Inhibitory Modulation of B Cell Receptor-mediated Ca2+ Mobilization by Src Homology 2 Domain-containing Inositol 5'-Phosphatase (SHIP)*

Akiko HashimotoDagger , Kenzo HiroseDagger , Hidetaka Okada§, Tomohiro Kurosaki§, and Masamitsu IinoDagger

From the Dagger  Department of Pharmacology, Faculty of Medicine, University of Tokyo, and CREST, Japan Science and Technology Corporation, Tokyo 113, Japan and the § Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) mediates inhibitory signals that attenuate intracellular Ca2+ mobilization in B cells upon B cell receptor (BCR) stimulation. To clarify the mechanisms affected by SHIP, we analyzed Ca2+ mobilization in the DT40 B cell line in which the SHIP gene was disrupted. In SHIP-deficient cells, Ca2+ transient elicited by BCR stimulation was more prolonged than that in control cells both in the presence and absence of extracellular Ca2+. Inositol 1,4,5-trisphosphate production following BCR stimulation was enhanced in SHIP-deficient cells. In SHIP-deficient cells in comparison with the control cells, BCR stimulation in the absence of extracellular Ca2+ induced a greater degree of Ca2+ store depletion and the Ca2+ influx upon re-addition of extracellular Ca2+ was also greater. However, store-operated Ca2+ influx (SOC) elicited by thapsigargin-induced store depletion was not affected by SHIP. These results indicate that the primary target pathway of SHIP is the Ca2+ release from the stores, and that Ca2+ influx by the SOC mechanism is secondarily controlled by the level of Ca2+ in the stores without direct inhibition of SOC. In this way, SHIP may play an important role in ensuring the robust tuning of Ca2+ signaling in B cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular calcium concentration ([Ca2+]i)1 regulates cellular functions in various types of cells (1). In B lymphocytes, [Ca2+]i controls cell proliferation, differentiation and apoptotic processes (2, 3). Cross-linking of B cell receptors (BCR) with specific antigens activates phospholipase Cgamma (PLCgamma ) through a series of tyrosine phosphorylations, resulting in hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Inositol 1,4,5-trisphosphate (IP3), a product of the hydrolysis of PIP2, then activates IP3 receptors to mobilize the intracellular Ca2+ stores. Ca2+ influx from the extracellular space is also activated in response to BCR cross-linking via the store-operated Ca2+ influx (SOC) pathway, which is activated by depletion of the intracellular Ca2+ stores (4, 5). BCR activation also results in the activation of phosphoinositide 3-kinase, which converts PIP2 to phosphatidylinositol 3,4,5-trisphosphate (PIP3).

In addition to BCR, B lymphocytes express another class of immunoreceptors, Fcgamma RIIB. Co-cross-linking of BCR and Fcgamma RIIB attenuates the BCR-induced Ca2+ response (6-10). The attenuation of the Ca2+ response requires the activation of the SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP) (11-14), which catalyzes dephosphorylation of PIP3 and inositol 1,3,4,5-tetrakisphosphate at their 5-position of the inositol ring in vitro (15, 16).

One of the possible target mechanisms of SHIP in the attenuation of the Ca2+ response is the SOC activity. Since the Fcgamma RIIB-mediated inhibition of Ca2+ signaling was more prominent in the presence of extracellular Ca2+ than in its absence (8-10), SHIP was postulated to attenuate Ca2+ influx via the SOC channel (11-13, 17). Another potential target of SHIP is the PLCgamma activity. Since PIP3 was reported to stimulate Bruton's tyrosine kinase (Btk), which in turn tyrosine-phosphorylates PLCgamma (18-20), degradation of PIP3 upon activation of SHIP by the co-cross-linking of Fcgamma RIIB would decrease PLCgamma activity and hence inhibit Ca2+ release from the stores. However, direct demonstration that SHIP affects SOC or Ca2+ release in Fcgamma RIIB-mediated signaling remains to be reported.

It has been shown that cross-linking of BCR alone leads to tyrosine phosphorylation of SHIP (21, 22). Furthermore, SHIP seems to inhibit the BCR-mediated Ca2+ response even without co-cross-linking of Fcgamma RIIB, because SHIP-deficient cells exhibit prolongation of the [Ca2+]i transient upon BCR activation (23). While SHIP is recruited to the immunoreceptor tyrosine-based inhibitory motif on the intracellular region of Fcgamma RIIB upon activation of this receptor (11), there is no immunoreceptor tyrosine-based inhibitory motif on the BCR complex. Therefore, SHIP is not a unique molecule for Fcgamma RIIB-mediated inhibitory signaling, but plays an important role in BCR-mediated signaling; the mechanisms of the recruitment of SHIP seem different in the absence or presence of Fcgamma RIIB-mediated signaling. It remains to be clarified how Ca2+ mobilization is attenuated by SHIP in BCR-mediated signaling.

