From the Division of Proteomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
Received for publication, February 4, 2003 , and in revised form, April 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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To propagate the immunoreceptor signal, adaptor proteins contribute to protein-protein and protein-lipid interactions through multiple domains and/or specific phosphotyrosine-containing sequences. Tyrosine phosphorylation of 3BP2
[PDB]
was observed in NK cells and mast cells by cross-linking FcR and Fc
RI, respectively (4, 5). To elucidate the function of 3BP2
[PDB]
, it is necessary to determine the protein-tyrosine kinase (PTK) that phosphorylates 3BP2
[PDB]
and its binding partner to assemble a signaling complex through specific phosphotyrosine-containing motifs in 3BP2
[PDB]
.
The Src family PTK Lyn is associated with FcRI
. Upon aggregation of Fc
RI, Lyn is critical for phosphorylating Fc
RI
and -
subunits on Tyr residues within the immunoreceptor tyrosine-based activating motif (ITAM) (68). By analogy with studies on Hck, Lyn is thought to be activated by the disassembly of the closed intramolecular interaction by (i) CD45-mediated dephosphorylation of C-terminal regulatory Tyr residue, (ii) binding to SH3 and SH2 ligands, and (iii) autophosphorylation of Tyr in the activation loop (9). What is the binding ligand of the SH3 and SH2 domains of Lyn in Fc
RI signaling pathway? Pull-down experiments using glutathione S-transferase (GST)-Lyn-SH2 fusion protein indicated that there were multiple phosphoproteins interacting with the Lyn-SH2 after the antigen stimulation of RBL-2H3 cells (10). In addition, although the displacement of intramolecular SH3 interaction is not well understood, it seems likely that some aggregation-induced change in an associated molecule provides a higher affinity SH3 ligand that binds to the Lyn-SH3 domain (11). The SH3 domain is directed toward the proline-rich region, but such a ligand has not been identified yet in the Fc
RI signaling.
We isolated nonreceptor type PTK Syk from porcine spleen (12). Syk is expressed in hematopoietic, epithelial, and endothelial cells (1316). When the ITAM of FcRI
subunits is phosphorylated by Lyn, Syk is recruited to the plasma membrane by binding its tandem SH2 domain and is autophosphorylated (17). Syk has multiple autophosphorylation sites. Tyr317, Tyr342, and Tyr346 are located in the linker region of Syk; Tyr519 and Tyr520 are in the activation loop of the kinase domain; and Tyr624 and Tyr625 are in the C-terminal region (18). Phosphorylation of Tyr519 and Tyr520 is critical for the enzymatic activation of Syk (19). Another member of the Syk family PTKs, ZAP-70, has a similar structural feature. However, there are differences in the binding molecules and mechanism of enzymatic activation between Syk and ZAP-70 (13, 14).
In the present study, we provide evidence that Tyr174, Tyr183, and Tyr446 on 3BP2 [PDB] are phosphorylated by a nonreceptor type PTK, Syk. Phosphorylation of 3BP2 [PDB] on Tyr446 creates a binding site for the Lyn-SH2 domain in vitro. Additional interaction between the proline-rich region and Lyn-SH3 may contribute to the constitutive codistribution of both molecules in RBL-2H3 cells. Furthermore, overexpression of 3BP2 [PDB] resulted in an enhancement of antigen-induced Lyn autophosphorylation in RBL-2H3 cells. On the other hand, overexpression of 3BP2 [PDB] -SH2 resulted in a suppression of Lyn autophosphorylation. Our results suggest that 3BP2 [PDB] is a potential ligand of Lyn-SH3/SH2 domains that positively regulates the autophosphorylation of Lyn in mast cells. Here, we are proposing the model of a novel Lyn-activating cycle in mast cells.
