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The Journal of Immunology, 2000, 165: 473-482.
Copyright © 2000 by The American Association of Immunologists

Differential Involvement of Src Family Kinases in Fc{gamma} Receptor-Mediated Phagocytosis1

Takeshi Suzuki*, Hajime Kono*, Naoto Hirose*, Masato Okada{dagger}, Tadashi Yamamoto{ddagger}, Kazuhiko Yamamoto* and Zen-ichiro Honda2,*

* Department of Allergy and Rheumatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; {dagger} Division of Protein Metabolism, Institute for Protein Research, Osaka University, Osaka, Japan; and {ddagger} Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The tyrosine phosphorylation cascade originated from Fc{gamma} receptors (Fc{gamma}Rs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc{gamma}R-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc{gamma}R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc{gamma}RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in Fc{gamma}R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phagocytosis of IgG-opsonized particles by macrophages is critical for clearance of pathogens as well as presentation of foreign and self Ags to T lymphocytes. This process is initiated by three classes of cell surface Fc receptors for IgG (Fc{gamma}Rs3), namely Fc{gamma}RI, Fc{gamma}RIIs, and Fc{gamma}RIIIs (1, 2, 3, 4, 5, 6). In murine macrophages, Fc{gamma}RI, Fc{gamma}RIIB, and Fc{gamma}RIIIA are expressed (3, 5, 7). Of these, Fc{gamma}RI and Fc{gamma}RIIIA are composed of an a subunit possessing IgG-binding sites and a dimer of the {gamma} subunit that contains a cell activation motif referred to as immunoreceptor tyrosine-based activation motifs (ITAMs) (5, 8, 9, 10, 11, 12). ITAM tyrosines in the {gamma} subunit are phosphorylated after the clustering of Fc{gamma}RI and Fc{gamma}RIIIA, and the tyrosine-phosphorylated ITAM creates sites for the assembly of Src homology 2 (SH2) domains, including that of Syk tyrosine kinase. (13, 14, 15, 16, 17, 18). Requirement of a {gamma} subunit for Fc{gamma}R-mediated phagocytosis was unequivocally established by the targeted disruption of the {gamma} subunit gene (19). The pivotal roles of Syk in connecting the early signaling to phagocytosis have also been shown by various approaches (4, 20, 21). Fc{gamma}RIIB does not associate with the {gamma} subunit, but it possesses a motif defined as immunoreceptor tyrosine-based inhibition motif (ITIMs) (22, 23). Coligation of Fc{gamma}RIIB with cell-activating Ag or Fc receptors has been shown to result in the down-regulation of inflammatory functions, presumably through the action of SH2-containing inositol 5'-phosphatase recruited to phosphorylated ITIM (5). Recent findings that Fc{gamma}RIIB-/- murine macrophages exhibit enhanced phagocytosis of IgG-coated particles are consistent with the idea (24).

The initial event of the phosphorylation of ITAM tyrosines is presumed to be catalyzed by Src family tyrosine kinases. This notion was first supported by pharmacological findings that herbimycin A, a tyrosine kinase inhibitor relatively specific for Src-family kinases, potently suppressed Fc receptor-mediated functions (25, 26). In addition, Src family members were found to physically associate with resting Fc receptors, and their catalytic activity was shown to be augmented by the receptor aggregation (27, 28, 29, 30, 31). To further dissect the roles of Src family kinases in FcR functions, targeted disruption of single or multiple Src family genes have been conducted (21, 32, 33, 34). Recently, Crowley et al. (21) found that Fc{gamma}R-mediated phagocytosis is delayed but preserved in Lyn-/-Hck-/-Fgr-/- macrophages. One of our laboratories (35) showed that mast cell degranulation mediated by Fc{epsilon}RI, a closely related member to Fc{gamma}Rs, is not diminished in Lyn-/- mast cells. These observations provided important information that Src family kinases possess overlapping roles in these FcR-mediated functions, but their significance is still left to be determined.

As an alternative approach, C-terminal Src kinase (Csk) has been utilized to suppress the activity of Src family kinases (36, 37, 38, 39, 40, 41). Src family kinases are assumed to be in an equilibrium between C-terminal tyrosine phosphorylated ("inactive") and dephosphorylated ("partially active") states and Csk shift the balance to the former by phosphorylating the negative regulatory tyrosine (42). We have previously shown that expression of membrane-anchored Csk mutant (mCsk) effectively down-regulates mast cell functions including Fc{epsilon}RI-mediated degranulation and integrin-mediated cell motility and that coexpression of selective Src family members lacking C-terminal negative regulatory tyrosine (termed a-Src kinases) could rescue these functions (37, 38). In the current study, we utilized the reconstitution strategy to investigate the roles of individual Src family kinase in Fc{gamma}R-mediated phagocytosis. We observed that Csk suppressed and mCsk almost abolished Fc{gamma}R-mediated Syk activation, filamentous actin (F-actin) assembly, and phagocytosis, but a kinase-defective mCsk (mCsk(-)) did not. Reconstitution experiments revealed that the impaired signaling cascade by mCsk was successfully restored by the coexpression of a-Lyn or by that of a-Hck but not by that of a-Src. These results strongly suggested that Src family kinases are required for Fc{gamma}R-mediated phagocytosis and that the function is catalyzed by selective Src family members including Lyn and Hck in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

All the culture media and Geneticin were purchased from Life Tech Oriental (Osaka, Japan). FCS was from Equitec Bio (Ingram, TX). Puromycin and HRP-conjugated cholera toxin B were from Sigma (St. Louis, MO). Rhodamine-conjugated phalloidin, fluorescein-conjugated Escherichia coli particles and fluorescein-conjugated zymosan particles were from Molecular Probes (Eugene, OR). Protein G-Sepharose was from Pharmacia-LKB (Uppsala, Sweden). SRBCs and rabbit anti-SRBC IgG were from InterCell Technologies (Hopewell, NJ). M-280 supraparamagnetic tosyl-activated beads and M-280 supraparamagnetic beads precoated with sheep anti-rabbit IgG (referred to as IgG-beads) were purchased from Dynal (Lake Success, NY).

Antibodies

Hybridoma producing 2.4G2, a rat anti-mouse Fc{gamma}RII/III mAb, was obtained from the American Type Culture Collection (Manassas, VA). Cultured supernatants of the hybridoma were applied to protein G-Sepharose to purify 2.4G2 mAb. FITC-conjugated 2.4G2 mAb was purchased from PharMingen (San Diego, CA). Anti-phosphotyrosine mAb, 4G10, was from ICN Biochemicals (Costa Mesa, CA). Polyclonal Abs against c-myc, Syk, c-Cbl, Vav, and Csk were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-mouse Fc{gamma}RIIB Ab was a generous gift from Dr. Toshiyuki Takai (Tohoku University, Sendai, Japan).