In an effort to understand the role of SHIP in BCR-mediated signaling, we addressed the following questions in this work: 1) how are the patterns of Ca2+ mobilization elicited by BCR stimulation modulated by SHIP, and 2) which Ca2+ mobilization pathway is affected by SHIP: Ca2+ release or SOC. To clarify these points, we compared the patterns of [Ca2+]i mobilization in control and SHIP-deficient DT40 cells at the single cell level. We found that the prolongation of [Ca2+]i transient in SHIP-deficient cells is due to enhanced Ca2+ release from the intracellular Ca2+ stores with enhancement of IP3 production. We also found that SHIP regulates SOC not by a direct interaction but through its effect on Ca2+ release. SHIP thus plays an important role in shaping the Ca2+ mobilization patterns after BCR stimulation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cell Lines and Culture-- Chicken B cells (DT40) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% chicken serum, 50 µM beta -mercaptoethanol, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO2 at 0.5-1 × 106 cells/ml. The generation of SHIP-deficient cells and transfection of either mouse wild-type SHIP or mutant SHIP with disrupted phosphatase activity (P671A, D675A, R676G) were described elsewhere (13).

Measurement of Intracellular Ca2+ Concentration-- About 30 min before the experiments, cells were attached to collagen-coated coverslips. Cells on the coverslips were incubated with 5 µM Fura-2 AM for 30 min at room temperature in physiological salt solution (PSS) (150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 5.6 mM glucose; pH adjusted to 7.4 with NaOH) containing 0.1% bovine serum albumin followed by rinsing with PSS. The coverslips with Fura-2-loaded cells were mounted on the stage of an inverted epifluorescence microscope (TMD 300, Nikon). Cells were examined under a 40× water immersion objective (numerical aperture: 0.7, Olympus). Pairs of fluorescence images with 340 and 380 nm excitations were collected using a cooled CCD camera (PXL-37, Photometrics,) at either 0.5 or 0.33 Hz. The ratio (R) between the fluorescence intensities at 340 and 380 nm excitations was converted to [Ca2+]i using the following equation (24); [Ca2+]i =Kd'(R - Rmin)/(Rmax - R), where Kd', Rmax, and Rmin are the dissociation constant and maximal and minimal R values, respectively. The values were determined in vitro under equivalent optical conditions. Rmax and Rmin were multiplied by a factor of 0.85 for viscosity correction (25). For the stimulation of the BCR, 2 µg/ml mouse anti-chicken IgM antibody, M4 (26), was applied onto the cells through a thin pipette. Other solutions were applied in the same manner. The Ca2+ free solution had the same composition as PSS except for the omission of CaCl2 and introduction of EGTA (5 mM).

Immunoblotting-- Cells were harvested and washed with phosphate buffered saline. The cells were precipitated in 10% trichloroacetic acid (2 × 107 cells/ml). The precipitates were dissolved in solution containing 100 mM Tris-HCl (pH 8.0), 30 mM NaCl, and 1% SDS. The samples were separated by SDS-polyacrylamide gel electrophoresis and were electrotransferred to polyvinylidene difluoride membranes (Trans-Blot, Bio-Rad). The membranes were incubated with the primary polyclonal antibody (anti-mouse SHIP, Upstate Biotechnology, Inc.) and then with biotin-labeled secondary antibody. The blots were detected with enhanced chemiluminescence (Renaissance, NEN Life Science Products).

IP3 Measurement-- Cells were harvested and washed with PSS. The BCR-stimulated cell suspension (2 × 106 cells) was mixed with 15% trichloroacetic acid for termination of the reaction. After centrifugation, the supernatant was extracted with water-saturated diethylether and the water phase was neutralized with NaHCO3 to pH 7.5. D-myo- [3H]Inositol-1,4,5-trisphosphate assay system (TRK1000, Amersham Pharmacia Biotech) was used for measurement of IP3 levels (27). Radioactivity was measured for 3 min in a beta -scintillation counter, and converted to IP3 concentration using the calibration curve.

