From the Department of Pathology and Cancer Research
and Treatment Center, University of New Mexico School of Medicine,
Albuquerque, New Mexico, 87107, § Department of Molecular
Pharmacology, Albert Einstein College of Medicine, Bronx, New York
10461, and ¶ Department of Biochemistry and Molecular Biology,
Monash University, Clayton 3168, Victoria, Australia
Received for publication, January 17, 2001, and in revised form, February 12, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fc Cross-linking of the high affinity receptor for IgE, Fc Despite demonstrated roles for PI 3-kinase in the activation of PLC Intracellular phosphoinositide levels are also regulated by a family of
inositol polyphosphate 5-phosphatases (18). Activation of
p150SHIP, a member of this family that removes phosphate
from the 5' position of PtdIns(3,4,5)P3 and the related
inositol phosphate, inositol 1,3,4,5-phosphate 4, down-regulates
Ca2+ signaling in RBL cells (19). In addition, bone
marrow-derived mast cells from p150SHIP In addition to regulating Ca2+ signaling, PI 3-kinase
activity regulates receptor-induced actin rearrangements in a number of cell types (22, 23). In the RBL-2H3 cell line, wortmannin-sensitive PI
3-kinase activity is required for the formation of large, actin-based, plasma membrane ruffles at the cell surface after Fc In this report, we show that PtdIns(3,4,5)P3 enhances the
activity of PLC Blocking Antibodies and Reagents--
Isoform-specific
inhibitory antibodies to the carboxyl termini of the p110 Cell Culture and Stimulation--
For immunoprecipitation-based
assays, RBL-2H3 cells were cultured on tissue culture-grade plastic in
minimal essential medium (Life Technologies, Inc., Grand Island, NY)
supplemented with 15% fetal calf serum (HyClone),
penicillin-streptomycin, and L-glutamine. For
microinjection experiments, cells were plated overnight onto glass
coverslips mounted in Teflon dishes. In all cases, IgE receptors were
primed with anti-DNP-IgE (1 µg/ml) for 12-20 h. Cells were washed to remove excess IgE and activated at 37 °C by the addition of polyvalent antigen (0.1 µg/ml DNP-BSA).
Phospholipase Activity--
Phospholipase activity was measured
as described previously (10), except that EGTA was omitted from
reaction buffers in experiments where pH was varied.
PI 3-Kinase Immunoprecipitation and Immunoblotting--
Adherent
cells (107 cells/sample) were stimulated with antigen,
washed with cold PBS, and scraped from plates into 1 ml of lysis buffer
C (50 mM Tris-HCl, pH 7.2, 1% Brij 96, 150 mM
NaCl, 1 mM NaVO4, and protease inhibitor
mixture from Boehringer Mannheim (Indianapolis, IN)). Lysates were
clarified by centrifugation (15,000 × g) for 5 min and
rocked for 2 h at 4 °C with 40 µl of protein A/G-Sepharose
bead mixture (1:1) (Pharmacia) prebound to 2 µg of rabbit polyclonal
anti-p85 antibodies (Upstate Biotechnology) or p110 isoform-specific
antibodies. Immunoprecipitates were washed three times in buffer C,
solubilized by heating to 95 °C for 5 min in 1× Laemmli buffer,
separated by SDS-PAGE on 7.5% acrylamide gels, and transferred to
nitrocellulose. Membranes were blocked with 5% immunoglobulin-free BSA
(Sigma) for 1 h at room temperature and incubated for 1 h at
room temperature with 1 µg/ml horseradish peroxidase-conjugated PY20
antibody (Transduction Laboratories, Lexington, KY). Membranes were
washed and incubated with enhanced chemiluminescence development
substrate (Pierce). Blots were then stripped and reprobed with
polyclonal p85 antibody, followed by horseradish peroxidase-conjugated
secondary antibodies and enhanced chemiluminescence development.
