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Division of Proteomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan

	Abstract

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Adaptor protein 3BP2 [PDB] positively regulates the high affinity IgE receptor (FcRI)-mediated activation of degranulation in mast cells. Genetic study identified the point mutations of 3BP2 gene in human-inherited disease cherubism. The multiple cysts in cherubism lesion of jaw bones are filled with the activated osteoclasts and stromal cells, including mast cells. By over-expression study using rat basophilic leukaemia RBL-2H3 mast cells, we have analysed the effect of the point mutations on the function of 3BP2 [PDB] protein, which plays a positive regulatory role on FcRI-mediated mast cell activation. Over-expression of 3BP2 [PDB] mutants suppressed the antigen-induced degranulation and cytokine gene transcription. Antigen-induced phosphorylation of Vav1, activation of Rac1, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), inhibitor of nuclear factor B kinase (IKK) and nuclear factor of activated T cells (NFAT) were all impaired in the cells over-expressing the cherubism mutants of 3BP2 [PDB] . Furthermore, cherubism mutations of 3BP2 [PDB] may abrogate the binding ability to interact with chaperone protein 14-3-3. These results demonstrate that over-expression of the mutant form of 3BP2 [PDB] inhibits the antigen-induced mast cell activation. It suggests that point mutations of 3BP2 gene cause the dysfunction of 3BP2 [PDB] in vivo.

	Introduction

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In the immune receptor signalling, the scaffold, anchoring and adaptor molecules have a role to connect the receptor-activating signals to the downstream effectors by assembling, targeting and regulating the signalling molecules (Pawson & Scott 1997; Burack et al. 2002). These molecules have multiple domains and motifs that allow binding to the other cytoplasmic molecules and therefore act as positive or negative regulators controlling the immune receptor-mediated intracellular signalling (Rudd 1999; Janssen & Zhang 2003). Among them, linker for activation of T cells (LAT), SH2-containing leucocyte phosphoprotein of 76 kDa (SLP-76) and Grb2-associated binder-2 (Gab2) have a critical role for FcRI-mediated anaphylactic reaction in vivo and mast cell activation (Pivniouk et al. 1999; Saitoh et al. 2000; Gu et al. 2001; Rivera 2002). Selective regulation of these adaptor molecules in mast cells might have therapeutic potential.

Molecular adaptor/scaffold 3BP2 [PDB] (c-Abl SH3 domain-binding protein-2) was originally isolated as a protein-tyrosine kinase (PTK) c-Abl-Src homology 3 (SH3) domain-binding protein of unknown function (Ren et al. 1993). 3BP2 [PDB] positively cooperates with Syk to stimulate T-cell receptor-mediated activation of transcription factors and cytokine promoters (Deckert et al. 1998). In natural killer (NK) cells, 3BP2 [PDB] is the substrate of PTK and positively regulates NK cell-mediated cytotoxicity through phosphorylation of Tyr¹⁸³ in 3BP2 [PDB] (Jevremovic et al. 2001). We previously demonstrated that adaptor protein 3BP2 [PDB] positively regulates FcRI-mediated mast cell activation (Sada et al. 2002). C-terminal SH2 domain of 3BP2 [PDB] binds to the LAT which regulates FcRI-mediated Ca²⁺ mobilization and activation of Ras. Over-expression of the truncated SH2 domain of 3BP2 [PDB] inhibits the FcRI-mediated degranulation in mast cells (Sada et al. 2002). 3BP2 [PDB] is tyrosine phosphorylated by non-receptor type of PTKs, in particular by Syk, on Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴⁴⁶. Among them, phosphorylation of Tyr⁴⁴⁶ appears to be important for the interaction with Lyn to regulate the early FcRI signals (Maeno et al. 2003). Recently, Ser²⁷⁷ in 3BP2 [PDB] was identified as a binding site for 14-3-3 to negatively regulate T-cell receptor-mediated activation of transcription factors (Foucault et al. 2003).

Several point mutations of 3BP2 [PDB] were identified in human-inherited disease cherubism (Ueki et al. 2001; Imai et al. 2003; Lo et al. 2003). All mutations were located in exon 9 and affect 3 amino acids within a 6-amino acid sequence, between the central Pro-rich region and C-terminal SH2 domain. Cherubism is characterized with the multiple symmetric cysts in the jaw bones. The cysts were filled with the multinucleated osteoclasts and stromal cells including mast cells. Both osteoclasts and mast cells express 3BP2 [PDB] (Ueki et al. 2001; Sada et al. 2002). Amino acid substitutions of 3BP2 [PDB] were expected to lead to a gain of function or to act in a dominant-negative manner in the process of differentiation or activation of these cells. In addition, 3BP2 [PDB] is located within the chromosome region 4p16.3 that is frequently deleted in bladder cancer and Wolf–Hirschhorn syndrome (Bell et al. 1997; Zollino et al. 2000).