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EXPERIMENTAL PROCEDURES |
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Construction of cDNAThe HA-tagged expression construct of mouse 3BP2
[PDB]
in pMT3 vector (pMT3-HA-3BP2) was kindly provided by Dr. A. Altman (La Jolla Institute). Point mutations of Tyr174, Tyr183, and Tyr446 of pMT3-HA-3BP2 cDNA to Phe were generated by the site-directed mutagenesis kit (Stratagene, La Jolla, CA). The loss of function mutation of the 3BP2
[PDB]
-SH2 domain (Arg486 Lys) was described previously (5). A loss-of-association mutation (deletion of proline-rich region plus Y446F mutation) was created by PCR. The wild type and mutations of 3BP2
[PDB]
used in this study were shown in Fig. 1, A and B. All mutations were verified by DNA sequencing. Expression constructs of porcine Syk wild-type, kinase-inactive mutation (Lys396
Arg) and SH2 domain deletion mutation that lacks two SH2 domains and interdomain A (Phe10Ala272) (Syk-
SH2) were described previously (20). The cDNAs encoding Lyn, HA-focal adhesion kinase (gifts from Dr. R. P. Siraganian, National Institutes of Health, Bethesda, MD), and CAK
/Pyk2 (gift from Dr. T. Sasaki, Sapporo Medical University) were subcloned into pSVL expression vector (Amersham Biosciences). pApuro-T7-tagged Btk was gift from Dr. T. Kurosaki (Kansai Medical University, Japan), and cDNA encoding human Syk was from Dr. B. Mueller-Hilke (Deutsches RheumaForschungs-Zentrum) and subcloned into pApuro expression vector.
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Cell Culture, Transfection of cDNA, and Cell ActivationRat basophilic leukemia RBL-2H3 cells and COS-7 cells were maintained as monolayer cultures in DMEM (Sigma) with 100 units/ml of penicillin and 10% (v/v) heat-inactivated fetal calf serum. HA-3BP2 wild type (WT) cDNA was stably transfected into RBL-2H3 cells by the LacSwitch II inducible mammalian expression system (Stratagene). Briefly, 40 µg of XmnI-cleaved LacI repressor-expressing vector (pCMVLacI) was transfected into 5 x 106 cells by electroporation (950 microfarads, 310 V) using Gene Pulser II (Bio-Rad). Cells resistant to 0.7 mg/ml of hygromycin B (Roche Applied Science) were screened by the level of LacI repressor protein expression by immunoblotting of total cell lysates with anti-LacI antibody (Stratagene). The cells expressing the highest level of LacI were used as parental cells for the secondary stable transfection. 40 µg of NheI-digested linearized HA-3BP2-WT cDNA in the operator vector (pOPRSVI/MCS) were transfected into 5 x 106 parental cells by electroporation (950 microfarads, 310 V). Cells were selected with 0.7 mg/ml hygromycin B and 0.2 mg/ml of active G418 (Invitrogen). Cloned lines were screened by the level of isopropyl--<I>DI>-thiogalactopyranoside (IPTG) (Nacalai, Osaka, Japan)-induced expression of HA-3BP2 using the immunoblotting of total cell lysates with anti-HA antibody. As an internal control, total cell lysates were also analyzed by immunoblotting with anti-Fc
RI
mAb, kindly provided by Dr. R. P. Siraganian. Finally, two positive clones were chosen for further experiments. The cloned lines were preincubated with 1 mM IPTG for 48 h prior to cell activation. The RBL-2H3 cells overexpressing HA-3BP2-SH2 were described previously (5).
For cell activation, RBL-2H3 cells were sensitized with anti-DNP IgE (1:5000). Cells were washed once with Tyrode-Hepes buffer (10 mM Hepes, pH 7.4, 127 mM NaCl, 4 mM KCl, 0.5 mM KH2PO4, 1 mM CaCl2, 0.6 mM MgCl2, 10 mM LiCl, 5.6 mM glucose, and 0.1% BSA) and then stimulated with 30 ng/ml antigen DNP-BSA in the same buffer for the indicated times.
For transient transfection of COS-7 cells, 1 µg of each cDNA and 6 µl of FuGENE 6 reagent (Roche Applied Science) were added to 105 cells seeded in a six-well plate, according to the manufacturer's instruction. 48 h after transfection, cells were used for the experiments.
Preparation of Cell Lysates, Immunoprecipitation, and ImmunoblottingFor immunoprecipitation, cells were washed with ice-cold PBS twice and solubilized in Triton X-100 lysis buffer (1% Triton X-100, 50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin). Cells were precleared by the centrifugation, and supernatants were incubated with the indicated antibody prebound to protein A-agarose beads. After rotation for 1 h at 4 °C, the beads were washed four times with the lysis buffer. Immunoprecipitated proteins were eluted by heat treatment at 100 °C for 5 min with 2x sample buffer. Aliquots of the supernatant of first centrifugation were used as detergent-soluble cell lysates.