5-Phosphatase Purification and Assay--
The cDNA encoding
murine Mr 45,000 5-phosphatase was subcloned
into the pTrcHis expression plasmid (Invitrogen). His-tagged 5-phosphatase fusion proteins were produced in Escherichia
coli after isopropyl-1-thio- Microinjection--
Microinjection was performed at room
temperature with an Eppendorf semiautomated microinjector and
micromanipulator (Eppendorf, Madison, WI) mounted on a Zeiss IM35
microscope equipped with a CO2 perfusion system. Injection
needles were pulled with a vertical pipette puller (David Kopf
Instruments, Tujunga, CA). Cells were allowed to recover for 30 min at
room temperature after microinjection, before warming to 37 °C and
the addition of antigen. Cells judged to contain morphological
abnormalities after the recovery period were excluded from further analysis.
Ca2+ Imaging--
As described above,
cells were microinjected with 5 mM solutions of fura-2-free
acid plus or minus inhibitory reagents. After recovery, ratio imaging
was performed at 37 °C on a Zeiss IM35 microscope equipped with a
stage heater and a CO2 perfusion system (Medical Systems
Corp., Greenvale, NY), a Ludl filter wheel, and a Dage-MTI intensified
video charge-coupled device camera interfaced to Simca imaging software
(Compix Inc., Cranberry Township, PA). Responses were measured
from groups of three to eight injected cells, in a single field of
view, per experiment. All Ca2+ experiments were performed
in Hank's balanced salt solution. Calibration of background-corrected
ratio values (350 nm/380 nm) against standard solutions in
vitro was done as described previously (29). Analysis of data and
graphing were performed with Graphpad Prism software.
Fluorescence Microscopy--
Microinjected, IgE-primed cells
were activated for 10 min with 100 ng/ml DNP-BSA and then fixed with
2% paraformaldehyde for 20 min. Cells were permeabilized with 0.1%
Triton X-100, followed by sequential staining with 1 µg/ml
fluorescein isothiocyanate-conjugated anti-rabbit IgG (to
identify injected cells; Cappell, Westchester, PA) and rhodamine
phalloidin (Molecular Probes) in PBS with 0.1% BSA. Cells were mounted
in Vectashield (Vector Laboratories, Burlington, CA) and viewed on a
Zeiss IM35 microscope. Images were captured with a Photometrics CH250
cooled charge-coupled device camera interfaced to Compix imaging
software. Figures were assembled with Adobe Photoshop software.
To confirm and extend our previous results demonstrating
enhancement of PLCRI-induced Ca2+ signaling
in mast cells is initiated by activation of cytosolic tyrosine kinases.
Here, in vitro phospholipase assays establish that
the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product,
phosphatidylinositol 3,4,5-triphosphate, further stimulates
phospholipase C
2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of
inhibiting class IA PI 3-kinases on Ca2+ responses after
Fc
RI cross-linking in RBL-2H3 cells. Injection of antibodies
to the p110
or p110
catalytic isoforms of PI 3-kinase, but not
antibodies to p110
, lengthens the lag time to release of
Ca2+ stores and blunts the sustained phase of the calcium
response. Ca2+ responses are also inhibited in cells
microinjected with recombinant inositol polyphosphate 5-phosphatase I,
which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3),
or heparin, a competitive inhibitor of the Ins(1,4,5)P3
receptor. This indicates a requirement for Ins(1,4,5)P3 to
initiate and sustain Ca2+ responses even when PI 3-kinase
is fully active. Antigen-induced cell ruffling, a calcium-independent
event, is blocked by injection of p110
and p110
antibodies, but
not by injection of 5-phosphatase I, heparin, or anti-p110
antibodies. These results suggest that the p110
and p110
isoforms
of PI 3-kinase support Fc
RI-induced calcium signaling by
modulating Ins(1,4,5)P3 production, not by directly
regulating the Ca2+ influx channel.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI, on
mast cells results in the activation of tyrosine kinases Lyn and Syk,
followed by tyrosine phosphorylation and activation of PLC
1 isoforms and other
downstream effectors (1). Subsequent hydrolysis of phosphatidylinositol
4,5-diphosphate by PLC
leads to the formation of
Ins(1,4,5)P3, the release of Ca2+ from internal
stores (2), and the influx of Ca2+ through store-operated
channels (3). Calcium influx, in turn, supports degranulation and the
release of inflammatory mediators (4). This scenario was complicated in
recent years by observations that PLC
activation and
Ca2+ responses and secretion are all diminished in mast
cells treated with inhibitors of PI 3-kinases (5, 6). It was
subsequently discovered that full activation of PLC
proteins in mast
cells requires both tyrosine phosphorylation, potentially mediated by Tec family kinases (7), and interaction with the lipid products of PI
3-kinase (8). PtdIns(3,4,5)P3 mediates membrane recruitment and phosphorylation of PLC
1 in RBL-2H3 cells (8) and increases the
activity of both PLC
1 and PLC
2 against lipid micelle substrates in vitro (9, 10).