Preparation of RAW 264.7 cell lines expressing Csk and its mutants

RAW 264.7 cells were maintained in DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in humidified 5% CO2 as described (37). cDNAs for rat Csk, a membrane-anchoring Csk mutant (mCsk) possessing myristoylation signal from rat c-Src, and its kinase-defective form (mCsk(-)) were described previously (37). These cDNAs were subcloned into an expression vector, pCXN2 harboring neomycin-resistant gene (43), and introduced into RAW 264.7 cells by electroporation. Geneticin-resistant cell lines were screened by immunoblotting with anti-Csk Ab, and independent cell lines expressing Csk, mCsk, or mCsk(-) were established.

Preparation of RAW 264.7 cell lines expressing mCsk in combination with mutated Src family kinases

To create c-myc-tagged Src family kinases lacking C-terminal negative regulatory tyrosine, C-terminal amino acids of rat c-Src (aa 527–536), human Lyn A (aa 505–512), and human p59hck (aa 498–505), corresponding to one another, were deleted and replaced with a c-myc epitope sequence (TSVDEQKLISEEDLN) as described (38). The resultant cDNAs, termed a-Src, a-Lyn, and a-Hck, respectively, were subcloned into an expression vector, pCAGGS (43), and stably introduced into mCsk expressing cells with the aid of a puromycin-resistant vector as described (38). Puromycin-resistant clones were screened by immunoblotting with anti-c-myc Ab and with anti-Csk Ab, and independent cell lines expressing a-Src, a-Lyn, or a-Hck in combination with mCsk were created.

Flow cytometry

To evaluate surface expression of Fc{gamma}RIIIA/IIB, cells were harvested by brief trypsinization, as described (38), and stained with 5 µg/ml of FITC-conjugated 2.4G2 mAb or an isotype-matched control Ab in PBS supplemented with 2% horse serum and 0.01% NaN3. Cells were washed three times with PBS, and fluorescence intensity was measured by EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA).

Rosetting assay

SRBCs (1%) were opsonized with rabbit anti-SRBC Ab for 30 min at 37°C at a subagglutinating concentration. Adherent RAW cells in ice-cold assay medium (DMEM containing 0.1% BSA and 10 mM HEPES-NaOH, pH 7.4) were loaded with precooled SRBCs and incubated for 60 min on ice to prevent SRBC internalization. Cells were washed twice with PBS, fixed with PBS containing 4% formaldehyde, and observed with light microscopy. Positive rosetting was defined as RAW cells binding three or more IgG-opsonized SRBCs.

Phagocytosis assay

M-280 tosyl-activated beads were covalently conjugated with 2.4G2 mAb (referred to as 2.4G2-beads) according to the manufacturer’s instructions. Fc{gamma}R-mediated phagocytosis was assayed by using the 2.4G2 beads or with sheep IgG-coated M-280 beads (IgG-beads). Cells were cultured on eight-well Falcon CultureSlides (Becton Dickinson, Franklin Lakes, NJ) overnight and incubated in the assay medium for 3 h for serum starvation. Then, cells were loaded either with IgG-beads or with 2.4G2-beads and incubated for 20 min at 4°C to allow beads to attach to the cell surface. Excess beads were removed by one gentle washing with ice-cold assay medium, and phagocytosis was initiated by incubating the cells at 37°C. After 1 h of incubation, beads outside the cells were stained with FITC-conjugated anti-IgG at 20°C for 5 min and fixed with PBS containing 4% formaldehyde. FITC-staining was observed and photographed by fluorescence microscopy as described (38). Beads within the cells were simultaneously photographed using visible light. Phagocytosis index was calculated as the number of ingested beads per 100 cells.

Phagocytosis of Escherichia coli, nonopsonized zymosan, and complement-opsonized zymosan (COZ) was performed by using fluorescein-conjugated particles as described previously (44). For complement-opsonization, zymosan particles were incubated for 1 h at 37°C in fresh FCS as described (26). Nonopsonized zymosan particles were added to adherent RAW cells and allowed to be ingested for 1 h at 37°C in assay medium. In the case of COZ phagocytosis, cells were pretreated with 200 nM PMA for 15 min at 37°C to elicit CD11b/CD18 receptor activation (26) and loaded with COZ for 1 h at 37°C. To examine E. coli phagocytosis, cells were loaded with E. coli in assay medium supplemented with 2 mM EDTA and 10% FCS for 1 h at 37°C to evaluate the CD14-dependent phagocytotic pathway (45). After the incubations, adherent cells were washed three times with PBS, and particles remaining on the outer cell surface were removed by treating cells with trypsin/EDTA for 1.5 h at 37°C, as described (44). The quantities of internalized fluorescent particles were determined by flow cytometry or visualized with fluorescence microscopy.

Fc{gamma}R clustering by 2.4G2 mAb and cell lysis

Cells (1 x 107) in 6-well plastic plates were serum starved overnight in assay medium. Cells were washed once with ice-cold assay medium, equilibrated with assay medium at 4°C, and reacted with 10 µg/ml 2.4G2 mAb for 30 min. After two washes with ice-cold assay medium, reaction was initiated by addition of assay medium at 37°C containing 30 µg/ml rabbit anti-rat IgG. Cells were incubated for the indicated periods at 37°C, medium was aspirated, and cells were solubilized with Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 20 mM ß-glycerophosphate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 0.2 mM PMSF). Insoluble materials were removed by centrifugation at 12,000 rpm for 10 min, and the supernatant was used as total cell lysate.

Immunoprecipitation and immunoblotting

In Fc{gamma}R stimulation experiments, total cell lysates were first incubated with 15 µl protein G-Sepharose beads alone (50% slurry) for 1 h at 4°C under continuous rotation to absorb 2.4G2 mAb- and rabbit anti-rat IgG-bound Fc{gamma}RIIB and Fc{gamma}RIIIA. Samples were centrifuged at 12,000 rpm at 4°C for 1 min, and the supernatant was saved. Beads were washed three times with 500 µl Nonidet P-40 lysis buffer, and bound materials were eluted with boiling 2% SDS sample buffer. The eluted proteins were used as 2.4G2 mAb immunoprecipitates. The saved supernatants were incubated first with various first Abs and then with 15 µl suspension of protein G-Sepharose beads for 1 h at 4°C under continuous rotation. The beads were washed, and bounded materials were eluted as described above.