For immunoblotting, samples were separated by SDS-PAGE and electronically transferred onto polyvinylidene difluoride membranes (0.45-µm pore size; Millipore Corp., Bedford, MA). The membranes were incubated with 5% milk in TBST (25 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Then membranes were washed once with TBST and incubated with the indicated primary antibodies in TBST for 1 h at 4 °C. After washing four times in TBST, the membranes were reacted with appropriate secondary antibodies (1:5000) for 30 min at room temperature and washed extensively four times in TBST. In all blots, proteins were visualized by the enhanced chemiluminescence (PerkinElmer Life Sciences).
Pull-down Experiments Using GST Fusion ProteinsThe GST-rat Lyn-SH2 domain expression construct was kindly provided by Dr. Reuben P. Siraganian (10). The Lyn-SH3 domain was amplified by PCR, using the primer 5'-GGAATTCAGAGGAGCAAGGGGACATTGTG-3' and 5'-GGCCGCTCGAGCTCTTCGGTTTCTAAGGTGTT-3' (the restriction enzyme digestion sites are underlined). The Pfu PCR product was subcloned into the pGEX-4T.3 (Amersham Biosciences) to make a domain in-frame with upstream GST.
The bacteria cells transformed by GST-fusion protein-expressing constructs were solubilized in Triton X-100 lysis buffer, and the cell lysates were clarified by the centrifugation. Resulting supernatants were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 1 h at 4 °C. The beads were washed four times with Triton X-100 lysis buffer and then washed twice with the binding buffer (1% Nonidet P-40, 50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na3VO4, and protease inhibitors). RBL-2H3 or COS-7 cells transfected with the different kinds of cDNA were solubilized in the binding buffer. The postnuclear cell lysates were incubated with the indicated GST fusion protein prebound to glutathione-Sepharose 4B beads for 1 h at 4 °C. The beads were washed four times with the binding buffer, and precipitated proteins were eluted by heat treatment at 100 °C for 5 min with 2x sample buffer.
In Vitro Protein Kinase AssayThe washed anti-Lyn immunoprecipitates were incubated in 40 µl of the kinase buffer (40 mM HEPES, pH 7.5, 10 mM MgCl2,4 µM ATP, 4 µCi of [-32P]ATP) at 30 °C for 5 min. Reactions were terminated by heat treatment at 100 °C for 5 min with 3x sample buffer, and proteins were separated by 8% SDS-PAGE. The gel was incubated with 1 N KOH for 1 h at 56 °C to remove phosphoserine and most of phosphothreonine. After gel drying, radiolabeled proteins were visualized by autoradiography using image analyzer FLA-3000GK (Fuji Film, Tokyo, Japan). Immunoprecipitated Lyn were confirmed by the immunoblotting with anti-Lyn antibody.
Immunofluorescence MicroscopyThe RBL-2H3 cells expressing HA-3BP2 were plated onto a coverslip in six-well plates (105 cells/well) and saturated with 1 mM IPTG. After a 24-h incubation at 37 °C, cells were sensitized with anti-DNP IgE (1:5000) and further incubated for 24 h (total of 48 h with IPTG) at 37 °C. Cells were washed two times with PBS, fixed with 4% paraformaldehyde in PBS for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Then cells were washed four times for 2 min in PBS and blocked with 3% BSA in PBS. For double staining, cells were reacted with rabbit anti-Lyn antibody and anti-HA mAb (Covance, Princeton, NJ) for 1 h, washed three times with 0.5% Triton X-100 in PBS, and then incubated with the secondary antibody (Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 594 goat anti-rabbit IgG (red) (Molecular Probes, Inc., Eugene, OR) for 30 min. After the extensive washing, cells were mounted using SlowFade-Light (Molecular Probes) and analyzed by using confocal imaging system LSM5Pascal (Carl Zeiss, Jena, Germany) (22).