,
its specific contribution to the temporal regulation of
Fc
RI-mediated Ca2+ signaling is unresolved. Although
wortmannin treatment can slightly increase the lag time leading to the
initial rise in calcium after antigen stimulation, it does not block
the initial response (10). The most dramatic effects of wortmannin
treatment are on the amplitude and duration of the sustained phase of
Ca2+ signaling (10). One possible interpretation of this
result is that tyrosine phosphorylation of the abundant PLC
2
isoform, which is unaffected by wortmannin treatment, is sufficient
stimulus for the initial Ins(1,4,5)P3 production required
for rapid release of stores. In this study, we test the hypothesis that
full activation of PLC
, mediated by PI 3-kinase, is particularly
important to sustain Ca2+ responses. This is consistent
with evidence in RBL-2H3 cells that activation of the store-operated
Ca2+ influx current, ICRAC, requires
concentrations of Ins(1,4,5)P3 higher than required to
initiate store depletion (11-13). It is important to note that several
alternative hypotheses could explain previous observations using
wortmannin. First, it is conceivable that PtdIns(3,4,5)P3
can regulate Ca2+ influx through PLC
-independent
mechanisms. Evidence in T cells suggests that
PtdIns(3,4,5)P3 can directly activate Ca2+
channels (14), and work in platelets and megakaryocytes implicates the
PtdIns(3,4,5)P3-sensitive Tec family kinase, Btk (15).
Second, Choi et al. (16) proposed that the second messenger
sphingosine-1-phosphate may regulate Fc
RI-mediated store release,
independent of Ins(1,4,5)P3 production. Third, PI
3-kinase-independent effects of wortmannin have been reported (17),
raising the possibility that other targets of the inhibitor might be
responsible for the inhibitory effects on the calcium responses.
/
mice show an increase in the amplitude of the Ca2+ response
in comparison to bone marrow-derived mast cells from their wild type
counterparts (20). Less well characterized is the role of the type I
5-phosphatase that regulates the levels of soluble phosphoinositides by
removing phosphate from the 5' position of Ins(1,4,5)P3 and
inositol 1,3,4,5-phosphate 4. In Chinese hamster ovary cells,
overexpression of this enzyme causes an oscillatory Ca2+
response and blocks sustained increases in intracellular
Ca2+ levels after purinergic receptor stimulation (21).
Recent work with nonhydrolyzable Ins(1,4,5)P3 analogs
suggests that this enzyme may limit activation of the store-operated
Ca2+ influx current, ICRAC, in RBL cells (13).
The type I 5-phosphatase is a potentially useful tool for examining the
effect of lowering Ins(1,4,5)P3 levels in cells under
conditions in which other phosphoinosotide signaling pathways are fully operational.
RI cross-linking (6). Although antigen-stimulated ruffling does not require extracellular Ca2+, it is mimicked by phorbol ester
treatment (24) and is thus potentially dependent on PLC
activation.