Immunoprecipitated proteins or the total cell lysate was subjected to SDS-PAGE, and separated proteins were electrotransferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Proteins were probed with first Abs and then reacted with HRP-conjugated second Abs. Signals were detected by chemiluminescence (ECL Western Blotting System, Amersham, Arlington Heights, IL), as described (37).

In vitro kinase assay

In vitro kinase assay of Syk and the mutated Src family kinases was performed as described previously (37, 46). Syk was immunoprecipitated using anti-Syk polyclonal Ab as described above. Beads were washed three times with Nonidet P-40 lysis buffer, washed three times with kinase buffer (50 mM HEPES-NaOH (pH 7.6), 10 mM MnCl2, 2 mM MgCl2, 10 µM Na3VO4, 1 mM 4-nitrophenyl phosphate), and resuspended in kinase buffer. Reaction was initiated by the addition of 2 µM ATP and 10 µCi [{gamma}-32P]ATP, proceeded for 10 min at 30°C. a-Src, a-Lyn, and a-Hck were immunoprecipitated with anti-c-myc Ab. Beads were washed twice with Nonidet P-40 lysis buffer and once with kinase buffer. Reaction was initiated by the addition of 1 µg enolase, 2 µM ATP, and 10 µCi [{gamma}-32P]ATP and proceeded for 1 min at 25°C. Under the reaction conditions, [{gamma}-32P]ATP incorporation into enolase was linear with time (37). Reactions were terminated by the addition of 2% SDS sample buffer. After boiling, samples were centrifuged, and the supernatants were subjected to SDS-PAGE. Gels were dried and subjected to phosphorimager analysis using a Fuji BAS 2000 image analyzer (Fujifilm Medical, Tokyo, Japan).

Detection of F-actin accumulation

To prepare a 2.4G2 mAb-coated surface, LAB-TEK chamber slides (Permanox, Nunc, Naperville, IL) were filled overnight with or without 10 µg/ml 2.4G2 mAb in carbonate buffer, pH 9.6, at 4°C. Wells were washed once with PBS, blocked with 20 mg/ml fatty acid-free BSA in PBS for 1 h at 37°C, and again washed twice with PBS and once with DMEM. RAW cells in assay medium were seeded onto the chamber slides and allowed to adhere to the 2.4G2 mAb-coated surface at 37°C for indicated periods. Accumulation of F-actin at the attachment sites was detected by staining with rhodamine-conjugated phalloidin and observed by fluorescence microscopy as described (38).

Sucrose density gradient centrifugation

Protein association with detergent-resistant membranes (DRMs) was analyzed by solubilizing cells with low concentration Triton X-100 followed by ultracentrifugation of cell lysates on sucrose density gradients according to the method of Field et al. (47). In brief, 4 x 106/ml cell suspension was solubilized with 0.05% Triton X-100, cell lysate was layered onto 80 to 10% discontinuous sucrose gradients prepared in a Hitachi 13 PA tube (1.5 x 9.6 cm), and centrifuged at 35,000 rpm at 4°C for 18 h (47). Aliquots (1 ml) of the gradients were collected, proteins were extracted following the methods of Wessel and Flugge (48), and the sample was subjected to Western blotting as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influences of Csk, mCsk, and mCsk(-) on Fc{gamma}R-mediated phagocytosis

As an approach to investigation of the roles of Src family kinases in Fc{gamma}R-mediated phagocytosis, we utilized Csk, mCsk (36, 37, 38), and mCsk(-), which functions as a dominant negative molecule (37). These Csk-based molecules were transfected into the RAW 264.7 macrophage cell line, and multiple independent clones stably expressing each of them were established. A representative immunoblot with anti-Csk Ab of RAW cells overexpressing Csk, mCsk, or mCsk(-) is shown in Fig. 1Go.



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  		FIGURE 1. Creation of RAW 264.7 cell lines expressing Csk, mCsk, or kinase-defective, mCsk(-). Representative immunoblot of WT-RAW and the cells overexpressing Csk, mCsk, or mCsk(-). Cells were solubilized with Nonidet P-40 lysis buffer and standardized for protein concentration; the cell lysates were subjected to immunoblotting with anti-Csk Ab. The migrated positions of Csk, mCsk, and mCsk(-) are indicated by arrows.

 
These cell lines were tested for the ability to ingest beads coated with sheep IgG (26) or with rat anti-Fc{gamma}RII/III mAb, 2.4G2. Cells were loaded with the beads and allowed to internalize them for 60 min at 37°C. As seen in Fig. 2GoA, 2.4G2-coated beads remaining on the cell surface were positively stained with FITC-conjugated anti-IgG (bright green particles), and internalized beads were observed as dark yellow particles. RAW 264.7 cells (WT-RAW) efficiently phagocytosed 2.4G2-beads. Csk-overexpressing cells ingested the beads less efficiently, and mCsk-overexpressing cells were virtually unable to ingest them. mCsk(-)-overexpressing cells phagocytosed the beads almost comparably with WT-RAW. Phagocytosis index (number of ingested beads per 100 cells) of the cell lines is shown as a bar graph in Fig. 2GoB. Csk and mCsk significantly suppressed the Fc{gamma}R-mediated phagocytosis, and the effects were kinase dependent. Surface expression of Fc{gamma}RIIB/Fc{gamma}RIIIA, as analyzed by fluorescent 2.4G2 mAb staining, and IgG-opsonized SRBC rosetting on the cells were not significantly altered by the expression of Csk or the mutated Csks (not shown). Therefore, those inhibitory effects of Csk and mCsk could not be ascribed to decreased density or affinity of Fc{gamma}RIIB and IIIA, but most likely to impaired postreceptor signaling pathway.



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  		FIGURE 2. Stepwise inhibition of Fc{gamma}R-mediated phagocytosis and F-actin rearrangement by Csk and by mCsk. A, Csk and mCsk inhibited phagocytosis of 2.4G2 mAb-coated beads in a kinase-dependent manner. Cells were loaded with microbeads coated with 2.4G2 and incubated at 37°C for 60 min. Beads outside cells were stained with FITC-conjugated second Ab and photographed by fluorescence microscopy (bright green microspheres). Ingested beads could be simultaneously photographed using visible light (dark yellow microspheres). Scale bar, 10 µm. B, Quantitative determination of Fc{gamma}R-mediated phagocytosis by WT-RAW and by cells expressing Csk-based molecules. Cells were loaded with sheep IgG-coated beads and incubated for 60 min at 37°C; ingested beads were counted as described in A. Number of ingested beads/100 cells was defined as phagocytosis index. Data are expressed as mean ± SE from independent experiments (WT-RAW, n = 9; others, n = 4). *, p < 0.003, **, p < 0.001 compared with parent RAW 264.7 cells (WT-RAW) by t test. C, Accumulation of F-actin at adhesion sites to 2.4G2-coated surface. Cells were allowed to adhere to 2.4G2-coated surface for 5 min at 37°C as described in Materials and Methods. Cells were permeabilized and stained with rhodamine-phalloidin. Ring-like F-actin staining was observed in WT- and mCsk(-)-expressing cells, but not in Csk- or mCsk-expressing cells. Scale bar, 10 µm.