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RESULTS |
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3BP2-Tyr446 Was a Major Binding Site to the Lyn-SH2 DomainTo examine the role of 3BP2
[PDB]
tyrosine phosphorylation, we utilized the RBL-2H3 cells in which the expression of HA-3BP2 is induced by the addition of IPTG. As we reported previously, aggregation of FcRI induced a rapid tyrosine phosphorylation of 3BP2
[PDB]
in RBL-2H3 cells (Fig. 2A) (5). Phosphorylation of 3BP2
[PDB]
was visible at 30 s after antigen stimulation and sustained to 10 min. To identify the binding molecule(s) of 3BP2
[PDB]
, pull-down experiments were carried out by using various GST-SH2 fusion proteins against mast cells expressing HA-3BP2 (data not shown). Among them, GST-Lyn-SH2 reacted with HA-3BP2 from cell lysates from antigen-stimulated cells, suggesting that antigen-induced tyrosine phosphorylation of 3BP2
[PDB]
creates the binding site for Lyn-SH2 in mast cells (Fig. 2B).
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To examine the mechanism of this interaction, a reconstitution system using COS-7 cells was utilized by transient transfection of cDNA. 3BP2
[PDB]
-WT, but not 3BP2
[PDB]
3F, was bound to Lyn-SH2 when it was coexpressed with Syk (Fig. 2C, lane 2 versus lane 4). Phosphorylation of 3BP2
[PDB]
by Lyn could not induce enough interaction with Lyn-SH2 in COS-7 cells, due to the low magnitude of 3BP2
[PDB]
tyrosine phosphorylation as shown in Fig. 1C (Fig. 2C, lane 6). Based on these observations, we then attempted to identify the corresponding Tyr residue(s) that associated with Lyn-SH2 by using this system. The 3BP2
[PDB]
-WT, 3BP2
[PDB]
3F, and 3BP2
[PDB]
mutants in which two Tyr residues were substituted for Phe (174Y, 183Y, and 446Y) (Fig. 1B) was cotransfected with Syk, and each cell lysate was incubated with GST-Lyn-SH2 (Fig. 2D). Among three Tyr residues, the binding of 3BP2
[PDB]
446Y for Lyn-SH2 was similar to that of wild type (Fig. 2D, lane 2 versus lane 10). 3BP2
[PDB]
183Y caused a weak binding with Lyn-SH2, and 3BP2
[PDB]
174Y did not associate with Lyn-SH2 (Fig. 2D, lanes 8 and 6, respectively). This result indicates that phosphorylation of Tyr446 by Syk creates binding site for Lyn-SH2. For further confirmation, another pull-down experiment was performed. 3BP2
[PDB]
-WT, 3BP2
[PDB]
3F, or 3BP2
[PDB]
mutants in which one Tyr residue was substituted for Phe (Y174F, Y183F, Y446F) (Fig. 1B) was cotransfected with Syk, and each cell lysate was incubated with GST-Lyn-SH2 (Fig. 2E). Compared with the 3BP2
[PDB]
-WT, expression of 3BP2
[PDB]
Y446F with Syk resulted in a decreased interaction with Lyn-SH2 (Fig. 2E, lane 2 versus lane 10). A point mutation of Tyr174 or Tyr183 into Phe did not decrease binding with Lyn-SH2 (Fig. 2E, lanes 6 and 8). In the experiments using COS-7 cells, the expression amounts of PTK and the different kinds of 3BP2
[PDB]
were confirmed by the immunoblotting of cell lysates (Fig. 2, CE, right panel). Therefore, these results suggest that antigen stimulation induces tyrosine phosphorylation of 3BP2
[PDB]
, causing the interaction with Lyn. Moreover, among three putative tyrosine phosphorylation sites in 3BP2
[PDB]
, phosphorylation of Tyr446 seemed to be the major site contributing to the interaction with Lyn-SH2 upon FcRI aggregation in mast cells.
Phosphorylation of Tyr446 but Not the SH2 Domain of 3BP2 [PDB] Contributed to the Interaction of Lyn with SykGST-Lyn-SH2 binds to Syk in antigen-stimulated RBL-2H3 mast cells (10). A pull-down experiment demonstrated that Syk was precipitated with GST-Lyn-SH2 when it was coexpressed with 3BP2 [PDB] -WT in COS-7 cells (Fig. 3A, lane 2). Kinase-inactive form of Syk was poorly precipitated with Lyn-SH2 (Fig. 3A, lane 3). Also, a point mutation of Tyr446 of 3BP2 [PDB] caused the same result, suggesting that phosphorylation of Tyr446 of 3BP2 [PDB] by Syk is required for the full-scale binding of Syk with Lyn-SH2 (Fig. 3A, lane 4). Thus, 3BP2 [PDB] is necessary for Syk to associate with Lyn-SH2 in COS-7 cells, because Syk alone could not phosphorylate the corresponding Tyr residue to interact with Lyn-SH2, unlike in antigen-stimulated mast cells (Fig. 3A, lane 1) (10). Interestingly, a point mutation of Arg486 into Lys in 3BP2 [PDB] -SH2 had no effect on the association with Syk, although 3BP2 [PDB] -SH2 was shown to directly associate with Syk in yeast (Fig. 3A, lane 5) (2).