2 that has already acquired the "uncapped" active conformation after in vivo tyrosine phosphorylation or
in vitro exposure to low pH. Inhibitory antibodies to the
class IA PI 3-kinase catalytic subunits, introduced by microinjection
into RBL-2H3 cells, provide further proof that PI 3-kinases mediate the
wortmannin-sensitive steps leading to Ca2+ signaling and
membrane ruffling in antigen-stimulated RBL-2H3 cells. Consistent with
recent evidence that the p110 subunits can have functionally distinct
roles in intracellular signaling (22, 25, 26), we find that antibodies
to p110
and p110
, but not to p110
, affect these two specific
antigen-stimulated responses, despite the fact that all three subunits
associate with tyrosine-phosphorylated proteins after receptor
stimulation. Finally, we compare inhibition of Ca2+
signaling in cells injected with PI 3-kinase-inhibitory
antibodies to that in cells microinjected with heparin, which blocks
binding of Ins(1,4,5)P3 to its receptor (27), or with
recombinant inositol polyphosphate 5-phosphatase I, which
hydrolyzes Ins(1,4,5)P3. The results show that
Fc
RI-mediated calcium responses are absolutely dependent on
Ins(1,4,5)P3-mediated gating of its receptors on intracellular stores. They are consistent with a model in which PI
3-kinase lipid products support maximal production of
Ins(1,4,5)P3 required for store depletion and do not
provide a PLC
-independent means to activate calcium influx in
RBL-2H3 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
p110
catalytic subunits of class IA PI 3-kinase (26) were prepared
as a 2.5 mg/ml solution in PBS. Commercial antibodies to the carboxyl
terminus of the p110
subunit (residues 1030-1044; PharMingen, San
Diego, CA) were dialyzed against 0.1× PBS to remove azide and
concentrated by freeze drying and resuspension at 2.5 mg/ml in
double-distilled H20. For experiments in which p110
and p110
antibodies were coinjected, freeze-dried p110
was resuspended in a solution of p110
to yield a preparation containing 2.5 mg/ml of each antibody. Low molecular weight heparin (~3000 average molecular weight; Sigma) was injected as a 25 mg/ml solution in PBS. Fura-2 pentapotassium salt (Molecular Probes, Eugene, OR) was added to injection solutions to a final concentration of 5 mM.
-D-galactopyranoside
induction and purified on Ni-NTAG-agarose (Qiagen) according to
the manufacturer's instructions. Purified protein was dialyzed against
phosphate buffer, yielding a preparation for microinjection containing
0.35 mg/ml (determined by the Bio-Rad Coomassie Blue protein assay kit)
and a single prominent band of Mr 51,000 by
SDS-PAGE. Phosphatase activity was confirmed in vitro, as
described by Ono et al. (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activity by the inclusion of
PtdIns(3,4,5)P3 in lipid micelle substrates (8),
we have investigated the effect of PtdIns(3,4,5)P3 on
PLC
2 activated in vivo by receptor stimulation and
in vitro by pH-dependent conformational change.
As demonstrated in Fig. 1A,
PtdIns(3,4,5)P3 is a weak stimulator of nonphosphorylated PLC
2 that has been immunoprecipitated from resting RBL-2H3 cells. However, even small amounts of PtdIns(3,4,5)P3 markedly
increase the activity of PLC
2 that was tyrosine-phosphorylated after
Fc
RI cross-linking and immunoprecipitated from activated cells.
Based on recent models that propose "uncapping" as the means by
which either tyrosine phosphorylation or low pH activates PLC
(30), we next tested the effect of adding PtdIns(3,4,5)P3 to
phospholipase reactions conducted at pH 5. Results in Fig.
1B show that PtdIns(3,4,5)P3 also augments
PLC
2 activated by low pH and suggest that
PtdIns(3,4,5)P3 activates PLC
2 by a mechanism distinct
from uncapping.
View larger version (29K):
[in a new window]
Fig. 1.
Activation of immunoprecipitated
PLC 2 by PtdIns(3,4,5)P3.
A, PLC
2 immunoprecipitates prepared from resting and
activated RBL-2H3 cells were incubated with 200 µM
[3H]phosphatidylinositol 4,5-diphosphate in the presence
of the indicated concentrations of PtdIns(3,4,5)P3.