The present experiments attempted to analyse the effect of point mutations of the 3BP2 gene on the function of 3BP2 [PDB] protein. The functional effects of the 3BP2 [PDB] mutations were analysed by the FcRI-mediated degranulation, cytokine production and intracellular signalling by using the rat basophilic leukaemia RBL-2H3 mast cells stably over-expressing the mutant forms of 3BP2 [PDB] .

	Results

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Generation of mast cell lines expressing the mutant form of 3BP2

As 3BP2 [PDB] -deficient mice were not available, we utilized the over-expression system using RBL-2H3 mast cells to analyse the functional defect of 3BP2 [PDB] with single amino acid substitutions identified in human-inherited disease cherubism (Ueki et al. 2001). The expression constructs of various mutants of 3BP2 [PDB] were generated (Fig. 1A) and then stably transfected into RBL-2H3 cells (Sada et al. 2002). Cloned lines were selected by 0.4 mg/mL of G418 and then screened by immunoblotting with anti-3BP2, anti-HA and anti-FcRI antibodies. For further analysis, we chose two clones of each mutant form of cDNA. Transfection of cherubism mutant forms of 3BP2 [PDB] [Arg⁴¹³ to Gln (R413Q), Pro⁴¹⁶ to His (P416H) and Gly⁴¹⁸ to Arg (G418R)] caused the eventual over-expression, whereas transfection of 3BP2 [PDB] with a point mutation of Ser²⁷⁷ to Ala (S277A), a binding site for 14-3-3 (Foucault et al. 2003), resulted in a little increase in the expression of total 3BP2 [PDB] (Fig. 1B). Densitometric analysis revealed that transfection of cherubism mutant forms of 3BP2 [PDB] caused 5–7-fold increase in 3BP2 [PDB] protein. We could not obtain the clones over-expressing 3BP2 [PDB] -S277A. To test the effect of the over-expression of 3BP2 [PDB] mutants on mast cell function, we examined degranulation and cytokine release in RBL-2H3 cells.

View larger version (26K): [in this window] [in a new window]   Figure 1  Generation of mast cell lines over-expressing cherubism mutant form of 3BP2 [PDB] . (A) Schematic diagram of HA-tagged mouse 3BP2 [PDB] constructs used in this study. Ser277 in the Pro-rich region, a putative binding site for 14-3-3 was substituted for Ala (S277A). Substitutions of Arg413 for Gln (R413Q), Pro416 for His (P416H) and Gly418 for Arg (G418R) correspond to the mutations identified in cherubism (Arg415, Pro418 and Gly420 in human 3BP2 [PDB] ). (B) Characterization of the cloned lines for further analysis. Total cell lysates (105) of non-transfected control RBL-2H3 cells and stable clones expressing the different kind of cDNA were analysed by immunoblotting with anti-3BP2, anti-HA and anti-FcRIß antibodies, respectively.

The antigen-induced degranulation was measured by the release of ß-hexosaminidase (Fig. 2). Control parental cells and cells expressing the different kinds of 3BP2 [PDB] were primed with IgE and then stimulated with the antigen at different concentrations. The antigen-induced release of ß-hexosaminidase was normalized by that induced by Ca²⁺ ionophore A23187 [GenBank] . Expression of 3BP2 [PDB] -S277A increased the antigen-induced degranulation, as expected by the evidence that the over-expression of 3BP2 [PDB] -S277A increases NFAT activity in Jurkat T cells (Foucault et al. 2003). In contrast, the over-expression of 3BP2 [PDB] -P416H or 3BP2 [PDB] -G418R decreased the antigen-induced degranulation, suggesting that these mutant forms of 3BP2 [PDB] act as the dominant negative form in RBL-2H3 cells. Over-expression of another cherubism mutant, 3BP2 [PDB] -R413Q, had no effect on the antigen-induced degranulation. We have previously demonstrated that over-expression of the truncated mutant of 3BP2 [PDB] (3BP2 [PDB] -SH2) results in a suppression of antigen-induced degranulation, Ca²⁺ mobilization, tyrosine phosphorylation of phospholipase C-1 and -2, suggesting that the endogenous 3BP2 [PDB] is a positive regulator of FcRI-mediated degranulation (Sada et al. 2002). Therefore, this result demonstrates that point mutations of 3BP2 [PDB] identified in cherubism cause the dysfunction of 3BP2 [PDB] on the FcRI-mediated degranulation pathway.