Statistical Analysis-- Statistical results are expressed as mean ± S.E. Statistical comparisons were made using the paired t test for the IP3 assay and the non-paired t test for all the other measurements.

Materials-- RPMI 1640 medium, glutamine, penicillin, and streptomycin were purchased from Life Technologies, Inc. Chicken serum, ionomycin, and thapsigargin were obtained from Sigma. Fura-2 AM was purchased from Molecular Probes. All other chemicals were of the highest reagent grade.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prolonged Ca2+ Mobilization in SHIP-deficient DT40 Cells-- We compared the time course of [Ca2+]i mobilization in control and SHIP-deficient cells at the single cell level. BCR was activated by the application of anti-µ mouse IgM monoclonal antibody, M4. Ca2+ mobilization in control cells showed either a single peak or attenuating oscillations with a few peaks, and [Ca2+]i returned to the resting value within 200 s (Fig. 1A). In contrast, SHIP-deficient cells showed a plateau-like response or oscillations persisting for over 200 s (Fig. 1B). Quantitative analysis of [Ca2+]i showed that the peak level of [Ca2+]i increase was slightly greater in SHIP-deficient cells (675 ± 29 nM, n = 140) than in control cells (556 ± 23 nM, n = 140; p < 0.002). The difference was more conspicuous in the late phase, and at 150 s after the BCR stimulation, [Ca2+]i remained at a higher level in SHIP-deficient cells (350 ± 31 nM, n = 70) than in control cells (110 ± 14 nM, n = 70; p < 0.0001) (Fig. 1, A and B; Fig. 2A).


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Fig. 1.   Prolongation of [Ca2+]i transient elicited by BCR stimulation in SHIP-deficient cells. Representative traces of changes in [Ca2+]i in control (A), SHIP-deficient cells (B), SHIP-deficient cells transfected with wild-type SHIP (C), and mutant SHIP lacking phosphatase activity (D). Arrows indicate the time point when mouse anti-IgM (M4) was applied to stimulate BCR. Immunoblotting analysis of transfected SHIP proteins expressed in SHIP-deficient cells (E). SHIP-deficient cells, and the cells transfected with wild-type SHIP or SHIP lacking phosphatase activity are indicated as SHIP(-), SHIP(-)/wt, and SHIP(-)/Delta P, respectively.


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Fig. 2.   Prolongation of [Ca2+]i transient elicited by BCR stimulation in SHIP-deficient cells in the presence and absence of extracellular Ca2+. The time course of changes in [Ca2+]i with (A) or without (B) extracellular Ca2+ in control cells (thick trace) or in SHIP-deficient cells (thin trace). The extracellular Ca2+ was removed 20 s before BCR stimulation. Arrows indicate the time point of M4 antibody application.

To confirm that the observed prolongation of Ca2+ mobilization in SHIP-deficient cells was due to the absence of SHIP, we transfected the SHIP-deficient cells with mouse SHIP or mutant SHIP lacking phosphatase activity. The expression of wild-type SHIP activity shortened the duration of [Ca2+]i increase (Fig. 1C), whereas transfection of SHIP lacking phosphatase activity was without effect (Fig. 1D). The levels of expression of the wild-type and mutant SHIP were almost the same (Fig. 1E). These results indicate that the shortening of the Ca2+ mobilization pattern by SHIP required its phosphatase activity.

Effect of SHIP on Ca2+ Release from Ca2+ Stores-- There exist two main pathways for Ca2+ mobilization: Ca2+ release from the Ca2+ stores and Ca2+ entry from the extracellular space. Both pathways are potential targets of SHIP. To determine the role of SHIP in the Ca2+ release pathway, we analyzed the time course of Ca2+ mobilization by BCR stimulation in the presence and absence of extracellular Ca2+. The prolongation of Ca2+ mobilization in SHIP-deficient cells in the presence of extracellular Ca2+ (Fig. 2A) was retained in the absence of extracellular Ca2+ (Fig. 2B). Although the peak [Ca2+]i rise in control cells and SHIP-deficient cells in the absence of extracellular Ca2+ showed no significant difference (control: 520 ± 34 nM, n = 120; SHIP-deficient: 523 ± 25 nM, n = 105; p > 0.4), the [Ca2+]i at 150 s after BCR stimulation was greater in SHIP-deficient cells (182 ± 14 nM, n = 60) than in control cells (80 ± 9 nM, n = 51; p < 0.0001). These results indicate that Ca2+ release from the Ca2+ stores is one of the target pathways of SHIP.

