BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell–independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca2+ mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.

Introduction

B cell development proceeds through sequential stages characterized by stage-specific surface marker expression (14). The B lineage cells in the bone marrow first initiate rearrangement of IgH locus and incorporate the resulting protein into the pre-B cell receptor (pre-BCR),* the expression of which is the hallmark of the development of pro-B cells into pre-B cells. Then, the rearrangement of IgL locus occurs and the BCR is subsequently assembled, marking the progression of pre-B cells to immature B cells. Immature B220+ B cells highly expressing IgM exit from the bone marrow and immigrate into the spleen. Once in the spleen, immature B cells, identified as IgMhiIgDlo population, become IgMhiIgDhi cells, which in turn develop into IgMloIgDhi mature B cells (5). These developmental progressions likely require the signaling ability of the pre-BCR and BCR complex, as exemplified by the block before the pre-B cell stage in mice lacking the tyrosine kinase Syk (6, 7).

The BCR utilizes sequential activation of three distinct families of nonreceptor protein tyrosine kinases (PTKs), such as Src, Syk, and Tec family, as initial activation (8). Indeed, deficiencies in any of these three families of PTKs result in defective or aberrant B cell function and development (811). Thus, characterization of the substrates of these activated PTKs is a prerequisite for understanding the mechanism of how such initial activation regulates the biological outcomes of B cell function and development.

The phosphorylation events that these PTKs catalyze both modulate the catalytic activity of effector enzymes and mediate protein–protein interactions that juxtapose critical signal transduction elements. In this context, it is increasingly appreciated that a group of cellular proteins called adaptor proteins regulate the interactions of effector enzymes with the BCR and their targets, thereby amplifying and integrating multiple signaling pathways (1214). In support of this, the importance of adaptor proteins in normal B lymphocyte development has emerged. For example, mutant mice lacking the adaptor protein B cell linker protein (BLNK; alternatively named SLP-65 or BASH) manifest defective maturation of pro-B to pre-B cells as well as impaired responses to T cell–independent (TI) antigens (1518).

BCAP has recently been isolated as an adaptor molecule that binds to the p85 subunit of phosphoinositide 3-kinase (PI3K; reference 19). It contains several characteristic structures, including an ankyrin repeat, coiled-coil regions, and proline-rich stretches, in addition to several candidate tyrosine phosphorylation sites that could mediate interactions with PI3K, Grb2, and Src homology 2 domain tyrosine phosphatase 2 (SHP-2). Experiments with BCAP-deficient DT40 B cells demonstrated that BCAP regulates BCR-mediated phospholipase C (PLC)-γ2 activation as well as PI3K activation (19).

In an effort to understand the role of BCAP in B cell development and function, we have generated mice deficient in BCAP. Here, we find that BCAP is required for B cell activation and maturation, and suggest that it causes these biological consequences, at least in part, by regulating Ca2+ flux.

Materials And Methods

Construction of BCAP Targeting Vector.

Partial BCAP genome in pBleoBAC11 vector was obtained from Genome Systems. A targeting vector was designed to replace a 2.4-kbp genomic fragment with neomycin resistance gene (neo) from pMC1-neo-poly(A) (Stratagene), deleting the BCAP exon harboring three YxxM motifs. A herpes simplex virus-thymidine kinase cassette (TK) mediating negative selection was inserted in the 5′ end of the BCAP-neo construct.

Generation of BCAP−/− Mice.

The SalI-linealized BCAP targeting construct was electroporated into E14.1 embryonic stem (ES) cells and the selection of ES cells was performed with G418 and gancyclovir as described previously (20). 550 ES cell colonies were screened by PCR. Nine PCR-positive ES clones were obtained, all of which gave the expected bands by Southern blot analysis. Six independent clones were injected into blastocysts and three of them gave rise to chimeric mice that transmitted the mutation to the germline. The mice were maintained under specific pathogen-free conditions. Genotyping of mice was performed by Southern blot analysis.

Northern Blot Analysis.

RNA was extracted from spleens using RNAzol B (Tel-Test Inc.). 20 μg of total RNA was separated on a 1% agarose formaldehyde gel, transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech), and probed with 32P-labeled mouse BCAP full-length cDNA or β-actin.

Flow Cytometric Analysis.

The expression of surface antigens was determined by standard flow cytometric methods. Single-cell suspensions from the indicated organs were depleted of erythrocytes by osmotic lysis and aliquots of 106 cells were stained with a combination of FITC-, PE-, and CyChrome-conjugated antibodies. The following mAbs used in the flow cytometric analyses were purchased from BD PharMingen: anti-CD4 (GK1.5), anti-CD5 (53–7.3), anti-CD8 (53–6.72), anti-CD11b (M1/70), anti-CD16 (2.4G2), anti-CD21 (7G6), anti-CD23 (B3B4), anti-CD43 (S7), anti-B220 (RA3–6B2), anti-heat stable antigen (HSA; M1/69), and anti-IgM (R6–60.2), and anti-IgD (1126) was obtained from Southern Biotechnology Associates, Inc. Data were collected on a FACScan™ flow cytometer (Becton Dickinson) and analyzed using CELLQuest™ software (Becton Dickinson).

Intracellular Staining Assays.