View larger version (31K): [in this window] [in a new window]   Figure 2  Analysis of ß-hexosaminidase release. The control cells and cloned lines were sensitized with anti-DNP IgE and then stimulated with the indicated concentration of antigen DNP-BSA (Ag) or 1 µM Ca2+ ionophore A23187 [GenBank] . The antigen-induced ß-hexosaminidase release was presented as a percentage of that induced by A23187 [GenBank] . The results are the mean values ± SD from three independent experiments. A23187 [GenBank] -induced average releases ± SD as a percentage of total activity were 28 ± 2% (control), 38 ± 2% (S277A-#6), 29 ± 2% (S277A-#10), 42 ± 2% (R413Q-#13), 35 ± 1% (R413Q-#17), 25 ± 1% (P416H-#1), 27 ± 2% (P416H-#8), 38 ± 2% (G418R-#16) and 30 ± 1% (G418R-#21), respectively.

View larger version (42K): [in this window] [in a new window]   Figure 3  Analysis of protein kinase activation. The control cells and cloned lines were sensitized with anti-DNP IgE and then stimulated with 30 ng/mL of antigen DNP-BSA (Ag) for the indicated times. Cell lysates (1–2 x 105) were analysed by immunoblotting with anti-phospho-JNK (pJNK) and JNK (A), phospho-ERK (pERK) and ERK (B), phospho-p38 (p-p38) and p38 MAPK (C) and phospho-IKK (pIKK) and IKK (D). Similar results were obtained when other cloned lines were examined. The results are representative of three independent experiments.

In vitro binding study using NK cell lysates demonstrated that Tyr¹⁸³ in 3BP2 [PDB] is a binding site to Vav1, a guanine nucleotide exchanging factor of Rac1 (Jevremovic et al. 2001). Genetic analysis demonstrated that expression of Vav1 is required for the antigen-induced mast cell activation (Manetz et al. 2001). Based on this evidence, we analysed the effect of the over-expression of various 3BP2 [PDB] mutants on Vav1 and Rac1 activation in mast cells (Fig. 4). In parental RBL-2H3 cells, antigen-stimulation increased tyrosine phosphorylation of Vav1 and GTP-binding form of Rac1 (Fig. 4A and B). Tyrosine phosphorylation of Vav1 and GTP-binding form of Rac1 were impaired by the over-expression of cherubism mutant forms of 3BP2 [PDB] . Over-expression of 3BP2 [PDB] -R413Q had no effect on antigen-induced tyrosine phosphorylation of Vav1. As expected, expression of 3BP2 [PDB] -S277A increases Rac1-GTP.

View larger version (46K): [in this window] [in a new window]   Figure 4  Analysis of Vav1 and Rac1 activation. (A) Tyrosine phosphorylation of Vav1. The control cells and cloned lines (107) were sensitized with anti-DNP IgE and then stimulated with 30 ng/mL of antigen DNP-BSA (Ag) for the indicated times. Cells were solubilized in the Triton lysis buffer and pre-cleared by centrifugation. The resulted supernatants were immunoprecipitated with anti-Vav1 antibody. Immunoprecipitates were analysed by immunoblotting with anti-pTyr and anti-Vav1 antibodies. Similar results were obtained when other cloned lines were examined. The results are representative of two independent experiments. (B) Pak1-PBD pull-down assay. The control cells and cloned lines (107) were sensitized with anti-DNP IgE and then stimulated without (–) or with 30 ng/mL of antigen DNP-BSA (Ag) for 1 min (+). Cells were solubilized in Mg2+ binding buffer and detergent-soluble lysates were pre-cleared by 20 µg of GST pre-bound to glutathione sepharose 4B beads. After centrifugation, the resulting supernatants were incubated with 20 µg of GST-Pak1-PBD pre-bound to glutathione sepharose 4B beads. The binding proteins and cell lysates were analysed by immunoblotting with anti-Rac1 mAb. Similar results were obtained when other cloned lines were examined. The results are representative of three independent experiments. (C) Vav1-SH2 domain pull-down assay. COS-7 cells were co-transfected with Syk, various 3BP2 [PDB] constructs. Forty-eight hours after transfection, cells (5 x 105) were solubilized in the binding buffer. Cell lysates were pre-cleared by GST and then incubated with 20 µg of GST-Vav1-SH2 domain pre-bound to glutathione sepharose 4B beads. The binding protein and cell lysates were analysed by immunoblotting with anti-HA mAb and anti-Syk antibody (upper panel). (C, right panel) Tyrosine phosphorylation of 3BP2 [PDB] mutants. COS-7 cells expressing Syk, together with various 3BP2 [PDB] constructs, were solubilized in the Triton lysis buffer and pre-cleared lysates were immunoprecipitated with anti-HA mAb. Immunoprecipitates and cell lysates were analysed by immunoblotting with anti-pTyr and anti-HA mAbs (lower panel). The results are representative of three independent experiments. vec, vector; WT, wild-type.

Over-expression of 3BP2 with a point mutation results in the suppression of cytokine synthesis in mast cells

The antigen-induced transcription of multiple cytokine mRNA was compared by RNase protection assay (Fig. 5A). Control parental cells and cells expressing the different kinds of 3BP2 [PDB] were primed with IgE and then stimulated with 30 ng/mL of the antigen. Compared with the wild-type, expression of cherubism mutant forms of 3BP2 [PDB] dramatically decreased the transcriptions of interleukin (IL)-3 and IL-4 (Fig. 5A).

