Go).

Recruitment of the crosslinked Fc{gamma}RII to DRMs was required, but not sufficient, for receptor phosphorylation. The phosphorylation was diminished by HMA, an agent that inhibits myristoylation of proteins, including Src family PTKs (Nadler et al., 1993Go). Under these conditions the integrity of the rafts was not disrupted as judged by CD55 location in low-density gradient fractions, although Lyn kinase was displaced from DRMs and its coimmunoprecipitation with crosslinked Fc{gamma}RII was impaired. At present, there is no direct evidence that Lyn kinase phosphorylates Fc{gamma}RII in U937 cells, and participation of other PTKs of the Src family in this process cannot be excluded. However, Lyn exhibited co-distribution with crosslinked Fc{gamma}RII in intact U937 cells and in DRMs as revealed by immunofluorescence and electron microscopy studies (Kwiatkowska and Sobota, 2001Go). In U937 cells Lyn is a predominant kinase of the Src family and, judging from immunoblotting analysis, we estimated that the level of its expression in these cells exceeds that of Hck by about 50 times. [Hck is another member of the Src family known to associate with Fc{gamma}RII in THP-1 cells (Ghazizadeh et al., 1994Go)]. Lyn was also found to associate with Fc{gamma}RII in THP-1, HL-60 monocytic cells and transfected mouse B-cells (Ghazizadeh et al., 1994Go; Katsumata et al., 2001Go; Bewarder et al., 1996Go) as well as in neutrophils (Ibarrola et al., 1997Go). In addition, in vitro studies indicated preferential phosphorylation of Fc{gamma}RIIA by Lyn (Bewarder et al., 1996Go) confirmed by potent Fc{gamma}RIIA phosphorylation in COS cells co-transfected with Lyn and the receptor (Cooney et al., 2001Go). Fc{gamma}RIIA expressed in non-hematopoietic BHK-21 cells, lacking Lyn, was likely to be phosphorylated by another member of the Src family of PTKs as indicated by the sensitivity of the process to PP1 and HMA.

Phosphorylation of Fc{gamma}RIIA was abrogated by substitution of a single tyrosine residue 298 by phenylalanine, which is in agreement with previous data on the crucial role of this tyrosine residue of the ITAM in receptor signaling (Mitchell et al., 1994Go; Bewarder et al., 1996Go; Ibarrola et al., 1997Go). The Y298F-substituted Fc{gamma}RIIA, when expressed in BHK cells, did not undergo translocation into cap-like structures after crosslinking and failed to promote spreading of the cells on the anti-Fc{gamma}RII-coated substratum. Similar inhibitory effects were achieved both in wild-type Fc{gamma}RIIA-expressing BHK cells and in U937 cells when activity of Src family PTKs was affected by PP1 and HMA or integrity of rafts was disrupted by cholesterol depletion. Concomitantly, phosphorylation of the crosslinked Fc{gamma}RII(A) in the cells was strongly impaired under the influence of the DRM- and PTK-targeting agents. Taken together, these data argue that phosphorylation of tyrosine residues of the ITAM in Fc{gamma}RIIA, catalyzed by raft-anchored PTKs of the Src family, triggers signaling pathways that target the actin cytoskeleton. It was previously demonstrated that actin and spectrin actively participate in formation of Fc{gamma}RII caps (Kwiatkowska and Sobota, 1999bGo), and sensitivity of cell spreading to cytochalasin B indicates that this process also relies on the participation of the actin-based cytoskeleton. The assembly of caps in U937 cells and other leukocytes is concomitant with their polarization as usually caps are formed at the posterior uropod of the cells (de Petris and Raff, 1972Go) (see Fig. 1). It is noteworthy that DRMs were recently shown to also play a pivotal role in the acquisition of the polarity needed for chemotaxis in adenocarcinoma cells and in T cells (Manes et al., 1999Go; Gomez-Mouton et al., 2001Go). By analogy with the assembly of Fc{gamma}RII caps and Fc{gamma}RIIA-promoted spreading of cells, polarization of T cells requires an involvement of the actin cytoskeleton, pointing again to an interaction between DRMs and the actin network (Gomez-Mouton et al., 2001Go). Despite extensive studies, the mechanism(s) of such interactions remain elusive and are likely to be complex (Pierini and Maxfiled, 2001). Recently, an involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells was reported (Föger et al., 2001Go). The presence of actin at clustered Fc{epsilon}RI was shown by immunoelectron microscopy (Wilson et al., 2000Go). Corresponding to our results, Harder and Simons reported that protein tyrosine phosphorylation by Src family PTKs, which accompanied patching of DRM components, was a prerequisite for the accumulation of actin filaments at the patches (Harder and Simons, 1999Go). On the basis of these data, the plasma membrane domains are thought to serve as centers for activation of immunoreceptors (Horejsi et al., 1999Go; Simons and Toomre, 2000Go) and for subsequent remodeling of the actin network, for example, by local generation of phosphatidylinositol 4, 5-bisphosphate and/or activation of Rho GTPase (Hirao et al., 1996Go; Pike and Miller, 1998Go; Rozelle et al., 2000Go; Caroni, 2001Go). Later on, when formation of Fc{gamma}RII caps proceeds, the actin cytoskeleton could be responsible for separation of the receptor and Src family kinases (Holowka et al., 2000Go; Wilson et al., 2000Go), facilitating the observed protein dephosphorylation. The persistent phosphorylation of wild-type Fc{gamma}RIIA detected in spreading BHK transfectants could result from progressive activation of subsequent receptors interacting with the substratum coated by ani-Fc{gamma}RII antibody.