 
We next examined F-actin accumulation at cytoplasmic surface of Fc{gamma}R-IgG contact sites, which is an early and obligatory step for Fc{gamma}R-mediated phagocytosis (49, 50). Cells were allowed to adhere onto 2.4G2-coated surfaces, and F-actin accumulation was detected by staining with rhodamine-phalloidin. As seen in Fig. 2GoC, a ring-like F-actin accumulation developed at ventral surfaces of cells in WT-RAW within 5 min. Such structures were not observed in Csk- or mCsk-overexpressing cells within 60 min, although those structures were clearly observed in mCsk(-)-overexpressing cells within 5 min (Fig. 2GoC). These findings were consistent with Csk-and mCsk-mediated inhibition of phagocytosis and indicated that Csk inhibits Fc{gamma}R-mediated phagocytosis at a step before F-actin rearrangement.

Differential inhibitory effects of Csk on the phagocytosis of E. coli, nonopsonized zymosan, and COZ

We next compared the effects of Csk-derived molecules on other phagocytosis pathways. Nonopsonized zymosan and COZ were shown to be internalized mainly via receptors for mannose-fucose and ß-glucan and via CD11b/CD18 integrin, respectively (6, 51, 52). Under the current experimental conditions, E. coli was shown to be ingested via the CD14-dependent pathway (45). Those particles conjugated with fluorescein were loaded onto RAW cell lines, and phagocytosis was evaluated by flow cytometry. As seen in Fig. 3Go, WT cells efficiently ingested these three materials. Csk and mCsk were found to exert differential effects on these phagocytotic activities: phagocytosis of nonopsonized zymosan was most severely affected by their expressions, while E. coli- and COZ phagocytosis was only marginally influenced. As compared with control WT cells, 97.7 and 89.8% of mCsk cells ingested E. coli, and COZ, respectively, whereas only 27.8% of mCsk cells phagocytosed nonopsonized zymosan. These findings suggested that increased Csk activity resulted in the suppression of selective phagocytotic receptor functions.



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  		FIGURE 3. Differential inhibitory effects of Csk on the phagocytosis of E. coli, zymosan, and serum-opsonized zymosan. Csk and mCsk potently suppressed zymosan phagocytosis but exerted minor effects on the phagocytosis of E. coli or serum-opsonized zymosan (COZ). Fluorescein-conjugated particles were loaded to adherent RAW cell lines for 1 h, and internalized fluorescence was measured by flow cytometry as described in Materials and Methods. Insets, representative cells observed under visible and fluorescent light. Internalized materials are recognized as bright green particles.

 
Csk inhibited Fc{gamma}RII/III-mediated Syk activation

Because Syk plays pivotal roles in Fc{gamma}R-mediated phagocytosis (20, 21), we next evaluated the effects of Csk and mCsk on Syk activation. Fc{gamma}RIIB and IIIA were aggregated with 2.4G2 and second Ab at 37°C, and cells were lysed 3 min after the receptor clustering. Syk was immunoprecipitated from the lysates and subjected to in vitro kinase assay or to immunoblotting. As seen in Fig. 4Go, top (IVK), Syk activity, as assessed by autophosphorylation, was clearly increased after Fc{gamma}RIIB and Fc{gamma}IIIA clustering in WT-RAW. Csk inhibited and mCsk more potently inhibited clustering-induced Syk activation. In mCsk(-) cells, Syk activation was almost preserved. As seen in Fig. 4Go, middle (pY blot), Syk was tyrosine phosphorylated under basal conditions in WT cells, and Fc{gamma}RIIB and Fc{gamma}IIIA clustering resulted in enhanced Syk tyrosine phosphorylation. As indicated by an asterisk, tyrosine-phosphorylated proteins of ~20 kDa, presumably corresponding to the {gamma} subunit (53), were coimmunoprecipitated with Syk, and the signals were increased by Fc{gamma}RII/III clustering. Csk expression suppressed basal and clustering-induced Syk tyrosine phosphorylation and decreased the signals of ~20 kDa tyrosine phosphorylation. mCsk expression almost abolished tyrosine phosphorylation of Syk and associated ~20 kDa tyrosine phosphorylation. In contrast, mCsk(-) expression did not appreciably inhibit these signals. These observation indicated that Csk and mCsk inhibited Fc{gamma}RII/III-mediated activation of Syk in a stepwise manner and that the effects were kinase dependent.



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  		FIGURE 4. Stepwise inhibition of Syk activation by Csk and by mCsk. Cells were left unstimulated (-) or were stimulated with 2.4G2 mAb and second Ab for 3 min at 37°C (+). Cells were lysed with Nonidet P-40 lysis buffer. The cell lysates were standardized for protein concentration, and Syk was immunoprecipitated. A portion of the immunoprecipitates was subjected to in vitro kinase assay (IVK), and the rest was to immunoblotting with anti-phosphotyrosine mAb, 4G10 (pY blot), or with anti-Syk Ab (Syk blot). Migrating positions of Syk, ~20 kDa signals presumably corresponding to tyrosine-phosphorylated {gamma} subunit (*), and Ig heavy (H) and light (L) chains are indicated on the right ordinate. Molecular mass markers shown on the left ordinate. Csk decreased and mCsk almost abolished clustering-induced Syk activation, but mCsk(-) did not (IVK). Csk and mCsk also decreased basal and clustering-dependent tyrosine phosphorylation of Syk and ~20 kDa proteins, in a stepwise manner, but mCsk(-) did not.