View larger version (22K): [in this window] [in a new window]   Figure 5  Analysis of cytokine gene transcriptions. (A) RNase protection assay. The control cells and cloned lines (5 x 106) were sensitized with anti-DNP IgE and then stimulated without (–) or with (+) 30 ng/mL of antigen DNP-BSA (Ag) for 1 h. Total RNA was extracted and hybridized with the 32P-labelled RNA probes of rat cytokines. After RNase treatment, the protected double-stranded RNA was separated by urea gel and analysed by autoradiography. Similar results were obtained when other cloned lines were examined. The results are representative of three experiments. (B) The reporter gene constructs were transiently transfected into control cells or cloned lines (107) by electroporation. Cells were primed with anti-DNP IgE and then stimulated without (–) or with (+) 30 ng/mL of antigen DNP-BSA (Ag). The relative luciferase activity of each reporter gene was normalized by the protein concentration and expressed as a fold of increase compared with activity without stimulation. The results are representative of three experiments.

Point mutations of 3BP2 in cherubism abrogate the interaction with 14-3-3

Recent findings revealed that phosphorylation of 3BP2 [PDB] on serine negatively regulates T-cell activation by binding with chaperone protein 14-3-3 (Foucault et al. 2003). Therefore, we tested the effect of cherubism mutations of 3BP2 [PDB] on the interaction with 14-3-3 (Fig. 6). Ectopic expression of 3BP2 [PDB] resulted in association with endogenous 14-3-3 in COS-7 cells, presumably by serine phosphorylation of 3BP2 [PDB] by protein kinase A, protein kinase C or Akt. As reported, substitution of Ser²⁷⁷ abrogated the interaction of 3BP2 [PDB] with 14-3-3 in COS-7 cells. Interestingly, all cherubism mutations of 3BP2 [PDB] failed to interact with 14-3-3. Therefore, this result suggests that point mutations of 3BP2 [PDB] in cherubism affect serine phosphorylation of 3BP2 [PDB] by protein serine/threonine kinase.

View larger version (58K): [in this window] [in a new window]   Figure 6  Analysis of interaction with chaperone protein 14-3-3. COS-7 cells were transfected with a different kind of 3BP2 [PDB] cDNAs. Forty-eight hours after transfection, cells (5 x 105) were solubilized in digitonin lysis buffer and cell lysates were immunoprecipitated with anti-14-3-3 antibody. Immunoprecipitates and detergent-soluble cell lysates were analysed by immunoblotting with anti-HA and anti-14-3-3 antibodies. The results are representative of three independent experiments.

Finally, we tested the biochemical characteristics of 3BP2 [PDB] mutants themselves (Fig. 7). We have shown that all cherubism mutations of 3BP2 [PDB] could not affect the in vitro binding with the SH2 domain of Vav1 because tyrosine phosphorylation of 3BP2 [PDB] mutants by Syk was identical with that of wild-type in COS-7 cells (Fig. 4C). Previously, we reported that phosphorylated Tyr⁴⁴⁶ and the Pro-rich region of 3BP2 [PDB] bind to the SH2 and SH3 domains of Lyn, respectively (Maeno et al. 2003). Therefore, we analysed the effect of cherubism mutations of 3BP2 [PDB] on the interaction with Lyn. In vitro binding study using 3BP2 [PDB] expressed in COS-7 cells revealed that all mutant forms of 3BP2 [PDB] could bind to the SH2 domain and SH3 domains of Lyn the same as 3BP2 [PDB] wild-type (Fig. 7A and B). This result suggests that the mutant forms of 3BP2 [PDB] posses the same binding ability to Lyn as the wild-type. As binding of 3BP2 [PDB] with Lyn appears to contribute to the enzymatic activation of Lyn, we examined the effect of the over-expression of cherubism mutant forms of 3BP2 [PDB] on FcRI-mediated activation of Lyn. Antigen-induced tyrosine phosphorylation of FcRIß and  subunits, which are the in vivo substrate of Lyn, was suppressed by the over-expression of 3BP2 [PDB] mutants (Fig. 7C). Consistent with this result, the in vitro protein kinase assay demonstrated that antigen-induced enzymatic activation of Lyn was suppressed by the over-expression of 3BP2 [PDB] mutants (Fig. 7D). Therefore, these results suggest that point mutations of 3BP2 [PDB] identified in cherubism inhibit the activation of Lyn, although these mutant forms of 3BP2 [PDB] possess the binding ability of the SH2 and SH3 domains of Lyn.