Single-cell suspensions from the spleen were stained with a combination of PE- and CyChrome-conjugated antibodies, washed, and fixed in PBS containing 3.7% paraformaldehyde. Subsequently, cells were washed and permeabilized in PBS containing 0.1% Triton X-100. After blocking with PBS with 10% fetal calf serum (staining buffer), cells were stained with an antiserum to BCAP (diluted at 1:1,000 in staining buffer) for 30 min at 4°C. Cells were washed and then stained with goat anti–rabbit IgG (Jackson ImmunoResearch Laboratories) at 0.2 μg/ml for 30 min at 4°C. Cells were washed and resuspended in ice-cold PBS for FACS® analysis.

ELISA and Immunizations.

Total serum levels of IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were determined using ELISA quantitation kits (Bethyl Lab.). Bound Ig was detected using TMB microwell peroxidase substrate system (Kirkegaard & Perry Lab.). Concentrations were calculated from curves constructed using mouse Ig standards ELISA quantitation kits contain. To evaluate TI-II responses, wild-type and BCAP−/− mice (five per group) were immunized intraperitoneally with 20 μg of the thymus-independent antigen TNP-Ficoll (Biosearch Technologies) in PBS. Mice were bled before and 7 d after immunization. To determine responses to a thymus-dependent antigen, wild-type and BCAP−/− mice (four per group) were immunized intraperitoneally with 20 μg TNP-KLH (Biosearch Technologies) in a 1:1 homogenate with complete Freund's adjuvant (Difco). Mice were bled before and 14 and 21 d after immunization. At day 14, a second immunization (boost) with 20 μg TNP-KLH was performed. Serum Abs specific for TNP were measured in Immuron® plates (Dynex) coated with TNP-BSA (Biosearch Technologies) using ELISA quantitation kits (Bethyl). Immunoabsorbance was read at 450 nm and titers were calculated relative to control sera from unimmunized mice.

Proliferative Response of Splenic B Cells.

The spleen cell suspensions were depleted of erythrocytes by osmotic lysis, and their B cells were purified with Dynabead (Dynal) in combination with antibodies against CD4, CD8, CD11b, and Gr-1 (all from BD PharMingen). The resulting preparations (hereafter referred to as splenic B cells) contained 93 and 90% B220+ cells from wild-type and BCAP−/− mice, respectively. Splenic B cells were cultured, either directly or immediately after sorting with a FACS Vantage™ (Becton Dickinson) using anti-B220 and anti-HSA antibodies, in 0.2 ml of RPMI 1640 (GIBCO BRL) containing 10% fetal calf serum, 50 μM 2-mercaptoethanol, 2 mM L-glutamine, penicillin, and streptomycin at a density of 1.5 × 105 cells/well in flat-bottom 96-well plates. The cells were stimulated with 15 μg/ml F(ab′)2 goat anti-IgM Ab (Jackson ImmunoResearch Laboratories), 10 μg/ml rat agonistic anti-CD40 mAb (clone 3/23; Serotec), or 10 μg/ml LPS (Sigma-Aldrich). For the last 8 h of the 48-h culture period the cultures were pulsed with 0.5 μCi/well [3H]thymidine. They were then harvested onto glass-fiber filters, and radioactivity was measured using a β counter.

Ca2+ Measurement.

Continuous monitoring of fluorescence from the total splenic B cells was performed using Fura-2/AM (Molecular Probes) as an indicator as described previously (21). Ca2+ mobilization in the subpopulations of splenic B cells was separately measured using a BD LSR (Becton Dickinson) as follows: erythrocyte-depleted splenocytes were loaded with 1.2 μM Indo-1AM (Sigma-Aldrich) and then incubated with anti-HSA-FITC and anti-B220-PE antibodies. Cells were washed and stimulated with 15 μg/ml F(ab′)2 goat anti-IgM Ab. Increases in intracellular Ca2+ were recorded in real time on live-gated cells.

Immunoprecipitation and Western Blot Analysis.

Immunoprecipitation (IP) and Western blotting were performed as described previously using the following antibodies: anti-phosphotyrosine (Upstate Biotechnology), anti-Syk (Santa Cruz Biotechnology, Inc.), anti-Btk (43–3B) provided by S. Arai (Hayashibara Biochemical Lab., Okayama, Japan) and S. Tsukada (Osaka University, Osaka, Japan), anti-PLC-γ2 (Santa Cruz Biotechnology, Inc.), and antisera to BCAP and BLNK, which we raised against GST-BCAP and GST-BLNK, respectively. To determine the kinase activity of Akt, in vitro kinase assays were done as described previously using histone (H2B) as a substrate (19).

Inositol-1,4,5-trisphosphate Generation.

Inositol-1,4,5-trisphosphate (IP3) generation in BCR-stimulated splenic B cells was measured with IP3 [3H] assay system (Amersham Pharmacia Biotech) according to manufacturer's instructions.

In Vitro PI3K Assay.

Splenic B cells were stimulated with 15 μg/ml F(ab′)2 goat anti-IgM Ab and were lysed in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris-Cl, pH 8.0, 1% NP-40, 10% glycerol, 1 mM Na3VO4, 1 μg/ml leupeptin, 0.1 μg/ml aprotinin, and 1 mM phenylmethlsulfonyl fluoride). After insoluble materials were removed by centrifugation, the supernatants were incubated with antisera recognizing PI3K p85 subunit (Upstate Biotechnology). Immunoprecipitates were recovered with protein A sepharose, washed sequentially with lysis buffer, wash buffer A (0.1 M Tris-Cl, pH 7.4, 5 mM LiCl, and 0.5 mM Na3VO4) and wash buffer B (10 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1 mM EDTA, and 0.5 mM Na3VO4), and were finally resuspended in 20 mM HEPES (pH 7.4). Kinase reactions were started by addition of 10 μCi [γ-32P]ATP, 15 μg of phosphatidylinositol (Sigma-Aldrich), MgCl2 (15 mM final), and ATP (40 μM final). Reactions were terminated with 1 M HCl after incubation at 37°C for 10 min. After organic extraction, aqueous samples were spotted onto TLC plates and phosphorylated products were separated in a CHCl3/methanol/4 M NH4OH (9:7:2) developing solvent. The incorporated radioactivity was measured using a Fuji FLA2000 bioimaging analyzer (Fuji Photo Film).