B, PLC
2 immunoprecipitates were incubated at either pH 5 or pH 6.8 with 200 µM
[3H]phosphatidylinositol 4,5-diphosphate and the
indicated concentrations of PtdIns(3,4,5)P3. Data represent
the average of duplicate assays in a single, representative experiment.
Experiments were repeated at least three times using cell lysates
prepared on separate occasions.
Results in Fig. 1 confirm a role for PI 3-kinase lipid products in the
direct activation of PLC but do not identify the specific form of PI
3-kinase involved in Fc
RI signaling. Because RBL-2H3 cells express
all three catalytic p110 isoforms of class IA PI 3-kinase, commercial
isoform-specific antibodies were used to immunoprecipitate PI 3-kinase
heterodimers from resting and antigen-stimulated cells.
Tyrosine-phosphorylated proteins associated with these immune complexes
were identified after SDS-PAGE and immunoblotting with
anti-phosphotyrosine antibodies. As shown in Fig.
2A, all three PI 3-kinases
isolated from resting cells associate with a prominent
tyrosine-phosphorylated protein migrating at an approximate molecular
weight of 100,000. Within 2 and 5 min after stimulation, new
tyrosine-phosphorylated species appear at Mr
~85,000, Mr 42,000, and
Mr 27,000. The profile of
tyrosine-phosphorylated proteins is similar but not identical in p85
immunoprecipitates. Blots were stripped and reprobed with anti-p85
antibodies (Fig. 2B). The amount of p85 precipitated with
anti-p110
antibodies was consistently less than that precipitated
with the other two isoform-specific antibodies, suggesting that p110
is the least abundant isoform in RBL-2H3 cells.
|
Several reports have described effective inhibition of catalytic
activity by antibodies raised to specific sequences in the carboxyl
termini of p110, p110
, and p100
(22, 26, 31). We introduced
anti-carboxyl-terminal inhibitory antibodies into RBL-2H3 cells to
identify the class IA PI 3-kinase p110 isoforms that regulate
Ca2+ signaling after Fc
RI cross-linking. Inhibitory
antibodies to p110
and p110
used here have been described
previously (22, 26). Antibodies to p110
, which were raised against
the same peptide as antibodies used to inhibit p110
in other studies
(31), were demonstrated to inhibit p110
activity in vitro
(data not shown). Fig. 3,
A
E, shows representative traces from control cells (Fig.
3A, cells injected with fura-2 alone) compared with cells
microinjected with the isoform-specific, anti-p110 antibodies (Fig. 3,
B-E). Whereas the blocking antibodies to p110
have no significant effect on the overall Ca2+ response (Fig.
3B), the presence of either p110
(Fig. 3C) or p110
(Fig. 3D) blocking antibodies results in diminished
Ca2+ responses, including a delayed onset after
stimulus, a blunted sustained phase and oscillations. Injection of the
combination of both p110
and p110
antibodies dramatically reduces
antigen-stimulated Ca2+ mobilization, resulting in a weak,
transient response (Fig. 3E). Fig. 3F reports the
average area under the curve for multiple experiments, where
Ca2+i is plotted against time after activation.
|
Coverslips containing antibody-injected cells were fixed immediately
after Ca2+ imaging had been performed. To determine the
effects of the microinjected antibodies on plasma membrane ruffling,
cells were permeabilized by a brief detergent treatment and stained
with rhodamine phalloidin (which labels filamentous actin in all cells)
and fluorescein isothiocyanate-conjugated anti-rabbit IgG (which
identifies injected cells). Fluorescence micrographs in Fig.