View larger version (42K): [in this window] [in a new window]   Figure 7  Biochemical characteristics of 3BP2 [PDB] mutations. COS-7 cells were transfected with various 3BP2 [PDB] constructs with (A) or without (B) Syk. Forty-eight hours after transfection, cells (5 x 105) were solubilized in binding buffer. Cell lysates were incubated with 20 µg of either GST-Lyn-SH2 domain, GST-Lyn-SH3 domain or control GST, pre-bound to glutathione sepharose 4B beads. The binding protein and cell lysates were analysed by immunoblotting with anti-HA mAb and anti-Syk antibody. (C and D) The control RBL-2H3 cells and cloned lines over-expressing cherubism mutant forms of 3BP2 [PDB] (5 x 106) were sensitized with anti-DNP IgE and then stimulated with 30 ng/mL of antigen DNP-BSA (Ag) for the indicated times. Cells were solubilized in the Triton lysis buffer and pre-cleared supernatants were immunoprecipitated with anti-FcRIß mAb or anti-Lyn antibody. Anti-FcRIß immunoprecipitates were analysed by immunoblotting with anti-pTyr and anti-FcRIß mAbs (C). Anti-Lyn immunoprecipitates were subjected to the in vitro protein kinase assay (D).

	Discussion

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We utilized mast cells as a model for analysing the dysfunction of 3BP2 [PDB] with point mutations identified in the human-inherited disease cherubism. Cherubism is characterized by the degradation of jaw bones with multiple cysts. Dysfunction of 3BP2 [PDB] protein by point mutations causes pathologic activation of osteoclasts in cherubism, suggesting that endogenous 3BP2 [PDB] has a regulatory role in the development and/or activation of osteoclasts in jaw bones. The maturation of osteoclasts requires osteoblasts and stromal cells releasing macrophage colony-stimulating factor (M-CSF) and the receptor for activation of nuclear factor B (NF-B) ligand (RANKL) that are essential and sufficient to promote osteoclastogenesis (Roodman 1999). Recent genetic study identified that expression of immunoreceptor-tyrosine-based activating motif (ITAM)-containing adaptor protein DAP12 and Syk are required for the development of functional osteoclasts from macrophages (Koga et al. 2004; Mocsai et al. 2004). These findings raised the idea that 3BP2 [PDB] , the substrate of Syk protein-tyrosine kinase, participates in the early development of osteoclasts from the haematopoietic lineage cells.

3BP2 is a substrate of protein-tyrosine kinase (Jevremovic et al. 2001; Sada et al. 2002; Maeno et al. 2003). The reconstitution experiments using COS-7 cells demonstrate that interaction with the Vav1-SH2 domain is not affected by the point mutations of 3BP2 [PDB] . However, effect of the over-expression of cherubism mutant forms of 3BP2 [PDB] on tyrosine phosphorylation of endogenous 3BP2 [PDB] was not determined because anti-3BP2 antibodies could not immunoprecipitate rat 3BP2 [PDB] (data not shown). Over-expression of cherubism mutant forms of 3BP2 [PDB] obviously suppressed the antigen-induced tyrosine phosphorylation of Vav1 and subsequent activation of Rac1 (Fig. 4). In addition, translocation of Vav1 into the lipid raft was also inhibited by the over-expression of cherubism mutant forms of 3BP2 [PDB] (data not shown). Analysis of Vav1-deficient mast cells revealed that Vav1 is necessary for the antigen-induced degranulation and cytokine synthesis (Manetz et al. 2001). Furthermore, antigen-induced tyrosine phosphorylated FcRIß and subunits itself was suppressed by cherubism mutant forms of 3BP2 [PDB] (Fig. 7). Our results have demonstrated that over-expression of cherubism mutant of 3BP2 [PDB] inhibits the endogenous 3BP2 [PDB] -mediated Lyn activation, Vav1-Rac1 signalling and subsequent mast cell activation. Therefore, our findings suggest that point mutations of 3BP2 [PDB] idenitified in human-inherited disease cherubism results in the loss of function.

Among three cherubism mutants of 3BP2 [PDB] , 3BP2 [PDB] -R413Q showed clear difference from other two mutants, 3BP2 [PDB] -P416H and 3BP2 [PDB] -G418R. Over-expression of 3BP2 [PDB] -R413Q did not affect the antigen-induced activation of Vav, ERK and degranulation, but inhibited the antigen-induced activation of Lyn, NFAT, IL-3 and IL-4 genes expression (Figs 2–5 and 7). The latter two mutants suppressed all of these cellular responses. Furthermore, 3BP2 [PDB] -R413Q stimulated the antigen-induced phosphorylation of IKK (Fig. 3D). Although the point mutations are restricted within the limited sequence of 3BP2 [PDB] , present results suggest the heterogeneous effect on mast cell signalling by the point mutations of 3BP2 [PDB] identified in cherubism. The in vitro binding experiments demonstrated that Syk could phosphorylate Tyr¹⁸³ and Tyr⁴⁴⁶ of 3BP2 [PDB] mutants to interact with Vav1-SH2 and Lyn-SH2 domains in COS-7 cells (Figs 4C and 7A). However, phosphorylation of these Tyr residues of 3BP2 [PDB] in intact RBL-2H3 cells could be suppressed because antigen-induced activation of Lyn and Vav1 was inhibited by the over-expression of 3BP2 [PDB] -P416H or 3BP2 [PDB] -G418R (Figs 4 and 7). Over-expression of 3BP2 [PDB] -R413Q could not affect the activation of Vav1, suggesting that the differential effects among 3BP2 [PDB] mutants could be explained by the effect on phosphorylation of Tyr¹⁸³ of 3BP2 [PDB] . Presumably, 3BP2 [PDB] -R413Q could suppress Tyr¹⁸³-mediated cellular signals which activate NFAT and cytokine genes transcriptions, but not Vav1, ERK and degranulation.