Effect of SHIP on IP3 Production-- We then compared the time course of IP3 production during BCR stimulation in control and SHIP-deficient cells (Fig. 3). The increase in IP3 concentration was significantly higher in SHIP-deficient cells at 180 s after BCR stimulation (p < 0.05, n = 5) than in control cells. These results suggest that SHIP regulates Ca2+ release through the attenuation of IP3 production.


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Fig. 3.   Enhancement of IP3 production in SHIP-deficient cells. BCR was stimulated with M4 antibody at 0 s, and the amount of IP3 at 0, 30, 60, and 180 s was measured using an IP3 binding protein assay system. The increase in IP3 with respect to the basal IP3 level in control (closed circles) and SHIP-deficient cells (open circles) is plotted against time.

Ca2+ Influx after BCR Stimulation-- We then evaluated the effect of SHIP on the Ca2+ influx mechanism. First, Ca2+ release from the Ca2+ stores was activated by BCR stimulation in the absence of extracellular Ca2+ for 600 s, during which period [Ca2+]i returned to the resting value. Reintroduction of extracellular Ca2+ resulted in an increase in [Ca2+]i due to influx of Ca2+ in both control and SHIP-deficient cells (Fig. 4, A and B, solid bar). The maximal level of [Ca2+]i reached during the extracellular application of Ca2+ was higher in SHIP-deficient cells than in control cells (Fig. 4C). These results indicate that Ca2+ influx as well as Ca2+ release is attenuated by SHIP.


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Fig. 4.   Ca2+ influx after BCR stimulation in control and SHIP-deficient cells. The BCR was stimulated with M4 antibody in control (A) and SHIP-deficient (B) cells at the time point indicated by the arrows. The open and closed bars below the traces shows the absence and presence of extracellular Ca2+, respectively. The average values and S.E. of [Ca2+]i increase during the extracellular Ca2+ application are shown in C. Numbers in parentheses indicate the number of analyzed cells.

Effect of SHIP on SOC Elicited by Thapsigargin-- Since we found that BCR-induced Ca2+ influx is inhibited by SHIP, we tested whether SHIP has a direct effect on the activity of the SOC mechanism that is activated by the depletion of Ca2+ stores. We used a sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inhibitor, thapsigargin to deplete the Ca2+ stores, and then assayed the extent of SOC after reintroduction of extracellular Ca2+ (Fig. 5, A and B) (28). The increase in [Ca2+]i in control cells was not statistically different from that in SHIP-deficient cells (Fig. 5C). The result suggests that the SOC is not affected by SHIP, at least under the conditions where BCR is not cross-linked.


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Fig. 5.   Store-operated Ca2+ influx elicited by thapsigargin in control and SHIP-deficient cells. Thapsigargin (1 µM) was applied to the control (A and D) or SHIP-deficient (B and E) cells as indicated by the striped bar in Ca2+-free solution (open bar), and then extracellular Ca2+ was introduced for 300 s (closed bar). In D and E, introduction of extracellular Ca2+ was preceded by BCR stimulation with M4 antibody (arrow). In F and C, the average and S.E. of [Ca2+]i increase during Ca2+ influx with or without BCR stimulation, respectively, are shown.

Then, we repeated the same experiment under BCR stimulation, by which SHIP is expected to be activated. After a 900-s application of thapsigargin to deplete the Ca2+ stores, BCR was stimulated (arrows in Fig. 5, D and E), and then extracellular Ca2+ was reintroduced. BCR stimulation elicited no [Ca2+]i rise after thapsigargin treatment, confirming the complete depletion of the stores after thapsigargin treatment. SOC during BCR stimulation was not affected by SHIP, and the amplitude of the [Ca2+]i rise due to SOC was the same in control and SHIP-deficient cells (Fig. 5F). These results indicate that the SOC mechanism is not a direct target of SHIP.

Content of Ca2+ in Ca2+ Stores after BCR Stimulation-- The extent of the Ca2+ store depletion has been postulated to control the extent of activation of SOC. Therefore, the difference in the activation of SOC after BCR stimulation in control and SHIP-deficient cells (Fig. 4) may reflect a possible difference in the extent of Ca2+ depletion. We tested this hypothesis by estimating the amount of Ca2+ in the Ca2+ stores by the application of ionomycin in the absence of extracellular Ca2+. Ionomycin treatment after BCR stimulation elicited a greater [Ca2+]i increase in control cells than in SHIP-deficient cells (Fig. 6, A-C). However, the peak size of [Ca2+]i rise elicited by ionomycin treatment without BCR stimulation in control and in SHIP-deficient cells did not show any significant differences (Fig. 6, D-F), indicating that the initial Ca2+ content within the Ca2+ stores was the same in control and SHIP-deficient cells. These results indicate that BCR stimulation resulted in only partial Ca2+ release from the stores in control cells and nearly complete release of Ca2+ in SHIP-deficient cells, and suggest that the enhancement of Ca2+ influx in SHIP-deficient cells was due to secondary activation of SOC by more profound Ca2+ depletion.