Results

Generation of BCAP−/− Mice.

We constructed a gene-targeting vector such that homologous recombination would replace an exon and its flanking introns with a neomycin resistance gene (neo), deleting three YxxM motifs that mediate binding to the p85 subunit of PI3K (Fig. 1 A). Nine ES clones with the appropriately targeted allele were obtained. Six clones were injected into C57BL/6 blastocysts and three of those yielded chimeric mice that transmitted the mutations to their offsprings. Southern blot analysis using an external probe confirmed the presence of the targeted locus (Fig. 1 B). Founder mice showing germline transmission were interbred to produce homozygous BCAP−/− mice, which were born at the expected Mendelian ratio. BCAP−/− mice appeared healthy and were fertile.

The expression of BCAP in the mutant mice was examined both at the RNA and protein levels. Northern blot analysis using the BCAP full-length cDNA as a probe revealed no messenger RNA in the homozygous mice (Fig. 1 C). We previously showed that mouse splenic B cells express two major BCAP isoforms (100- and 98-kD long isoforms and 72- and 70-kD short isoforms) presumably by alternative initiation or splicing (19). Western blot analysis of splenic B cells with an antiserum to BCAP (19) displayed no expression of either BCAP isoform in the mutant mice in accord with Northern blot data (Fig. 1 D).

Defective B Cell Development in BCAP−/− Mice.

BCAP expression, as determined by intracellular flow cytometric staining, was detected in B220+ B cells, but not in CD3+ T cells (Fig. 2 A). Cellularity in the thymus of BCAP−/− mice was equivalent to that in wild-type mice, and thymocyte development was normal, as assessed by expression of CD4 and CD8 (Table I and Fig. 2 B). Then, B cell development in BCAP−/− mice was examined by multiparameter flow cytometry. In the bone marrow of BCAP−/− mice, no gross developmental arrest was found except for a reduction of pre-B cell subset compared with wild-type mice (Table I and Fig. 2 B). The number of B220+ cells in the bone marrow did not differ significantly between wild-type and the mutant mice. In the spleen, the total number of cells was reduced by 20–30% in the mutant mice. Reflecting this decrease, the number of B220+ cells in the spleen of BCAP−/− mice was reduced by 20–30% relative to that in wild-type littermate controls (Table I). Moreover, transition from IgMhiIgDhi cells to IgMloIgDhi mature B cells was considerably blocked, resulting in the lowered ratio of IgMloIgDhi to IgMhiIgDhi cells in the spleen of BCAP−/− mice compared with that of wild-type mice (0.75 ± 0.20 versus 2.25 ± 0.20; Fig. 2 B). The percentages of marginal zone (MZ) B cells (CD21hi CD23loB220+), which are thought to contribute to TI immune responses (22, 23), were comparable in the spleens between BCAP−/− and wild-type mice. In contrast, a slight decrease of follicular B cells (CD21intCD23hiB220+) was observed in BCAP−/− mice, presumably reflecting a blocked development beyond the immature B cell stage (Fig. 2 B). As in the spleen, the lymph nodes of BCAP−/− mice also had a reduced subpopulation of mature IgMloIgDhi B cells. In addition, CD5-expressing B1 B cells, usually found in larger amounts in the peritoneal cavity, were virtually absent in BCAP−/− mice (Fig. 2 B). Hence, we conclude that BCAP is indispensable for the production of B1 B cells, but not for MZ B cells, and contributes to the development of conventional B cells, particularly to the transition from immature to mature B cells.

BCAP transcript was detected in macrophage cell lines as well as B cell lines (19). We thus determined whether the lack of BCAP affected macrophage development. Although the number of CD11b+CD16+B220 cells containing the macrophage lineage was marginally decreased in the spleens of BCAP−/− mice relative to wild-type littermate controls, no difference in the proportion of this subset was found between these mice (Table I and Fig. 2 B). Similarly, the number of CD3+ T cells in the spleen from BCAP−/− mice was marginally decreased (Table I).

Attenuated Humoral Immune Responses in BCAP−/− Mice.

To further assess the consequences of BCAP deficiency, we first measured Ig production in unimmunized mice. Decreased titers of both IgM and IgG3 were seen in BCAP−/− mice (Fig. 3 A), corresponding to a lack of CD5-expressing B1 B cells in their peritoneal cavities. A slight reduction in IgG2b was also seen. In contrast, serum concentrations of IgG1, IgG2a, and IgA were not different from those in littermate controls.

Then, to determine whether BCAP−/− mice can respond to TI and T cell–dependent (TD) antigens, we immunized mice intraperitoneally with TI type II (TI-II, TNP-Ficoll) and TD (TNP-KLH) antigens. 7 d after immunization with TNP-Ficoll, we measured serum titers of the hapten-specific immunoglobulins by ELISA. BCAP−/− mice produced TNP-specific immunoglobulins of any of the isotypes to a lesser extent than wild-type mice (Fig. 3 B).