3BP2 is a substrate of protein-serine/threonine kinases (Foucault et al. 2003). The reconstitution experiments using COS-7 cells demonstrated that all cherubism mutations of 3BP2 [PDB] (R413Q, P416H and G418R) abrogate the binding with 14-3-3 (Fig. 6). All three cherubism mutant forms of 3BP2 [PDB] may affect the conformation of full-length 3BP2 [PDB] to be phosphorylated by protein-serine/threonine kinase, such as protein kinase C, or to interact with 14-3-3. In Jurkat T cells, 3BP2 [PDB] -S277A shows increased capacity to stimulate T-cell receptor-mediated NFAT transcriptional activities, suggesting that 14-3-3 binding to 3BP2 [PDB] negatively regulates 3BP2 [PDB] adaptor function in T cells (Foucault et al. 2003). In RBL-2H3 cells, the expression of 3BP2 [PDB] -S277A resulted in an enhancement of FcRI-mediated degranulation, activation of ERK, JNK, p38 MAPK, IKK and Rac1 in mast cells (Figs 2–4). The enhanced mast cell activation by the expression of 3BP2 [PDB] -S277A could be explained by the loss of interaction with 14-3-3. However, the over-expression of cherubism mutant forms of 3BP2 [PDB] also suppressed mast cell activation, although they lost the ability to interact with 14-3-3 as expected from the results in COS-7 cells. It suggests that phosphorylation of 3BP2 [PDB] on Ser²⁷⁷ is not simply implicated in the negative regulation of FcRI signalling in mast cells. One possible explanation is that activation of Ser/Thr kinase is downstream of Vav1-Rac1. Negative regulation by phosphorylation of Ser²⁷⁷ on 3BP2 [PDB] could be a secondary event through the process of mast cell activation.

Present analysis demonstrated the biochemical relevance of individual point mutation of 3BP2 [PDB] identified in cherubism in mast cells. This study raised the hypothesis that cherubism mutations of 3BP2 [PDB] cause the dysfunction of 3BP2 [PDB] in stromal cells to participate in the pathologic hyperactivation of osteoclasts in cherubism lesions.

	Experimental procedures

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Materials

Anti-3BP2, anti-p38 MAPK, anti-IKK, anti-Vav1, anti-Syk, anti-14-3-3 and anti-Lyn antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-haemagglutinin epitope (HA) mAb was obtained from Covance (Princeton, NJ). Anti-phospho-p44/42 ERK (extracellular signal regulated kinase) (Thr²⁰²/Tyr²⁰⁴) and anti-p44/42 ERK antibodies were from New England Biolabs (Beverly, MA). Anti-phospho-JNK (c-Jun N-terminal kinase) (Thr¹⁸³/Tyr¹⁸⁵), anti-phospho-specific p38 MAP kinase (Thr¹⁸⁰/Tyr¹⁸²) and anti-phospho-IKK (Ser¹⁸⁰)/IKKß (Ser¹⁸¹) antibodies were from Cell Signalling Technology (Beverly, MA). Anti-JNK antibody, anti-phosphotyrosine (pTyr) mAb and anti-Rac1 mAb were from Upstate Biotechnology (Lake Placid, NY). Anti-FcRIß mAbs were kindly provided by Dr Juan Rivera (Rivera et al. 1988) and Dr Reuben P. Siraganian (National Institutes of Health, MD, USA). Reporter plasmid pNFAT-luciferase was a gift from Dr Gerald R. Crabtree (Stanford University, CA).

