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INTRODUCTION |
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 C
(PLC
) 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, Fc
RIIB. Co-cross-linking of BCR and Fc
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
Fc
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 PLC
activity. Since PIP3 was reported to
stimulate Bruton's tyrosine kinase (Btk), which in turn
tyrosine-phosphorylates PLC
(18-20), degradation of
PIP3 upon activation of SHIP by the co-cross-linking of
Fc
RIIB would decrease PLC
activity and hence inhibit
Ca2+ release from the stores. However, direct demonstration
that SHIP affects SOC or Ca2+ release in Fc
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 Fc
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 Fc
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 Fc
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 Fc
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.
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EXPERIMENTAL PROCEDURES |
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
-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
-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.
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RESULTS |
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( )/ 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.
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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.
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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.
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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.
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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.
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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).
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DISCUSSION |
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 Fc
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 PLC
(18-20). In other studies, accumulation of
PIP3 was reported to lead to PH domain-mediated membrane
targeting of PLC
(29, 30). In either case, PIP3
functions as a potent activator of PLC
. Therefore, degradation of
PIP3 by SHIP is expected to inhibit PLC
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 Fc
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 Fc
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 Fc
RI-stimulated RBL-2H3 cells, SHIP was
reported to be phosphorylated and recruited to the immunoreceptor
tyrosine-based activation motif of Fc
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.