14 d after immunization with TNP-KLH, TNP-specific immunoglobulin titers were measured. TNP-specific IgG1, IgG2a, and IgG2b were yielded in BCAP−/− mice equivalently to wild-type mice, although the production of both IgM and IgG3 was reduced (Fig. 3 C). Reimmunization of wild-type mice with TNP-KLH 14 d after primary immunization induced a robust secondary TD response. Similarly, BCAP−/− mice upon this boosting mounted a remarkable secondary response to TD antigens, nonetheless, with reduced titers of both IgM and IgG3. Therefore, BCAP−/− mice are able to respond to TD antigens, in contrast to their attenuated TI-II responses.

Impaired BCR Signaling in BCAP−/− Splenic B Cells.

The basis of humoral response is the ability of individual B cells to respond to an array of antigens, which is tightly regulated by signals through the BCR complex (24, 25). Thus, we directly assessed the growth response of BCAP−/− B cells to various stimulations in vitro. As shown in Fig. 4 A, BCAP−/− B cells did respond poorly to antigen receptor stimulation with F(ab′)2 anti-IgM Ab compared with wild-type B cells. Interestingly, the ability of BCAP−/− B cells to respond to either CD40 ligation or LPS stimulation was reduced to almost 50% of that of wild-type B cells. Given that mature B cells proliferate upon BCR engagement whereas immature B cells are particularly susceptible to BCR-triggered apoptosis (26, 27), the reduced thymidine uptake observed in BCAP−/− B cells might reflect the lowered ratio of mature/immature B cells in the spleen of mutant mice (Fig. 2 B). We thus used HSA as a maturation marker to separate splenic B cells into B220+HSAhi immature B and B220+HSAlo mature B cells (28, 29). As shown in Fig. 4 B, both fractions of splenic B cells from the mutant mice responded poorly to BCR triggering.

To further evaluate the defects of BCR signals in BCAP−/− B cells, we measured the mobilization of intracellular calcium, as Ca2+ flux is implicated in both B cell maturation and BCR-induced B cell proliferation (21, 30). BCR-elicited Ca2+ flux in BCAP−/− B cells was reduced in amplitude compared with that in wild-type B cells (Fig. 5 A). This overall change cannot be ascribed to developmental differences between wild-type and BCAP−/− mice; both immature and mature subsets of BCAP−/− B cells exhibited attenuated Ca2+ flux (Fig. 5 B). As IP3 is a critical mediator for BCR-induced calcium mobilization, the reduced Ca2+ flux in BCAP−/− B cells could be explained, at least partly, by impaired activation of PLC-γ2. To test this possibility, we directly measured IP3 generation upon BCR stimulation. Indeed, BCAP−/− B cells yielded 20–30% less IP3 than wild-type controls (Fig. 5 C). Accordingly, we conclude that PLC-γ2 is not sufficiently activated in the absence of BCAP, thereby leading to insufficient calcium flux.

To examine the molecular mechanism underlying the insufficient PLC-γ2 activation in BCAP−/− B cells, we first compared the BCR-mediated tyrosine phosphorylation status of PLC-γ2 in mutant versus wild-type B cells. PLC-γ2 was normally tyrosine phosphorylated in BCAP−/− B cells after BCR ligation, as was seen in wild-type B cells (Fig. 6 A). Consistent with this observation, the BCR-mediated tyrosine phosphorylation status of BLNK, Syk, and Btk, molecules responsible for PLC-γ2 phosphorylation (31), did not significantly differ between BCAP−/− and wild-type B cells (Fig. 6 A).

Not only upstream PTKs but PI3K is implicated in PLC-γ2 activation. Phosphatidylinositol-3,4,5-trisphosphate (PIP3) generated by PI3K is thought to mediate membrane targeting of both Btk and PLC-γ2 via its interactions with PH domains, thereby participating in PLC-γ2 activation (3234). Thus, it is possible that BCAP indirectly facilitates PLC-γ2 activation by activating PI3K. To address this possibility, we directly measured PI3K activity. BCAP−/− B cells exhibited PI3K activation similarly to wild-type B cells (Fig. 6 B). In support of this, Akt activity was not significantly affected by the loss of BCAP (Fig. 6 C). As a control, treatment with the PI3K inhibitor LY294002 reduced the Akt kinase activity to the basal levels in both BCAP−/− and wild-type B cells. Taken together, BCAP is likely involved in PLC-γ2 activation independently of tyrosine phosphorylation and PI3K activation.

Discussion

We show here that a lack of BCAP, which is a substrate of BCR-activated PTKs, leads to defects in B cell development and humoral immune responses. Although the BCAP protein has been detected in macrophage cell lines (data not shown), BCAP is unlikely to play an essential role in macrophage development in that macrophage numbers are only slightly decreased in spleens of BCAP-deficient mice. Moreover, overall T cell development appears normal in BCAP−/− mice, which is consistent with no detectable expression of BCAP in CD3+ T cells (Fig. 2, A and B).