Construction of cDNA

The HA-tagged expression construct of mouse 3BP2 [PDB] in pMT3 vector (pMT3-HA-3BP2) was a gift from Dr A. Altman (La Jolla Institute, CA). Substitutions of Ser²⁷⁷ to Ala (S277A), Arg⁴¹³ to Gln (R413Q), Pro⁴¹⁶ to His (P416H) and Gly⁴¹⁸ to Arg (G418R) in mouse 3BP2 [PDB] cDNA were generated by the site-directed mutagenesis (Fig. 1A). Each mutation of mouse 3BP2 [PDB] cDNA corresponded to that in human 3BP2 [PDB] identified in cherubism (Ueki et al. 2001). Substitutions of Tyr¹⁸³ to Phe (Y183F) and Tyr⁴⁴⁶ to Phe (Y446F) of mouse 3BP2 [PDB] were previously described (Maeno et al. 2003). The cDNA for 3BP2 [PDB] -Pro which lacks Pro²⁰¹-Pro³⁸⁴ was created by PCR. All mutations were verified by DNA sequencing.

Cell culture and cDNA transfection

Rat basophilic leukaemia RBL-2H3 cells and COS-7 cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM, Sigma, St Louis, MO) with 100 U/mL of penicillin and 10% (v/v) heat-inactivated foetal calf serum. Various mutant forms of 3BP2 [PDB] cDNAs were stably transfected into RBL-2H3 cells by electroporation (Sada et al. 2000, 2001, 2002; Maeno et al. 2003; Qu et al. 2004). Clones were selected by 0.4 mg/mL of G418 (Invitrogen, Rockville, MD) and screened by immunoblotting with anti-3BP2, anti-HA, anti-FcRIß antibodies. For preparation of total cell lysates, cell monolayers were washed once with phosphate-buffered saline (PBS) and directly resolved in 2 x sample buffer at 100 °C for 40 min. Two positive clones in which the expression level of 3BP2 [PDB] was highest were chosen for further experiments (Fig. 1B).

For transient transfection of COS-7 cells, 1 µg of each cDNA and 6 µL of FuGENE 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN) were added to 10⁵ cells seeded in 6-well plates, according to the manufacturer's instruction. Cells were used for the experiments 48 h after transfection.

Analysis of ß-hexosaminidase release

Degranulation of different stable mast cell lines was determined by the measurement of ß-hexosaminidase release (Sada et al. 2002; Qu et al. 2004). RBL-2H3 cells, or cells expressing the mutant forms of 3BP2 [PDB] , were sensitized with mouse monoclonal anti-dinitrophenyl IgE (anti-DNP IgE, clone SPE-7) (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 either 1–1000 ng/mL of the antigen DNP-BSA (2,4-dinitrophenylated bovine serum albumin, LSL, Tokyo, Japan) or 1 µM Ca²⁺ ionophore A23187 [GenBank] (Sigma) in the same buffer for 1 h. The cell culture medium and total cell lysates (obtained by the addition of 1% NP-40 in Tyrode-HEPES buffer) were incubated with substrate p-nitrophenyl-N-acetyl-ß-D-glucopyranoside (Nacalai, Kyoto, Japan) at 37 °C for 1 h and the product 4-p-nitrophenol was monitored by the absorbance at 405 nm by using microplate reader (Model 550, Bio-Rad, Hercules, CA). The antigen-induced ß-hexosaminidase release was expressed as a percentage of the maximal release induced by A23187 [GenBank] .

Cell activation, immunoprecipitation and immunoblotting

RBL-2H3 cells, or cells expressing the mutant forms of 3BP2 [PDB] , were sensitized with anti-DNP IgE and then stimulated with 30 ng/mL of antigen for the indicated times. For the immunoprecipitation of Vav1, cells were solubilized in the Triton 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 phenylmethylsulphonyl fluoride (PMSF) and 2 µg/mL of aprotinin). Pre-cleared cell lysates were incubated with anti-Vav1 or anti-FcRIß antibody pre-bound to protein A-agarose beads (Sigma) for 1 h at 4 °C. The beads were then washed four times with the Triton lysis buffer. Immunoprecipitated proteins were eluted by heat treatment at 100 °C for 5 min with 2x sample buffer. Detergent-soluble lysates and immunoprecipitates were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and electronically transferred onto polyvinylidene difluoride membranes (0.45 µm pore size, Millipore, Bedford, MA). After blocking with 5% milk, the membranes were reacted with the indicated antibodies. In all blots, proteins were visualized by the horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad, Hercules, CA) and the enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA). For the immunoprecipitation of HA-3BP2 from COS-7 cells, cells were solubilized in the Triton lysis buffer and cell lysates were incubated with anti-HA mAb pre-bound to protein A-agarose beads. Detergent-soluble lysates and immunoprecipitates were analysed as described above.

For the immunoprecipitation of 14-3-3, COS-7 cells transfected with the different kinds of cDNA were solubilized in the digitonin (Wako, Osaka, Japan) lysis buffer (1% digitonin, 50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF and 2 µg/mL of aprotinin). Pre-cleared cell lysates were incubated with anti-14-3-3 antibody and analysed as described above.