 
Creation of RAW cell lines coexpressing mutated Lyn, Hck, or c-Src with mCsk

The above findings that Csk and mCsk inhibited Fc{gamma}RII/III-mediated Syk activation, actin cytoskeleton reorganization, and phagocytosis suggested that Src family kinases play pivotal roles in the signaling pathway. To further confirm the requirement of Src family kinases and to investigate the roles of individual Src family kinase for the functions, we created Src family kinases in which C-terminal sequences containing negative regulatory tyrosine were replaced with c-myc epitope (Fig. 5GoA). These partially active constructs, termed a-Src kinases, were reconstituted in mCsk cells and tested for the ability to reconstruct Fc{gamma}RII/III-mediated signaling. In this study, we focused on Lyn, Hck, and c-Src. Among the Src family members, Lyn and Hck are expressed predominantly in hemopoietic cells, whereas c-Src is ubiquitously expressed. RAW 264.7 cells express these molecules (not shown). a-Lyn and a-Src, in which the corresponding C-terminal amino acid sequences (aa 505–512 for Lyn A (p56lyn), aa 527–536 for c-Src (p60c-src)) were replaced with a c-myc epitope tag sequence were described previously (38). a-Hck, in which aa 498–505 were replaced with c-myc epitope, was created as described in Materials and Methods (Fig. 5GoA). Puromycin-resistant vector alone or in combination with each a-Src kinase construct was transfected into mCsk cells, and multiple mCsk cell lines coexpressing comparable levels of a-Lyn, a-Hck, or a-Src were obtained. Representative immunoblots of the clones with anti-c-myc Ab and with anti-Csk Ab are shown in Fig. 5GoB. Expression of these a-Src kinases did not affect surface expression of Fc{gamma}RIIB and Fc{gamma}RIIIA, as assessed by fluorescent 2.4G2 staining (not shown).



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  		FIGURE 5. Co-expression of a-Lyn, a-Hck, or a-Src in mCsk-overexpressing RAW 264.7 cells. A, Schematic representation of the structures of a-Lyn, a-Hck and a-Src. In these a-Src kinases, C-terminal short sequences including negative regulatory tyrosine (boxed amino acids in sequence alignment) were replaced with a c-myc epitope sequence. B, Representative immunoblots of RAW 264.7 cell lines expressing mCsk in combination with vector alone (puro/mCsk), a-Lyn (a-Lyn/mCsk), a-Hck (a-Hck/mCsk), or a-Src (a-Src/mCsk). In each lane, 2.5 µg of proteins were applied. Migrated positions of a-Lyn, a-Hck, and a-Src and those of Csk and mCsk are on the right. C, Catalytic activity of a-Lyn, a-Hck, and a-Src. Quiescent cells were lysed with Nonidet P-40 lysis buffer, standardized for protein concentration, and a-Lyn, a-Src, and a-Hck was immunoprecipitated with anti-c-myc Ab. The immunoprecipitates were divided and subjected to in vitro kinase assay using enolase as an external substrate (IVK) and to immunoblotting with anti-c-myc Ab (myc blot). Migrating positions of a-Lyn, a-Src, a-Hck, enolase, and Ig heavy chain (H) are indicated on the right.

 
To evaluate kinase activity of a-Src, a-Lyn, and a-Hck in the cell lines, a-Src kinases were immunoprecipitated with anti-c-myc Ab, and catalytic activity toward enolase was assayed at 25°C for 1 min. Under the reaction conditions, 32P incorporation into enolase was linear within 2 min (not shown). As seen in Fig. 5GoC, all the constructs efficiently phosphorylated enolase to roughly comparable levels, although activity of a-Src exceeded those of a-Lyn and a-Hck. We could not find the reason for the difference, but it might be ascribed to different intrinsic kinase activities of c-Src, Lyn, and Hck.

Fc{gamma}R-mediated phagocytosis and F-actin rearrangement were reconstructed by a-Lyn or by a-Hck, but not by a-Src

We compared Fc{gamma}RII/III-mediated phagocytosis in the cells expressing mCsk or mCsk with a-Src kinases. Cells were loaded with 2.4G2-beads or sheep IgG-beads for 60 min at 37°C, and beads internalized in the cells were observed and counted as described above. As seen in Fig. 6GoA, vector control cells (puro/mCsk cells) failed to ingest 2.4G2-beads, as expected. Intriguingly, expression of a-Lyn or that of a-Hck in mCsk cells clearly reconstructed the ability to ingest 2.4G2-beads (Fig. 6GoA). In contrast, a-Src expression did not efficiently restore the phagocytosis. As seen in the bar graph of the calculated phagocytosis index (Fig. 6GoB), expression of a-Lyn or that of a-Hck significantly increased phagocytosis index above vector control, to almost comparable levels in WT cells, but expression of a-Src did not. Fc{gamma}R-mediated F-actin assembly was also examined by using 2.4G2-coated surface. The ring-like structures of accumulated F-actin at the contact sites were clearly observed in a-Lyn/mCsk cells and in a-Hck/mCsk cells, but these structures were barely detectable in a-Src/mCsk cells (Fig. 6GoC).



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  		FIGURE 6. Fc{gamma}R-mediated phagocytosis and F-actin rearrangement were reconstructed by a-Lyn and by a-Hck, but not by a-Src. A, Phagocytosis of 2.4G2-coated beads were successfully reconstructed by a-Lyn and by a-Hck, but not by a-Src. Ingested beads and uningested ones are visualized as dark yellow microspheres and as bright green ones, respectively. See legend for Fig. 2GoA for detail. B, Quantitative determination of phagocytosis of sheep IgG-coated beads. The number of the beads ingested by 100 cells was defined as phagocytosis index. See legend to Fig. 2GoB for details. Data are expressed as mean ± SE from independent experiments (WT-RAW, n = 9; puro/mCsk, n = 6; others, n = 4). *, p < 0.001 compared with puro/mCsk by t test. C, Accumulation of F-actin at adhesion sites to a 2.4G2-coated surface. See also legend to Fig. 2GoC. Ring-like F-actin assembly was observed in a-Lyn/mCsk cells and a-Hck/mCsk cells, but not in puro/mCsk control cells or in a-Src/mCsk cells.