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Truncation of the SH2 domain of Syk resulted in an increase in the kinase activity and tyrosine phosphorylation of Syk (24). Unlike the wild type, the SH2 domain-truncated form of Syk (Syk-SH2) was capable of associating with Lyn-SH2, independent of 3BP2
[PDB]
tyrosine phosphorylation (Fig. 3B, lanes 1 and 2). The multiple bands of Syk-
SH2 seem to be due to its autophosphorylation, since Syk-
SH2 possesses seven putative autophosphorylation sites (Tyr317, Tyr342, Tyr346, Tyr519, Tyr520, Tyr624, and Tyr625) (Fig. 3B, lanes 1 and 2, lower panel) (18, 25). This suggests that Lyn-SH2 could bind to the autophosphorylation site of Syk-
SH2 in COS-7 cells. To interact with Lyn-SH2, wild type Syk requires 3BP2
[PDB]
; however, Syk with multiple phosphorylation does not require 3BP2
[PDB]
. The expression amounts of Syk, 3BP2
[PDB]
, and their mutants were confirmed by the immunoblotting of cell lysates (Fig. 3, A and B, right panel). Therefore, these results suggest that 3BP2
[PDB]
stimulates tyrosine phosphorylation of Syk, indirectly or directly. Phosphorylation of Tyr446 is critical for this hypothetical function of 3BP2
[PDB]
.
Proline-rich Region of 3BP2
[PDB]
Associated with Lyn-SH3 Domain3BP2 was first isolated as a c-Abl-SH3 domain-binding protein via its proline-rich region (1). Also, 3BP2
[PDB]
was capable of binding to SH3 domain of Grb2, Nck, Src, or Fyn (1, 2). Since we have demonstrated that 3BP2
[PDB]
binds to the Lyn-SH2 domain, we then tested whether the SH3 domain of Lyn could interact with 3BP2
[PDB]
. The binding of 3BP2
[PDB]
with the Lyn-SH3 domain was observed when GST-Lyn-SH3 domain reacted with cell lysates from both unstimulated and antigen-stimulated mast cells expressing HA-3BP2 (Fig. 4A). The expression of HA-3BP2 was confirmed by the immunoblotting of cell lysates (Fig. 4A, bottom panel). To examine the mechanism of this interaction, a reconstitution system using COS-7 cells was utilized. Whereas the Lyn-SH2 bound to 3BP2
[PDB]
when it was coexpressed with Syk, Lyn-SH3 could bind to 3BP2
[PDB]
without phosphorylation by Syk (Fig. 4B). Substitution of Tyr446 did not affect the interaction of both molecules. The interaction between 3BP2
[PDB]
and Lyn-SH3 was dramatically decreased by the deletion of the proline-rich region in 3BP2
[PDB]
(Fig. 4C). 3BP2
[PDB]
with the deletion of the proline-rich region and a Y446F mutation (Pro Y446F) was poorly bound to Lyn-SH3. Therefore, this result indicated that proline-rich region could associate with Lyn-SH3 domain. The expression amounts of Syk and the different kinds of 3BP2
[PDB]
were confirmed by the immunoblotting of cell lysates (Fig. 4, B and C, right panel). Similarly, both Lyn-SH2 and SH3 domains bound to the endogenous 3BP2
[PDB]
(Fig. 4D). The binding of the endogenous 3BP2
[PDB]
with Lyn-SH2 was observed when the cells were stimulated with the antigen, whereas the interaction with Lyn-SH3 domain was also detected both in nonstimulated and stimulated cells. Immunohistochemical analysis by laser confocal microscopy revealed that 3BP2
[PDB]
and Lyn were colocalized in the plasma membrane in unstimulated and antigen-stimulated RBL-2H3 cells (Fig. 4E). Since 3BP2
[PDB]
was not tyrosine-phosphorylated in unstimulated cells, the interaction of the 3BP2
[PDB]
proline-rich region with Lyn-SH3 may contribute to the colocalization of both molecules in mast cells.