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Fig. 6.   Ca2+ remaining in the Ca2+ stores evaluated by ionomycin treatment after BCR stimulation. BCR stimulation with M4 antibody (arrow) for 600 s was followed by 600-s application of 1 µM ionomycin (closed bar) in control (A) and SHIP-deficient cells (B). Extracellular Ca2+ was removed 20 s before the BCR stimulation (open bar). Control experiments were performed under the same protocol without BCR stimulation in control (D) and SHIP-deficient cells (E). C and F show the average and S.E. of [Ca2+]i increase during the ionomycin application with and without BCR stimulation, respectively.

Effect of SHIP on Ca2+ Extrusion Mechanism-- We finally tested whether SHIP affects the Ca2+ extrusion mechanism via the plasma membrane, which potentially controls [Ca2+]i in concert with the Ca2+ mobilization mechanism. We estimated the level of activity of the Ca2+ extrusion mechanism from the initial rate of decline of [Ca2+]i after termination of SOC. The rate was obtained from the slope of the line, which was fitted to the data points of [Ca2+]i for 2 min after removal of extracellular Ca2+ (Fig. 7, inset), and was plotted against the [Ca2+]i just before the removal of extracellular Ca2+ (Fig. 7). The plots from control and SHIP-deficient cells were superimposable, indicating no difference in the level of activity of the Ca2+ extrusion mechanism between control and SHIP-deficient cells.


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Fig. 7.   The Ca2+ extrusion rates in control and SHIP-deficient cells. The Ca2+ extrusion rate, which was represented by the initial rate of decrease in [Ca2+]i upon removal of extracellular Ca2+, was plotted against the [Ca2+]i just before the Ca2+ removal. The closed and open circles are data from control and SHIP-deficient cells, respectively. The initial rate of Ca2+ extrusion was estimated from the results of experiments shown in Fig. 5 (D and E) as the slope of the dotted line that was fitted to the data points for 120-s period after the removal of extracellular Ca2+ (inset).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We examined the Ca2+ mobilization patterns in SHIP-deficient B cells at the single cell level and showed that the main role of SHIP in BCR-mediated Ca2+ signaling was to abbreviate the duration of Ca2+ mobilization. SHIP prevented continuous Ca2+ release from the intracellular Ca2+ stores via inhibition of IP3 production. Furthermore, we showed that SHIP regulates SOC not by a direct effect on the SOC mechanism but by controlling the depletion level of the Ca2+ stores. This inhibitory cascade of SHIP is shared by the Fcgamma RIIB pathway despite the difference in the way for recruitment of SHIP.

In B lymphocytes, accumulation of PIP3 via the activation of phosphoinositide 3-kinase was reported to stimulate Btk, which then phosphorylates PLCgamma (18-20). In other studies, accumulation of PIP3 was reported to lead to PH domain-mediated membrane targeting of PLCgamma (29, 30). In either case, PIP3 functions as a potent activator of PLCgamma . Therefore, degradation of PIP3 by SHIP is expected to inhibit PLCgamma and hence Ca2+ release from the Ca2+ stores. We now show direct evidence that IP3 production in BCR-mediated signaling is enhanced in SHIP-deficient cells (Fig. 3).