 
Fc{gamma}RII/III-mediated Syk activation was restored by a-Lyn or by a-Hck but not by a-Src

To investigate the mechanisms underlying the differential restoration of phagocytosis by a-Src kinases, we next examined Fc{gamma}RII/III clustering-induced Syk activation. As seen in Fig. 7Go, top, Syk autophosphorylation activity in puro/mCsk control cells was almost undetectable, as seen in parent mCsk cells (Syk ip IVK). In a-Lyn/mCsk cells and in a-Hck/mCsk cells, receptor clustering resulted in clear increase in the kinase activity (Fig. 7Go). In contrast, receptor clustering did not induce detectable Syk activation in a-Src/mCsk cells (Fig. 7Go). As seen in Fig. 7Go, middle (pY blot), Syk tyrosine phosphorylation was almost undetectable before and after Fc{gamma}RII/III clustering in puro/mCsk cells. In a-Lyn/mCsk and a-Hck/mCsk cells, basal Syk tyrosine phosphorylation was clearly observed, and Fc{gamma}RII/III clustering resulted in intense Syk tyrosine phosphorylation. In addition, the ~20 kDa signal, presumably corresponding to the tyrosine-phosphorylated {gamma} subunit, was coimmunoprecipitated with Syk under basal conditions, and the intensity of the signals was increased after the receptor clustering. In a-Src/mCsk cells, Syk tyrosine phosphorylation under resting conditions was barely detectable, and it was increased to a lesser extent than those in a-Lyn/mCsk and a-Hck/mCsk cells after the receptor clustering. Therefore, Syk activation was successfully reconstructed by a-Lyn or a-Hck. a-Src weakly tyrosine phosphorylate Syk upon clustering but could not restore Syk activation.



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  		FIGURE 7. Fc{gamma}RII/III-mediated Syk activation was restored by a-Lyn or by a-Hck but not by a-Src. Cells were stimulated (+) or not (-) for 3 min at 37°C with 2.4G2 and second Ab, lysed, and subjected to in vitro kinase assay of Syk (IVK) and to immunoblotting with anti-phosphotyrosine Ab (pY blot) or with anti-Syk Ab (Syk blot). Migrating positions of Syk, ~20 kDa signal(s) presumably corresponding to tyrosine-phosphorylated {gamma} subunit (*), and Ig heavy (H) and light (L) chains are indicated on the right. Clustering-induced Syk activation was clearly observed in a-Lyn/mCsk and a-Hck/mCsk cells, but almost undetectable in puro/mCsk control cells and in a-Src/mCsk cells (IVK). As seen in pY blot, a-Lyn- and a-Hck expression augmented basal and clustering-induced tyrosine phosphorylation of Syk and ~20 kDa protein(s). a-Src did not significantly increase basal tyrosine phosphorylation of these proteins but modestly augmented it after Fc{gamma}R clustering.

 
Relative abilities of a-Lyn, a-Hck, and a-Src to restore tyrosine phosphorylation of signaling molecules upon Fc{gamma}RII/III clustering

The above findings indicated that a-Lyn and a-Hck almost fully reconstructed Fc{gamma}RII/III-mediated signaling leading to phagocytosis, whereas a-Src could only weakly transmit Syk tyrosine phosphorylation. To further explore their differential abilities to transduce Fc{gamma}RIIIA/IIB signaling, we surveyed basal and clustering-induced tyrosine phosphorylation of signaling molecules including c-Cbl, Vav, and Fc{gamma}RIIB. Vav transmits positive signals through GTP/GDP exchange of Rac, which leads to JNK activation and the regulation of actin cytoskeleton (54, 55). c-Cbl and Fc{gamma}RIIB seem to function as negative regulators, through the down-regulation of Syk activity and/or the degradation of Syk molecule via ubiquitination (56, 57), and by the recruitment of SH2 containing inositol phosphatase, respectively (58, 59). As seen in Fig. 8GoA (c-Cbl ip), all three a-Src kinases commonly increased basal and clustering induced c-Cbl tyrosine phosphorylation above control levels (puro/mCsk cells). In a-Hck/mCsk cells, clustering-induced signal appeared weak, but significantly reduced c-Cbl recovery after clustering (see c-Cbl blot) suggested that it was presumably due to a-Hck association with detergent-insoluble cytoskeletal fraction (60, 61). They also in common augmented basal and clustering-induced Vav tyrosine phosphorylation (Fig. 8GoA, Vav ip). The smaller clustering effects in a-Lyn/mCsk and a-Hck/mCsk cells than that in a-Src/mCsk cells presumably reflected intense constitutive tyrosine phosphorylation. In contrast to c-Cbl and Vav tyrosine phosphorylation, Fc{gamma}RIIB tyrosine phosphorylation was reconstructed only by a-Lyn and a-Hck, but not by a-Src (Fig. 8GoB, pY blot); a-Lyn and a-Hck expression resulted in detectable basal Fc{gamma}RIIB tyrosine phosphorylation and intense Fc{gamma}RIIB tyrosine phosphorylation after clustering, whereas a-Src expression did not exert such effects.



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  		FIGURE 8. Relative abilities of a-Lyn, a-Hck, and a-Src to restore tyrosine phosphorylation of signaling molecules upon Fc{gamma}RII/III clustering. A, Cells were left unstimulated (-) or stimulated for 3 min at 37°C with 2.4G2 and second Ab (+), lysed, and subjected to immunoprecipitation with anti-c-Cbl Ab (c-Cbl ip) or to anti-Vav Ab (Vav ip) followed by immunoblotting with anti-phosphotyrosine Ab (pY blot). See also legend for Fig. 3Go for details. Those membranes were reprobed with anti-c-Cbl (c-Cbl blot) and anti-Vav Ab (Vav blot), respectively. Migrating positions of c-Cbl and Vav are on the right. Expression of a-Lyn, a-Hck, and a-Src increased basal and clustering-induced tyrosine phosphorylation of c-Cbl and Vav above control levels in puro/mCsk cells. Also c-Cbl recoveries (c-Cbl blot) decreased after clustering in a-Lyn/mCsk cells and in a-Hck/mCsk cells. B, Cells were stimulated or not as above, and cross-linked Fc{gamma}RII/III was precipitated with protein G-Sepharose (Fc{gamma}R-II/III ip). Precipitated proteins were subjected to anti-phosphotyrosine immunoblot (pY blot), and the same membrane was reprobed with anti-Fc{gamma}RIIB Ab (Fc{gamma}RIIB blot). Migrated positions of Fc{gamma}RIIB are on the right. Expression of a-Lyn and that of a-Hck modestly increased basal Fc{gamma}RIIB tyrosine phosphorylation and markedly augmented it after clustering. Expression of a-Src did not increase basal or clustering-induced tyrosine phosphorylation of Fc{gamma}RIIB.