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Overexpression of 3BP2 [PDB] -stimulated Autophosphorylation of LynFinally, we attempted to examine the functional role of the interaction of 3BP2 [PDB] with Lyn. Cells with or without an overexpression of 3BP2 [PDB] wild type were stimulated with an antigen, and cell lysates were immunoprecipitated with anti-Lyn antibody. Anti-Lyn immunoprecipitates were subjected to an in vitro protein kinase assay. Overexpression of 3BP2 [PDB] resulted in an increase in the autophosphorylation of Lyn upon antigen stimulation in RBL-2H3 cells (Fig. 5A). The densitometric analysis revealed that IPTG-induced expression of 3BP2 [PDB] resulted in a 1.7-fold increase in the kinase activity of Lyn after antigen stimulation compared with that of unstimulated cells, whereas the value was 1.2-fold in control cells (Fig. 5A). Furthermore, overexpression of 3BP2 [PDB] -SH2, the dominant-negative form of 3BP2 [PDB] , resulted in the decrease (0.30.7-fold) in the autophosphorylation of Lyn (Fig. 5B). Expression of HA-3BP2-WT and HA-3BP2-SH2 was confirmed by the immunoblotting of cell lysates (Fig. 5, A and B, lower panels). Therefore, these results indicated that overexpression of 3BP2 [PDB] -WT enhances the autophosphorylation of Lyn to increase in its kinase activity. Interaction of Lyn with the endogenous 3BP2 [PDB] positively regulates the kinase activity of Lyn.
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DISCUSSION |
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Aggregation of FcRI induces activation of Lyn, which then phosphorylates tyrosine resides in the ITAM of Fc
RI
and -
subunits to recruit and activate downstream Syk. Similarly, genetic studies have demonstrated that an expression of Lyn is necessary to activate Syk to trigger calcium mobilization in avian B-cells (30). These observations indicate that Lyn is upstream of Syk (Fig. 6B, left) (31). It is expected that 3BP2
[PDB]
is the downstream substrate of Syk. When 3BP2
[PDB]
is tyrosine-phosphorylated by Syk, it mediates feedback regulation of the Fc
RI-mediated signaling pathway by stimulating or sustaining the kinase activity of Lyn (Fig. 6B, left). Therefore, 3BP2
[PDB]
may function to multiply the antigen-induced mast cell-activating signal. Interestingly, there is an evidence of the alternative activation mechanism of Syk (Fig. 6B, right). Syk is associated with the T-cell receptor prior to the stimulation, and CD3 engagement induces activation of Syk, independent of Lck or CD45 (32, 33). In addition, Syk augments tyrosine phosphorylation of CD3
to stimulate the kinase activity of Syk by itself (34). We have demonstrated that 3BP2
[PDB]
is preferentially tyrosine-phosphorylated by Syk in COS-7 cells. Phosphorylation of Tyr446 of 3BP2
[PDB]
by Syk probably functions as a molecular switch to activate Lyn (Fig. 6A). Some of Syk in the different compartmentalized fractions could have a regulatory role for Lyn, since B-cell receptor aggregation may directly stimulate the activity of the receptor-associated Syk (35). Collectively, our findings raise the possibility that Lyn could be regulated by Syk in Fc
RI signaling in mast cells (Fig. 6B, right). 3BP2
[PDB]
may contribute the alternative Syk-mediated activation mechanism of Lyn in mast cells.