Pull-down assay

The cDNA for human Pak1-PBD (p21-binding domain in serine/threonine kinase Pak1) (Lys⁶⁷-Ala¹⁵⁰) was isolated by RT-PCR from Jurkat T lymphocytes. The resulting PCR product was subcloned into the pGEX-4T.3 (Amersham Biosciences, Piscataway, NJ) to make a domain in-frame with upstream glutathione S-transferase (GST) and verified by DNA sequencing. For the pull-down assay, RBL-2H3 cells, or cells expressing the mutant forms of 3BP2 [PDB] (10⁷), were solubilized in the Mg²⁺ binding buffer [1% NP-40, 25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 2% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 µg/mL of aprotinin]. Cell lysates were incubated with 20 µg of GST protein pre-bound to glutathione sepharose 4B beads (Amersham) for 30 min at 4 °C. After centrifugation, pre-cleared cell lysates were reacted with 20 µg of GST-Pak1-PBD fusion protein pre-bound to glutathione sepharose 4B beads for 1 h at 4 °C. The beads were then washed four times with the Mg²⁺ binding buffer and precipitated proteins were eluted by heat treatment at 100 °C for 5 min with 2x sample buffer. The binding proteins were analysed by immunoblotting with anti-Rac1 mAb.

The cDNA for rat Vav1-SH2 domain (Met⁶⁷⁴ to Leu⁷⁵⁹) was isolated by RT-PCR from RBL-2H3 cells. The cDNA for rat Lyn-SH2 and Lyn-SH3 domains was previously described (Maeno et al. 2003). For pull-down assay, COS-7 cells expressing the different kinds of 3BP2 [PDB] and Syk (5 x 10⁵) were solubilized in the binding buffer (1% NP-40, 50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na₃VO₄ and protease inhibitors) (Maeno et al. 2003). Pre-cleared cell lysates were reacted with 20 µg of either GST-Vav1-SH2, GST-Lyn-SH2 or GST-Lyn-SH3 domain fusion protein pre-bound for glutathione sepharose 4B beads and analysed as described above using anti-HA mAb.

Analysis of multiple cytokine mRNA transcription

The production of multiple cytokine mRNA was quantitatively measured by using Multi-probe RNase protection assay kit (BD Biosciences, San Jose, CA) (Qu et al. 2004). ³²P-labelled RNA probes were synthesized by using rat cytokine templates (rCK1) and in vitro transcription kit (BD Biosciences). The synthesized probes were hybridized with total RNA from the either unstimulated cells or cells stimulated by 30 ng/mL antigen DNP-BSA (5 x 10⁶) for 1 h. After the RNase treatment, the protected double-stranded RNAs were separated by the urea gel and visualized by the autoradiography.

Reporter gene assay

The reporter gene constructs (5 µg) were transiently transfected into RBL-2H3 cells or cells expressing the mutant forms of 3BP2 [PDB] (10⁷) by electroporation using Nucleofector device (Amaxa GmbH, Cologne, Germany). Thirty-six hours after transfection, cells were collected and primed with anti-DNP IgE for further 12 h. Forty-eight hours after transfection, cells were stimulated with 30 ng/mL of antigen for 6 h. The luciferase activities were determined by the Luciferase Assay System (Promega, Madison, WI) using the luminometer (Lumat LB9501, Berthold, Bad Wildbad, Germany). The activity of each reporter gene was normalized by the protein concentration and expressed as a fold of increase compared with that in non-stimulated cells.

In vitro protein kinase assay

RBL-2H3 cells, or cells expressing the mutant forms of 3BP2 [PDB] , were sensitized with anti-DNP IgE and then stimulated with 30 ng/mL of antigen for the indicated times. Cells were solubilized in the Triton lysis buffer and cell lysates were immunoprecipitated with anti-Lyn antibody. Anti-Lyn immunoprecipitates were washed twice with the kinase buffer without ATP, then incubated with 40 µL of the kinase buffer (40 mM HEPES, pH 7.5, 10 mM MgCl₂, 4 µM ATP, 4 µCi [-³²P]ATP) and 2.5 µg of acid-treated enolase (Sigma, St Louis, MO) as the exogenous substrate at 30 °C for 5 min. Reactions were terminated by the heat treatment at 100 °C for 5 min with 2x sample buffer. Proteins were separated by SDS–PAGE and gels were incubated with 1 N KOH at 56 °C for 1 h to remove phosphoserine and most of phosphothreonine. After gel drying, radiolabelled proteins were visualized by autography and the immunoprecipitation of Lyn was shown by immunoblotting (Qu et al. 2004).

	Acknowledgements

 
This study was supported in part by research funding from the 21st Century COE Program and the Grant-in-Aids for the Scientific Research from Japan Society for the Promotion of Science, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Charitable Trust Osaka Cancer Researcher-Fund. We are grateful to Dr Koichiro Maeno for assistance.

	Footnotes

 
Communicated by: Kozo Kaibuchi

^* Correspondence: E-mail: ksada{at}med.kobe-u.ac.jp

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