 
Differential localization of a-Src kinases at DRMs

Recent studies have revealed that localization of Src family kinases at specialized low density membrane domain, called DRMs or sphingolipid-cholesterol rafts (62), is critical for Ag receptor signal transduction (47, 63, 64, 65, 66, 67). We thus examined the association of a-Lyn, a-Hck, and a-Src with DRMs by sucrose density gradient centrifugation (47, 65). a-Src kinases were detected with anti-c-myc Ab, and GM1 ganglioside, a marker of DRMs (62), with cholera toxin B. As seen in Fig. 9Go, a-Lyn and a-Hck distributions exhibited separated peaks at high density (fractions 2–6) and low density (fractions 8 and 9) fractions, and the latter colocalized with GM1 (a marker of DRMs). In contrast, a-Src was recovered mainly from high density fractions (fractions 2–7), and its association with DRMs was minimal. These findings revealed that a-Lyn and a-Hck were in part associated with DRMs and that a-Src was almost excluded from DRMs.



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  		FIGURE 9. Differential localization of a-Src kinases at DRMs. Quiescent cells expressing mCsk and a-Src kinases were solubilized and subjected to discontinuous sucrose gradient ultracentrifugation as described in Materials and Methods. Fractions were analyzed by Western blotting. a-Lyn, a-Hck, and a-Src were detected with anti-c-myc Ab (anti-myc), and GM1 with cholera toxin B (ChTx). Molecular mass markers were shown on the left, and migration positions of a-Src kinases and GM1 are shown on the right. Under the experimental conditions, BSA was nonspecifically stained (N.S.). See text for details.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Src family tyrosine kinases have been implicated in the initiation of Fc{gamma}R-mediated signaling. Several lines of evidence indicated that the earliest event after Fc{gamma}R clustering is phosphorylation of ITAM tyrosines in the {gamma} subunit and that this modification is prerequisite for transducing downstream signaling (16, 68, 69). Impaired Syk activation in Fc{gamma}R-stimulated Lyn-/-Hck-/-Fgr-/- murine macrophages further evidenced the close proximity of Src family kinases to Fc{gamma}Rs (21). However, the findings that Fc{gamma}R-mediated phagocytosis is delayed but preserved in the Lyn-/-Hck-/-Fgr-/- cells still left the roles of Src family kinases to be determined. Furthermore, the specificity of Src family kinases in transducing phagocytosis signaling has not been elucidated. As an approach to complementing the knowledge obtained from gene targeting studies, we designed reconstitution experiments using membrane-anchored Csk that functions as a gain-of-function molecule (37).

Through the analysis of RAW 264.7 murine macrophage cell line overexpressing Csk, mCsk, or mCsk(-), it was first observed that Fc{gamma}R-mediated phagocytosis was inhibited in Csk-expressing cells and almost abolished in mCsk-expressing cells under our experimental conditions, and that the inhibitory effects of mCsk was kinase dependent (see Fig. 2Go, A and B). Several lines of evidence indicate that Syk tyrosine kinase is indispensable for Fc{gamma}R-mediated phagocytosis: Syk-/- murine macrophages are incapable of Fc{gamma}R-mediated phagocytosis (21), and clustering of a Fc{gamma}R-Syk chimera is sufficient to induce phagocytosis when expressed in COS cells (20). It is also established that F-actin assembly around phagosomes is an early and obligatory process in the phagocytosis (49, 50). We observed that these early signals were also inhibited by Csk and by mCsk in a stepwise manner and that the inhibitory effects were kinase dependent. These findings strongly indicate that Src family kinases are indispensable upstream regulators for Fc{gamma}RII/III-mediated phagocytosis, and they confirm the idea that Src family kinases lies upstream of Syk activation (21). It might be also possible that Src family kinases regulate common downstream signaling (e.g., actin polymerization) and that the inhibitory effects of mCsk are not specific for selective receptors. This hypothesis seems unlikely because mCsk exerted differential effects on nonopsonized zymosan, COZ, and E. coli phagocytosis. Marginal effects of mCsk on COZ and E. coli internalization are consistent with previous reports showing that herbimycin A did not affect COZ phagocytosis (26) and that CD14-mediated signaling was essentially normal in Lyn-/-Hck-/-Fgr-/- triple knockout macrophages (70). Mechanisms of nonopsonized zymosan, presumably mediated by receptors for mannose-fucose and ß-glucan (6, 51, 52), have not been fully elucidated (6), but our findings suggested essential roles of Src family kinases in these pathways.

The prominent inhibitory effects of mCsk on the phagocytosis of IgG-coated beads are apparently contradictory to the data from Lyn-/-Hck-/-Fgr-/- primary macrophages challenged with IgG-coated SRBC (21). The delayed kinetics of phagocytosis in the triple knockout macrophages is consistent with our data indicating positive regulatory roles of Src family kinases. However, net phagocytosis was unchanged in the triple knockout cells after long incubation periods (21), whereas it clearly decreased in mCsk-expressing RAW cells. One possible explanation for the discrepancy is that Fc{gamma}R-mediated phagocytosis in Lyn-/-Hck-/-Fgr-/- primary macrophages was compensated by other Src family kinases including Yrk, as suggested by the authors (70), and that mCsk blocked the activity of the residual kinases. Alternatively, it might be ascribed to different carriers, SRBC in the study by Crowley et al. and artificial beads in this study. Although quantitative data were not available, Lowell et al. (32) showed that uptake of IgG-coated beads was reduced but that IgG-SRBC phagocytosis was normal in Hck-/-Fgr-/- double knockout macrophages, findings suggestive of the influence of carrier particles. RBCs express several ligands for phagocytotic receptors including scavenger receptors and asialoglycoprotein receptor, especially at senescent or physically damaged states (6, 71, 72, 73, 74). Therefore, it may also be possible that the slow phagocytosis of IgG-SRBC in Lyn-/-Hck-/-Fgr-/- cells is catalyzed via ligands on SRBC surfaces.

The clear suppression of Fc{gamma}R-mediated phagocytosis in mCsk-overexpressing cells led us to test the abilities of individual Src family kinase to reconstruct the functions by coexpression procedures. Basal tyrosine phosphorylation of Syk and ~20 kDa protein(s) corresponding to {gamma} subunit were decreased in Csk cells and almost vanished in mCsk cells, but not in mCsk(-) cells (Fig. 4Go, pY blot). Similar observations were made in Lyn-/- mast cells (34) and in Lyn-/-Hck-/-Fgr-/- primary macrophages (21). These findings strongly indicated that under basal conditions, Src family kinases are partly in an active (C-terminal tyrosine dephosphorylated) conformation, and the mCsk stronly reduced the probability for the kinases to take the active conformation. We thus tested the effects of the coexpression of C-terminal tyrosine-deleted a-Src kinases in mCsk cells on Fc{gamma}RII/III-mediated phagocytotic signaling.