Elimination of BCAP does not result in the severe early B cell developmental arrest. Rather, BCAP−/− mice manifest mainly defects in peripheral B cell populations. Splenic B cells are present, albeit somewhat reduced in number, and are skewed toward an immature phenotype, with an over-representation of IgMhiIgDlo cells and a decrease of IgMloIgDhi fraction (Fig. 2 B). Immature B cells that emigrate from the bone marrow to the periphery are referred to as transitional B cells. Only 10–30% of transitional B cells enter the long-lived mature peripheral B cell compartment (27, 35, 36). This entering mechanism is not entirely clear, but BCR signaling appears to be an important determinant in this transition. Indeed, loss of BCR expression by conditional IgM ablation aborts all further development from the transitional to mature B cell stage (37). Hence, the developmental arrest at the immature B cell stage in BCAP−/− mice could be explained by two mechanisms; (a) the BCAP mutation affects BCR signaling, thereby interfering with differentiation to mature B cells; (b) although this maturation occurs in BCAP−/− mice, albeit at low levels, the survival of mature B cells requires BCAP.

B1 cells often express germline-encoded self-reactive BCRs and favoring the B1 cell development could be a function of BCR signaling and positive selection (38). MZ B cells may be selected similarly to the B1 population, as there are shared characteristics between B1 and MZ B cells (39). For example, B cells harboring a bias for self-reactive BCRs are also enriched in the MZ subset (40). BCAP−/− mice display a lack of CD5-expressing B1 B cells (found primarily in the peritoneal cavity), while MZ B cell population, defined as CD21hiCD23lo B cells, appears normal (Fig. 2 B). Assuming that BCAP functions as a positive regulator in BCR signaling, the straightforward explanation of these data is that the decreased BCR signaling by loss of BCAP is still sufficient to promote the maturation and/or survival of the MZ subset, but not the B1 subset. These results nevertheless allow for the alternative possibility that qualitative differences in BCR signaling determine MZ or B1 cell fate.

TI-II antigens, which are typically derived from polysaccharides, usually consist of complex repeating units that drive B cell responses by extensive cross-linking of specific BCRs. Both B1 and MZ B cells are thought to be responsible for a TI-II response (41). Thus, an impaired TI-II response in BCAP−/− mice could be simply explained by the lack of CD5-expressing B1 B cells. However, considering the importance of MZ B cells in the TI-II response (22, 23), dysfunction of MZ B cells in BCAP−/− mice, in addition to B1 deficiency, could contribute to the impaired TI-II response. In this regard, we would like to speculate that despite the dispensability of BCAP for MZ B cell development, BCAP could be required for MZ B cells to transduce extensive BCR engagement and thereby generate TI-II responses. In contrast to an impaired response to TI-II antigens, BCAP−/− mice mounted a robust response to a TD antigen (Fig. 3 C). These data, together with normal germinal center formation observed in BCAP−/− mice (data not shown), suggest that BCAP is not normally involved in the formation and activation of memory B cells.

The phenotype of BCAP−/− mice resembles that of mice lacking Btk (xid) (42, 43). Both mutant mice manifest reduced levels of mature B cells; an increase in transitional B cells; CD5+ B1 cell deficiency; low serum IgM and IgG3 levels; defective humoral responses to TI-II antigens; poor proliferative responses after stimulation via the BCR, anti-CD40, and LPS; and reduced Ca2+ responses upon BCR engagement. A number of signaling molecules whose disruption causes a phenotype similar to xid have now been identified, including BLNK (1518), Vav1/Vav2 (44, 45), PLC-γ2 (21, 30), and PI3K p85α (46, 47). A common property of all these molecules including BCAP is their regulation of calcium flux.

Given the importance of PI3K in calcium flux, we expected that the reduced Ca2+ response in BCAP−/− B cells was attributable to an impairment of BCR-mediated PI3K activation, because we previously showed that BCR-mediated PI3K activation was partly blocked by loss of BCAP in chicken DT40 B cells (19). However, elimination of mouse BCAP does not significantly interfere with PI3K activity upon BCR cross-linking (Fig. 6, B and C). This apparent discrepancy might simply reflect differences between transformed and primary B cells. A recent study has provided the possibility that PI3K pathway is not negatively regulated under a normal fashion in transformed lymphocytes. Indeed, the Jurkat T cell line is shown to be devoid of PTEN, a negative regulator of PI3K signaling (48). Thus, if so in DT40 B cells, the PI3K pathway in this transformed cell line might not reflect the normal regulatory manner in naive B cells. Alternatively, this disparity might reflect species differences between chicken and mouse. In this regard, there exist at least two possibilities. First, mouse B cells might utilize other PI3K binding proteins, such as CD19 (49, 50) and Gab1 (51), rather than BCAP, to activate PI3K pathway upon BCR engagement. Second, chickens have only one isoform of BCAP, whereas mice might have several BCAP isoforms that could function redundantly in BCR-mediated PI3K activation.

Therefore, apparently normal PI3K activation in mouse BCAP−/− B cells raises the question of how BCAP is involved in PLC-γ2/calcium pathway. Calcium signals are triggered by the second messenger IP3, the product of the hydrolyzing action of PLC-γ2 on phosphatidylinositol-4,5-bisphosphate (PIP2). The attenuated IP3 generation in BCAP−/− B cells could be accounted for by the decreased availability of the PLC-γ2 substrate PIP2 and/or by the decreased hydrolysis of PIP2 through insufficient PLC-γ2 activation. In this context, if the former mechanism is dominantly operating, we predicted that BCR-mediated PI3K activation was similarly attenuated, as PI3K utilizes the same substrate, PIP2, to generate PIP3. Thus, normal Akt activation in BCAP−/− B cells could place the latter possibility more likely.