The kinase activity of Src family PTKs has been thought to be regulated by C-terminal Src kinase (Csk) and CD45 in hematopoietic cells. The present study suggests the existence of the four-step model of Lyn activation cycle shown in Fig. 6C. First, genetic study by Yanagi et al. (36) demonstrated that disruption of the CD45 gene in avian B cells resulted in an increase in phosphorylation of both tyrosine residues in the C-terminal regulatory site and in the activation loop. Kinase activity of Lyn was suppressed in the CD45-negative cells, although both of the tyrosine residues were hyperphosphorylated (36). In mast cells, genetic studies demonstrated that CD45 is not required for FcRI-mediated degranulation (37). These observations indicate that CD45 is a prerequisite for Lyn prior to activation, presumably by dephosphorylation of both autophosphorylation and C-terminal negative regulatory tyrosines to displace an intramolecular interaction (Fig. 6C) (11, 36). Second, we have demonstrated that 3BP2
[PDB]
is a ligand of SH3/SH2 domains of Lyn. Inhibition of the function of Lyn by its SH3 domain binding peptide demonstrates the importance of the Lyn-SH3 domain in mast cell activation (38). Immunohistochemical analysis by laser confocal microscopy reveals that 3BP2
[PDB]
is distributed in the plasma membrane together with Lyn prior to stimulation (Fig. 4E). In the resting state, the Lyn-SH3 domain can be associated with the proline-rich region of 3BP2
[PDB]
. It is expected that 3BP2
[PDB]
interacts with the target molecule via its proline-rich region, which allows for the efficient and rapid activation of Lyn upon Fc
RI aggregation. Third, phosphorylation of Tyr446 might create a high affinity binding site to the Lyn-SH2 domain, and presumably this binding causes the conformational change of Lyn, leading to the autophosphorylation. Syk, Lyn itself, or Btk is a candidate PTK to phosphorylate Tyr446 of 3BP2
[PDB]
. Our results suggest the possibility that Lyn could be downstream of some other PTKs at the initial state of mast cell activation. Autophosphorylated Lyn elevates its kinase activity for propagating the downstream signals. Fourth, the activation of Lyn was terminated by the phosphorylation of C-terminal tyrosine by Csk. Lyn with a double tyrosine phosphorylation is inactive (36). Alternatively, some of the activated Lyn was modified by multiubiquitinations by c-Cbl after Fc
RI stimulation.2 Taken together, we concluded that 3BP2
[PDB]
might be one of the regulatory factors of the Lyn activation cycle in Fc
RI signaling.
Although the phenotype of 3BP2 [PDB] knockout mice has not been reported yet, there is evidence that mutation of the 3BP2 gene cause the human disease cherubism. Cherubism is an autosomal dominant inherited disease characterized by multiple symmetrical cysts in the mandible and the maxilla, excessive bone degradation, and typical facial swelling. The cysts are filled with a fibrous tissue mass containing multinucleated, osteoclastic giant cells. Studies of 12 families of this disease detected a single amino acid mutation in exon nine of the 3BP2 gene causing amino acid substitution in Arg415, Pro418, or Gly420 of the 3BP2 [PDB] protein (39). 3BP2 [PDB] could be implicated in the balance of osteoclast and osteoblast activities that are essential for normal tooth eruption. Although the abnormal signals in cherubism have not yet been identified, it is expected that Tyr446-mediated function of 3BP2 [PDB] could be affected by these mutations, since all mutations identified are located near Tyr446. Although 3BP2 [PDB] was preferentially detected in spleen, peripheral blood leukocyte, and thymus, the exact role of 3BP2 [PDB] in hematopoietic/lymphoid cell signaling remains unclear (2, 3). So far, immunological defects due to the mutations of 3BP2 [PDB] observed in cherubism have not yet been reported.
The present study has demonstrated that 3BP2
[PDB]
interacts with Lyn and may possibly promote the catalytic activity of Lyn. In addition to integrating signaling molecule at the appropriate location, an "adaptor" protein 3BP2
[PDB]
has a regulatory role in PTK as a ligand binding to the SH3/SH2 domains of Lyn, similar to the function of FcRI
as a ligand of Syk PTK in mast cells.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 81-78-382-5404; Fax: 81-78-382-5419; E-mail: ksada{at}med.kobe-u.ac.jp.
1 The abbreviations used are: SH, Src homology; FcRI, high affinity IgE receptor; PTK, protein-tyrosine kinase; ITAM, immunoreceptor tyrosine-based activating motif; GST, glutathione S-transferase; HA, hemagglutinin epitope; IPTG, isopropyl-
-<I>DI>-thiogalactopyranoside; DNP, dinitrophenyl; mAb, monoclonal antibody; BSA, bovine serum albumin; WT, wild type; PBS, phosphate-buffered saline.
2 S. Kyo, K. Sada, and H. Yamamura, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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