Reconstitution experiments showed that Fc{gamma}R-mediated phagocytosis was restored by a-Lyn or by a-Hck, but not by a-Src. The successful reconstruction of the phagocytosis by the two hemopoietic Src family kinases, together with Csk-mediated inhibition of the phagocytosis, further strengthened the idea that Src family kinases are indispensable for Fc{gamma}R-mediated phagocytosis. In addition, the differential abilities of a-Src kinases strongly suggested that Fc{gamma}R-mediated phagocytosis is catalyzed by selective Src family members. The inability of a-Src to restore the phagocytosis was not due to low expression level of the kinase (see Fig. 5GoB), or to defective catalytic activity of a-Src construct (Fig. 5GoC). Preserved surface expression of Fc{gamma}RII/III and IgG-opsonized SRBC rosetting in a-Src/mCsk cells excluded the possibility that a-Src expression altered ligand-Fc{gamma}R binding. We thus concluded that a-Src could not drive postreceptor signaling. Consistent with the notion, clustering-mediated activation of Syk and accumulation of F-actin on a 2.4G2-coated surface were reconstructed by a-Lyn and by a-Hck, but not by a-Src. The inability of a-Src to restore Fc{gamma}R-mediated phagocytosis is most likely ascribed to its inability to induce clustering-mediated Syk activation. Although it should be taken into account that these a-Src kinases are not in normal equilibrium, the above findings strongly suggest that Fc{gamma}R-mediated phagocytosis could be driven by selective Src family members including Lyn and Hck. As an alternative explanation, it may be possible that a-Src kinases conversely down-regulated mCsk activity or displaced it from a proper position and that Fc{gamma}R signaling was initiated by endogenous Src family kinases, but not by a-Src kinases. These possibilities should be examined by further studies. However, it was noted that Fc{gamma}RIIIA/IIB-clustering induced physical association of the receptor complexes with a-Lyn, but not with a-Src (H. Kono and Z.-i. Honda, unpublished observation). These observations suggest that a-Lyn positively participates in the signal initiation rather than nonspecifically down-regulating mCsk functions.

The mechanisms underlying the specificity should be elucidated by future studies, but one potential clue is that N-terminal palmitoylation sites were found in Lyn and Hck but not in c-Src (75). It has become increasingly clear that palmitoylation of Src family kinases is required for kinases to associate with functional membrane subdomains variously called detergent-resistant membranes (DRMs) or sphingolipid-cholesterol rafts (62). Association of Src family kinases with DRMs seems to be essential for kinases to transduce signaling originated from aggregated TCR (63, 64). Recently, we provided evidence that early Fc{epsilon}RI signaling is catalyzed by Lyn and Fyn, but not by c-Src, and that creation of palmitoylation site in c-Src rendered it competent to transmit Fc{epsilon}RI signaling (76). In the current study, we confirmed that a-Lyn and a-Hck partially associated with DRMs, whereas a-Src did not. The differential localization may in part explain the specificity of Src family kinases in Fc{gamma}RIIIA/IIB-mediated phagocytosis.

Although a-Src could not restore Fc{gamma}RIIIA/IIB-mediated phagocytosis, a-Src did not seem to be completely separated from the Fc{gamma}RIIIA/IIB-signaling pathway. Expression of a-Src up-regulated clustering-induced tyrosine phosphorylation of c-Cbl and Vav. Syk tyrosine phosphorylation was also weakly up-regulated, but Syk activation was not detectable in a-Src/mCsk cells, suggesting that activation loop tyrosine was not efficiently phosphorylated. Concerning the roles of c-Src for other Fc receptor functions, physical association of c-Src with Fcµ receptor and Fcµ- and Fc{epsilon}RI-mediated c-Src activation have been noted (27, 77). These data including ours argue for involvement of a-Src in Fc-receptor signaling, albeit that it could transduce merely abortive signaling. Considering that the Fc receptor {alpha} subunit localizes at the outside of DRMs before clustering (47), it might be possible that FcR signaling could be initiated at the outside of DRMs, but that sufficient amplification of signaling to induce biological functions (i.e., phagocytosis) could be catalyzed only by DRM-associated Src family kinases.

Intriguingly, Fc{gamma}RIIB tyrosine phosphorylation was not increased by a-Src expression, while it was intensely augmented by a-Lyn and a-Hck expression. The striking specificity is of interest, because Fc{gamma}RIIB is implicated in negative signaling in a variety of Ag and Fc receptors (5, 22, 23, 59, 78, 79). One of our laboratory showed that tyrosine phosphorylation of Fc{gamma}RIIB ITIM after coligation with surface IgM is almost exclusively catalyzed by Lyn in murine B cells (34). The current findings revealed through a different approach that Fc{gamma}RIIB could be tyrosine phosphorylated by DRM-associated Lyn and Hck. We are now investigating the mechanisms how Fc{gamma}RIIB tyrosine phosphorylation is catalyzed solely by DRM-associated kinases.

Because distributions and functions of Src family kinases are highly overlapping, it is crucial to define selective Src family kinases in inflammatory signaling pathways. The current study provided further evidence that Src family kinases are important upstream regulators of Fc{gamma}R-mediated phagocytosis signaling, and for the first time suggested that the phagocytosis signaling could be driven by Lyn and by Hck, but only incompletely by c-Src. To further define the roles of other Src family kinases and to elucidate the submolecular structures responsible for the Fc{gamma}R functions are the next problems that should be investigated.

Since the submission of the paper, Fitzer-Attas et al. (80) showed that phagocytosis of IgG-coated SRBC in Lyn-/-Hck-/-Fgr-/- primary macrophages was not only delayed but also substantially decreased. These findings are consistent with our observations that mCsk almost abolished phagocytosis of IgG-coated beads in RAW cells.


   Acknowledgments
 
We thank Dr. Toshiyuki Takai for providing us with anti-Fc{gamma}RIIB Ab and H. Ota-Ichijo and M. Saka for excellent technical assistance.


   Footnotes
 
1 This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture and by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government. Back

2 Address correspondence and reprint requests to Zen-ichiro Honda, Department of Allergy and Rheumatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Back

3 Abbreviations used in this paper: Fc{gamma}R, Fc{gamma} receptor; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; Csk, C-terminal Src kinase; mCsk, membrane-anchored C-terminal Src kinase; mCsk(-), kinase-defective, membrane-anchored C-terminal Src kinase; F-actin, filamentous actin; COZ, complement-opsonized zymosan; DRM, detergent-resistant membrane; SH2, Src homology 2; WT, wild type; WT-RAW, wild-type RAW 264.7 cells. Back

Received for publication November 8, 1999. Accepted for publication April 12, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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