BCAP appears to participate in PLC-γ2 activation through phosphorylation-independent mechanisms, because of the normal tyrosine phosphorylation status of PLC-γ2 in BCAP−/− B cells. Thus, at least two possibilities might account for the manner by which BCAP regulates PLC-γ2 activation. First, as PLC-γ2 should be targeted to subdomains of the plasma membrane known as rafts for its activation (52), BCAP might contribute to this recruitment. Given that BLNK is required for PLC-γ2 targeting (53), BCAP could act in concert with BLNK, thereby ensuring sufficient targeting of PLC-γ2. Second, considering the requirement for conformational changes of PLC-γ2 (54), in addition to its targeting, in its full activation, BCAP might be involved in forming the active conformation of PLC-γ2. Indeed, a recent report has demonstrated that binding of PLC-γ1 to multiple adaptors, including linker for activation of T cells (LAT), Gads, and SLP-76, is required for its optimal activation (55, 56). Although it is clear that the insufficient PLC-γ2 activation in BCAP−/− B cells contributes to the decreased calcium flux, this mechanism may not entirely account for the effect of BCAP on calcium mobilization. For example, BCAP could directly regulate a calcium influx channel on the plasma membrane. Hence, it might be reasonable to anticipate that BCAP is an important molecule for forming a spatially compact signaling complex including PLC-γ2 and calcium channels on the plasma membrane (“the signalosome”) (57, 58).

The data presented here support the concept that quantitative differences in intracellular Ca2+ can have clear effects on subsequent activation of downstream signaling events, which in turn affect B cell development. Extracellular signal–regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38 cascades, and the Ca2+-dependent transcription factors nuclear factor of activated T cells (NFAT) and NF-κB could be potential targets of calcium signaling (5961). In addition, given the importance of calcium concentration for Rag2 expression and apoptosis in immature B cells (62), BCAP might contribute to B cell tolerance by modulating receptor editing and/or cell deletion.

Acknowledgments

We thank N. Tsuji and M. Nishi for blastocyst injections; M. Kurosaki for Ca2+ measurement; K. Inabe and M. Turner for in vitro PI3K assay; K. Sumi for sorting; S. Arai and S. Tsukada for anti-Btk Ab (43-3B); R.P. Trible and M. Hikida for critical reading of this manuscript; M. Matsumoto for discussion.

This work was supported by grants to T. Yamazaki and T. Kurosaki from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and to T. Kurosaki from the Uehara Foundation and the Human Frontier Science Program.

*

Abbreviations used in this paper: BCR, B cell receptor; BLNK, B cell linker protein; ES, embryonic stem; HSA, heat-stable antigen; IP, immunoprecipitation; IP3, inositol-1,4,5-trisphosphate; MZ, marginal zone; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; PTK, protein tyrosine kinase; TD, T cell–dependent; TI, T cell–independent.