Fc{gamma}RIIA-mediated phagocytosis, cell spreading and receptor capping are triggered by common events, such as clustering and phosphorylation of the receptor. Despite this, it seems that various signaling pathways can lead from activated Fc{gamma}RIIA to the actin cytoskeleton. During Fc{gamma}RII-mediated phagocytosis, activation of Syk and PI 3-kinase takes place (Cooney et al., 2001Go). Chimeric proteins composed of an extracellular domain of Fc{gamma}Rs and an intracellular domain containing either Syk or the p85 subunit of PI3-kinase were sufficient to mediate phagocytosis of IgG-coated particles (Greenberg et al., 1996Go; Lowry et al., 1998Go). Fc{gamma}R-mediated phagocytosis was abrogated in Syk-deficient monocytes and macrophages (Matsuda et al., 1996Go; Crowley et al., 1997Go; Kiefer et al., 1998Go). Similarly, a chimera of Fc{gamma}R-truncated p85, unable to bind the p110 catalytic subunit of PI 3-kinase, did not trigger phagocytosis (Lowry et al., 1998Go). Other reports showed that inhibition of Syk and PI 3-kinase activity by piceatannol and wortmannin, respectively, markedly diminished phagocytosis mediated by Fc{gamma}Rs, including Fc{gamma}RIIA (Ninomiya et al., 1994Go; Araki et al., 1996Go; Cooney et al., 2001Go), confirming that both kinases are indispensable for the process. It was considered that Syk controls actin assembly at ingested particles (Greenberg, 1999Go). However, in Syk-deficient macrophages (also in wortmannin-treated cells), formation of actin cups surrounding IgG-coated particles occurred (Araki et al., 1996Go; Crowley et al., 1997Go). By contrast, in hck-/- fgr-/- lyn-/- macrophages, formation of actin cups was delayed (Fitzer-Attas et al., 2000Go). On the basis of the comparison of the phenotypes of hck-/- fgr-/- lyn-/- and syk-/- cells it was inferred that the Src family PTKs can govern actin polymerization in phagocytic cups not only through Syk but also without Syk engagement (Fitzer-Attas et al., 2000Go). Our data showing that the activity of Syk and PI 3-kinase is less important for assembly of Fc{gamma}RII caps than activity of Src family PTKs are in line with those of Fitzer-Attas et al. (Fitzer-Attas et al., 2000Go). During phagocytosis the activity of PI 3-kinase is proposed to coordinate insertion of intracellular vesicles, providing the membrane for pseudopod extension (Greenberg, 1999Go; Lennartz, 1999Go). Extensive studies of the capping mechanism carried out between 1980 and 1990 indicated that translocation of protein patches in the plane of the plasma membrane is driven by the actin-based cytoskeleton and the process of vesicle flow does not essentially contribute to cap assembly (Heath and Holifield, 1991Go). This provides a clue as to why the activity of PI 3-kinase is not significant for capping.

Studies of Karnovsky's group demonstrated that capping of surface immunoglobulins on B cells was controlled by the cellular cholesterol level (Hoover et al., 1983Go). This capping was inhibited by removal of lymphocyte cholesterol with liposomes and restored after cholesterol repletion. Following these changes in cholesterol content, lipids of the plasma membrane were reversibly shifted from a rigid to fluid-like state. The authors suggested that the `gel-like lipid domain' of the plasma membrane was a place where `protein(s) involved in capping were located'. Disordering this domain, evoked by diminishing the cholesterol level, led to the attenuation of capping ability, whereas addition of cholesterol led to restoration of the gel-like nature of the domain and enabled capping to occur (Hoover et al., 1983Go). Our results indicate that the gel-like lipid domain proposed by Karnovsky can correspond to DRMs, serving as sites of Lyn kinase concentration, the activity of which is crucial for Fc{gamma}RII capping to proceed.


    Acknowledgments
 
We thank Helen L. Yin of the UT Southwestern Medical Center, Dallas TX for critical reading of the manuscript, Vaclav Horejsi of Institute of Molecular Genetics, Prague, Czech Republik for mouse anti-CD55 IgG (clone IA10) and mouse anti-CD55 IgM (clone MEM-118) and Jean-Luc Teillaud of Unite INSERM 255, Center de Recherches Biomedicals des Cordeliers, Paris, France, for the gift of rabbit anti-Fc{gamma}RII serum. We also thank Kazimiera Mrozinska for excellent technical assistance. This work was supported by grant KBN 0999/PO4/2000/18 from the Polish State Committee for Scientific Research.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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