In Fcgamma RIIB-mediated inhibitory signaling, SHIP was implicated not only in IP3 production but also in the inhibition of SOC, or store depletion-induced Ca2+ influx (11, 13). We, therefore, examined the effects of SHIP on Ca2+ influx elicited after BCR activation, and found that Ca2+ influx in SHIP-deficient cells was indeed greater than that in control cells (Fig. 4). SOC has been postulated to be a mechanism of Ca2+ influx in B lymphocytes, as is the case in other nonexcitable cells (4, 31). Thus, we also examined the effect of SHIP on SOC after store depletion by thapsigargin, which is a potent inhibitor of SERCA and depletes the Ca2+ stores without IP3 production (28). However, thapsigargin-induced SOC was not affected by the presence of SHIP either with or without BCR cross-linking (Fig. 5). These results clearly indicate that SHIP does not have a direct inhibitory effect on either SOC channel activity or the SOC activation mechanism itself in BCR-mediated signaling. Then, how did SHIP inhibit the Ca2+ influx after BCR stimulation? The activation of SOC was shown to be regulated by the level of depletion the Ca2+ stores (32-34). We therefore postulated that the effect of SHIP on Ca2+ influx resulted from the difference in the extent of Ca2+ store depletion in control and SHIP-deficient cells after BCR cross-linking. To test this hypothesis, we evaluated the amount of Ca2+ remaining in the Ca2+ stores after BCR cross-linking. In SHIP-deficient cells, the amount of Ca2+ remaining in the stores was much smaller than that in control cells (Fig. 6). The results indicate that SHIP causes early termination of Ca2+ release making SOC activation minimal in normal BCR signaling. On the other hand, the Ca2+ stores were likely to be depleted enough to activate SOC in thapsigargin-treated cells and in BCR-stimulated SHIP-deficient cells. A similar inhibitory cascade after Fcgamma RIIB co-cross-linking was shown recently (20).

The differential effects of SHIP on Ca2+ influx elicited by BCR cross-linking and SOC elicited by thapsigargin could also be accounted for by postulating two independent Ca2+ influx pathways: the BCR-regulated and SHIP-sensitive influx, and the thapsigargin-elicited and SHIP-insensitive pathways. However, this possibility can be readily ruled out. If this were the case, cells treated with both BCR cross-linking and thapsigargin would allow Ca2+ influx via both components. The total Ca2+ influx should also be affected by SHIP as a result from the effect on the SHIP-sensitive Ca2+ influx. However, reintroduction of Ca2+ elicited the same extent of Ca2+ influx in control and SHIP-deficient cells (Fig. 5, D and E), arguing against the hypothesis. This conclusion was supported by the results obtained from the DT40 cells in which all three subtype genes of the IP3 receptor were disrupted. In these IP3 receptor-deficient cells, there was no Ca2+ response to BCR stimulation, although thapsigargin-elicited Ca2+ increase was clearly observed (31). These results suggest the absence of a Ca2+ influx pathway other than the SOC in BCR signaling in DT40 cells.

SHIP has been found in a variety of hematopoietic cells other than B cells and may play a role in cell signaling in these cells (21, 35). For example, SHIP may be involved in thrombin-induced platelet activation (36, 37). In Fcepsilon RI-stimulated RBL-2H3 cells, SHIP was reported to be phosphorylated and recruited to the immunoreceptor tyrosine-based activation motif of Fcepsilon RI (38, 39). However, the role of SHIP in these cells is unclear. Therefore, it will be important to examine whether SHIP plays the same inhibitory role in Ca2+ signaling in different cell types as shown here in B cells.

Accumulating evidence suggests that the cellular responses are more often controlled by the temporal pattern of Ca2+ mobilization than by the peak or average levels of [Ca2+]i (1, 34, 40-42). Although the mechanism underlying the Ca2+ mobilization pattern-mediated control of cell function is not fully understood, the temporal pattern of [Ca2+]i increase has been implicated in the differential activation of subsets of proteins and/or genes due to different Ca2+-mediated activation kinetics. Our present finding indicates that the Ca2+ mobilization is curtailed by the inhibition of IP3 production by SHIP activity during BCR stimulation. Because the extent of activation of SOC steeply depends on the extent of Ca2+ store depletion (32-34), this control mechanism may provide the cells with sharp on- and off-regulation of Ca2+ influx.

    ACKNOWLEDGEMENTS

We thank Mari Kurosaki, Toshiko Yamazawa, and Tomoya Miyakawa for helpful discussion.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and the Toray Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 813-5802-8687; Fax: 813-3815-9360 (after May 1, 1999: Tel.: 813-5841-3687; Fax: 813-5841-3390); E-mail: iino{at}m.u-tokyo.ac.jp.

    ABBREVIATIONS

The abbreviations used are: [Ca2+]i, intracellular calcuim concentration; BCR, B cell receptor; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; SOC, store-operated calcium influx; PIP3, phosphatidylinositol 3,4,5-trisphosphate; SH, Src homology; SHIP, Src homology 2 domain-containing inositol 5'-phosphatase; Btk, Bruton's tyrosine kinase; PSS, physiological salt solution; SERCA, sarco(end)plasmic reticulum Ca2+-ATPase.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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