References

References
1
Meffre, E., R. Casellas, and M.C. Nussenzweig.
2000
. Antibody regulation of B cell development.
Nat. Immunol.
1
:
379
–385.
2
Hardy, R.R., and K. Hayakawa.
2001
. B cell development pathways.
Annu. Rev. Immunol.
19
:
595
–621.
3
Benschop, R.J., and J.C. Cambier.
1999
. B cell development: signal transduction by antigen receptors and their surrogates.
Curr. Opin. Immunol.
11
:
143
–151.
4
Rajewsky, K.
1996
. Clonal selection and learning in the antibody system.
Nature.
381
:
751
–758.
5
Loder, F., B. Mutschler, R.J. Ray, C.J. Paige, P. Sideras, R. Torres, M.C. Lamers, and R. Carsetti.
1999
. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.
J. Exp. Med.
190
:
75
–89.
6
Cheng, A.M., B. Rowley, W. Pao, A. Hayday, J.B. Bolen, and T. Pawson.
1995
. Syk tyrosine kinase required for mouse viability and B-cell development.
Nature.
378
:
303
–306.
7
Turner, M., P.J. Mee, P.S. Costello, O. Williams, A.A. Price, L.P. Duddy, M.T. Furlong, R.L. Geahlen, and V.L. Tybulewicz.
1995
. Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.
Nature.
378
:
298
–302.
8
DeFranco, A.L.
1997
. The complexity of signaling pathways activated by the BCR.
Curr. Opin. Immunol.
9
:
296
–308.
9
Reth, M., and J. Wienands.
1997
. Initiation and processing of signals from the B cell antigen receptor.
Annu. Rev. Immunol.
15
:
453
–479.
10
Tamir, I., and J.C. Cambier.
1998
. Antigen receptor signaling: integration of protein tyrosine kinase functions.
Oncogene.
17
:
1353
–1364.
11
Kurosaki, T.
1999
. Genetic analysis of B cell antigen receptor signaling.
Annu. Rev. Immunol.
17
:
555
–592.
12
Campbell, K.S.
1999
. Signal transduction from the B cell antigen-receptor.
Curr. Opin. Immunol.
11
:
256
–264.
13
Leo, A., and B. Schraven.
2001
. Adapters in lymphocyte signalling.
Curr. Opin. Immunol.
13
:
307
–316.
14
Kelly, M.E., and A.C. Chan.
2000
. Regulation of B cell function by linker proteins.
Curr. Opin. Immunol.
12
:
267
–275.
15
Jumaa, H., B. Wollscheid, M. Mitterer, J. Wienands, M. Reth, and P.J. Nielsen.
1999
. Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65.
Immunity.
11
:
547
–554.
16
Pappu, R., A.M. Cheng, B. Li, Q. Gong, C. Chiu, N. Griffin, M. White, B.P. Sleckman, and A.C. Chan.
1999
. Requirement for B cell linker protein (BLNK) in B cell development.
Science.
286
:
1949
–1954.
17
Hayashi, K., R. Nittono, N. Okamoto, S. Tsuji, Y. Hara, R. Goitsuka, and D. Kitamura.
2000
. The B cell-restricted adaptor BASH is required for normal development and antigen receptor-mediated activation of B cells.
Proc. Natl. Acad. Sci. USA.
97
:
2755
–2760.
18
Xu, S., J.E. Tan, E.P. Wong, A. Manickam, S. Ponniah, and K.P. Lam.
2000
. B cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice.
Int. Immunol.
12
:
397
–404.
19
Okada, T., A. Maeda, A. Iwamatsu, K. Gotoh, and T. Kurosaki.
2000
. BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation.
Immunity.
13
:
817
–827.
20
Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, and S. Akira.
1996
. Essential role of Stat6 in IL-4 signalling.
Nature.
380
:
627
–630.
21
Hashimoto, A., K. Takeda, M. Inaba, M. Sekimata, T. Kaisho, S. Ikehara, Y. Homma, S. Akira, and T. Kurosaki.
2000
. Cutting edge: essential role of phospholipase C-γ2 in B cell development and function.
J. Immunol.
165
:
1738
–1742.
22
Guinamard, R., M. Okigaki, J. Schlessinger, and J.V. Ravetch.
2000
. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response.
Nat. Immunol.
1
:
31
–36.
23
Schiemann, B., J.L. Gommerman, K. Vora, T.G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew, and M.L. Scott.
2001
. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway.
Science.
293
:
2111
–2114.
24
Fearon, D.T., and M.C. Carroll.
2000
. Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex.
Annu. Rev. Immunol.
18
:
393
–422.
25
Tedder, T.F., M. Inaoki, and S. Sato.
1997
. The CD19-CD21 complex regulates signal transduction thresholds governing humoral immunity and autoimmunity.
Immunity.
6
:
107
–118.
26
Sandel, P.C., and J.G. Monroe.
1999
. Negative selection of immature B cells by receptor editing or deletion is determined by site of antigen encounter.
Immunity.
10
:
289
–299.
27
Rolink, A.G., J. Andersson, and F. Melchers.
1998
. Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity.
Eur. J. Immunol.
28
:
3738
–3748.
28
Allman, D.M., S.E. Ferguson, and M.P. Cancro.
1992
. Peripheral B cell maturation. I. Immature peripheral B cells in adults are heat-stable antigenhi and exhibit unique signaling characteristics.
J. Immunol.
149
:
2533
–2540.
29
Allman, D.M., S.E. Ferguson, V.M. Lentz, and M.P. Cancro.
1993
. Peripheral B cell maturation. II. Heat-stable antigenhi splenic B cells are immature developmental intermediates in the production of long-lived marrow-derived B cells.
J. Immunol.
151
:
4431
–4444.
30
Wang, D., J. Feng, R. Wen, J.C. Marine, M.Y. Sangster, E. Parganas, A. Hoffmeyer, C.W. Jackson, J.L. Cleveland, P.J. Murray, and J.N. Ihle.
2000
. Phospholipase C-γ2 is essential in the functions of B cell and several Fc receptors.
Immunity.
13
:
25
–35.
31
Kurosaki, T., and S. Tsukada.
2000
. BLNK: connecting Syk and Btk to calcium signals.
Immunity.
12
:
1
–5.
32
Bolland, S., R.N. Pearse, T. Kurosaki, and J.V. Ravetch.
1998
. SHIP modulates immune receptor responses by regulating membrane association of Btk.
Immunity.
8
:
509
–516.
33
Scharenberg, A.M., O. El-Hillal, D.A. Fruman, L.O. Beitz, Z. Li, S. Lin, I. Gout, L.C. Cantley, D.J. Rawlings, and J.P. Kinet.
1998
. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals.
EMBO J.
17
:
1961
–1972.
34
Falasca, M., S.K. Logan, V.P. Lehto, G. Baccante, M.A. Lemmon, and J. Schlessinger.
1998
. Activation of phospholipase C-γ by PI 3-kinase-induced PH domain-mediated membrane targeting.
EMBO J.
17
:
414
–422.
35
Osmond, D.G.
1991
. Proliferation kinetics and the lifespan of B cells in central and peripheral lymphoid organs.
Curr. Opin. Immunol.
3
:
179
–185.
36
Forster, I., and K. Rajewsky.
1990
. The bulk of the peripheral B-cell pool in mice is stable and not rapidly renewed from the bone marrow.
Proc. Natl. Acad. Sci. USA.
87
:
4781
–4784.
37
Lam, K.P., R. Kuhn, and K. Rajewsky.
1997
. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death.
Cell.
90
:
1073
–1083.
38
Hayakawa, K., and R.R. Hardy.
2000
. Development and function of B-1 cells.
Curr. Opin. Immunol.
12
:
346
–353.
39
Martin, F., and J.F. Kearney.
2001
. B1 cells: similarities and differences with other B cell subsets.
Curr. Opin. Immunol.
13
:
195
–201.
40
Martin, F., and J.F. Kearney.
2000
. Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk.
Immunity.
12
:
39
–49.
41
Martin, F., A.M. Oliver, and J.F. Kearney.
2001
. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens.
Immunity.
14
:
617
–629.
42
Kerner, J.D., M.W. Appleby, R.N. Mohr, S. Chien, D.J. Rawlings, C.R. Maliszewski, O.N. Witte, and R.M. Perlmutter.
1995
. Impaired expansion of mouse B cell progenitors lacking Btk.
Immunity.
3
:
301
–312.
43
Khan, W.N., F.W. Alt, R.M. Gerstein, B.A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Muller, A.B. Kantor, L.A. Herzenberg, et al.
1995
. Defective B cell development and function in Btk-deficient mice.
Immunity.
3
:
283
–299.
44
Doody, G.M., S.E. Bell, E. Vigorito, E. Clayton, S. McAdam, R. Tooze, C. Fernandez, I.J. Lee, and M. Turner.
2001
. Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation.
Nat. Immunol.
2
:
542
–547.
45
Tedford, K., L. Nitschke, I. Girkontaite, A. Charlesworth, G. Chan, V. Sakk, M. Barbacid, and K.D. Fischer.
2001
. Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling.
Nat. Immunol.
2
:
548
–555.
46
Suzuki, H., Y. Terauchi, M. Fujiwara, S. Aizawa, Y. Yazaki, T. Kadowaki, and S. Koyasu.
1999
. Xid-like immunodeficiency in mice with disruption of the p85α subunit of phosphoinositide 3-kinase.
Science.
283
:
390
–392.
47
Fruman, D.A., S.B. Snapper, C.M. Yballe, L. Davidson, J.Y. Yu, F.W. Alt, and L.C. Cantley.
1999
. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85α.
Science.
283
:
393
–397.
48
Shan, X., M.J. Czar, S.C. Bunnell, P. Liu, Y. Liu, P.L. Schwartzberg, and R.L. Wange.
2000
. Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.
Mol. Cell. Biol.
20
:
6945
–6957.
49
Tuveson, D.A., R.H. Carter, S.P. Soltoff, and D.T. Fearon.
1993
. CD19 of B cells as a surrogate kinase insert region to bind phosphatidylinositol 3-kinase.
Science.
260
:
986
–989.
50
Buhl, A.M., and J.C. Cambier.
1999
. Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor- mediated activation of Bruton's tyrosine kinase.
J. Immunol.
162
:
4438
–4446.
51
Ingham, R.J., L. Santos, M. Dang-Lawson, M. Holgado-Madruga, P. Dudek, C.R. Maroun, A.J. Wong, L. Matsuuchi, and M.R. Gold.
2001
. The Gab1 docking protein links the B cell antigen receptor to the phosphatidylinositol 3-kinase/Akt signaling pathway and to the SHP2 tyrosine phosphatase.
J. Biol. Chem.
276
:
12257
–12265.
52
Aman, M.J., and K.S. Ravichandran.
2000
. A requirement for lipid rafts in B cell receptor induced Ca2+ flux.
Curr. Biol.
10
:
393
–396.
53
Ishiai, M., M. Kurosaki, R. Pappu, K. Okawa, I. Ronko, C. Fu, M. Shibata, A. Iwamatsu, A.C. Chan, and T. Kurosaki.
1999
. BLNK required for coupling Syk to PLC-γ2 and Rac1-JNK in B cells.
Immunity.
10
:
117
–125.
54
Homma, Y., and T. Takenawa.
1992
. Inhibitory effect of src homology (SH) 2/SH3 fragments of phospholipase C-γ on the catalytic activity of phospholipase C isoforms. Identification of a novel phospholipase C inhibitor region.
J. Biol. Chem.
267
:
21844
–21849.
55
Zhang, W., R.P. Trible, M. Zhu, S.K. Liu, C.J. McGlade, and L.E. Samelson.
2000
. Association of Grb2, Gads, and phospholipase C-γ1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell antigen receptor-mediated signaling.
J. Biol. Chem.
275
:
23355
–23361.
56
Yablonski, D., T. Kadlecek, and A. Weiss.
2001
. Identification of a phospholipase C-γ1 (PLC-γ1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-γ1 and NFAT.
Mol. Cell. Biol.
21
:
4208
–4218.
57
DeFranco, A.L.
2001
. Vav and the B cell signalosome.
Nat. Immunol.
2
:
482
–484.
58
Fruman, D.A., A.B. Satterthwaite, and O.N. Witte.
2000
. Xid-like phenotypes: a B cell signalosome takes shape.
Immunity.
13
:
1
–3.
59
Healy, J.I., R.E. Dolmetsch, L.A. Timmerman, J.G. Cyster, M.L. Thomas, G.R. Crabtree, R.S. Lewis, and C.C. Goodnow.
1997
. Different nuclear signals are activated by the B cell receptor during positive versus negative signaling.
Immunity.
6
:
419
–428.
60
Dolmetsch, R.E., R.S. Lewis, C.C. Goodnow, and J.I. Healy.
1997
. Differential activation of transcription factors induced by Ca2+ response amplitude and duration.
Nature.
386
:
855
–858.
61
Dolmetsch, R.E., K. Xu, and R.S. Lewis.
1998
. Calcium oscillations increase the efficiency and specificity of gene expression.
Nature.
392
:
933
–936.
62
Melamed, D., R.J. Benschop, J.C. Cambier, and D. Nemazee.
1998
. Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection.
Cell.
92
:
173
–182.