4 show that antibodies to p110 and
p110
both inhibited the formation of actin-based plasma membrane
ruffles in activated cells, whereas antibodies to p110
had little or
no effect. Injection of inhibitory antibodies did not prevent the
formation of actin plaques on the ventral surface of activated cells
(data not shown), in agreement with results obtained using the PI
3-kinase inhibitor wortmannin (6).
|
To investigate the Ins(1,4,5)P3 dependence of
antigen-stimulated calcium responses, RBL-2H3 cells were microinjected
with recombinant inositol polyphosphate 5-phosphatase I. The
5-phosphatase was produced as a His-tagged protein in E. coli and purified by nickel chromatography. The purity of the
enzyme was greater than 90%, based on analysis of Coomassie
Blue-stained SDS-PAGE gels, and inositol 5-phosphatase activity was
confirmed using [3H]inositol 1,3,4,5-tetrakis-phosphate
as a substrate (data not shown). Representative traces of
Ca2+ responses after FcRI cross-linking in
control-injected and 5-phosphatase I-injected cells are shown in Fig.
5, A and B. These
results demonstrate that high levels of 5-phosphatase inhibit the
sustained phase of Fc
RI-induced Ca2+ signaling,
resulting in a single initial spike of calcium release (Fig.
5B). The overall magnitude of the Ca2+ response
in 5-phosphatase-injected cells was significantly inhibited compared
with the response of control cells (Fig. 5C). The inhibition of Ca2+ responses caused by 5-phosphatase injection was
similar to that seen by injection of inhibitory antibodies to p110
and p110
(Fig. 3). However, direct observation of injected cells
after Ca2+ imaging revealed that 5-phosphatase injection
did not affect the plasma membrane ruffling response (data not
shown).
|
Heparin is a competitive inhibitor of the Ins(1,4,5)P3
receptor that blocks Ins(1,4,5)P3-mediated store release
(27). Ca2+ responses in heparin-microinjected RBL-2H3 cells
were either completely ablated (Fig.
6B, top panel) or reduced to a
single initial spike (Fig. 6B, bottom panels), presumably
reflecting a brief pulse of intracellular store release. In this series
of experiments, complete inhibition of the Ca2+ signal was
seen in ~30% of heparin-injected cells. The SERCA inhibitor
thapsigargin was added 10 min after stimulation with antigen to
passively deplete internal stores and rule out possible direct effects
of heparin on the store-operated influx pathway. Thapsigargin induced a
sustained Ca2+ response in heparin-injected cells,
demonstrating that the store-operated pathway was functionally intact
and apparently ruling out any direct role for binding of
Ins(1,4,5)P3 to its receptor in the store-operated influx
pathway. Thapsigargin induced a secondary response in
antigen-stimulated control cells, suggesting that some refilling of
internal Ca2+ stores can occur during continuous antigen
stimulation. Direct observation of injected cells after
Ca2+ imaging revealed that heparin did not affect the
ruffling response induced by FcRI cross-linking, even in cells
displaying no Ca2+ response (data not shown). These results
demonstrate that neither heparin nor 5-phosphatase I interferes with
global events downstream of PI 3-kinase and support earlier
observations that membrane ruffling is not dependent on elevations in
intracellular Ca2+ (24).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tyrosine phosphorylation of PLC isoforms was first demonstrated
to occur downstream of epidermal growth factor receptor activation (32). This led to a simple model for PLC
activation downstream of
tyrosine kinase signaling cascades and, in the Fc
RI system, was
supported by early evidence that PLC
proteins were rapidly tyrosine-phosphorylated after antigen stimulation (1) (33). Unexpectedly, we showed that PI 3-kinase inhibitors markedly
reduced the antigen-stimulated production of Ins(1,4,5)P3
(6), an observation explained in part by inhibition of the
translocation and tyrosine phosphorylation of the less abundant PLC
1
isoform (8) and by direct activation of both PLC
isoforms by the PI
3-kinase lipid product PtdIns(3,4,5)P3 (9, 10). Here, we
compared the ability of PtdIns(3,4,5)P3 to enhance
enzymatic activity of PLC
2 immunoprecipitated from resting and
stimulated cells, demonstrating that PtdIns(3,4,5)P3
is a more effective stimulator of tyrosine-phosphorylated PLC
than of enzyme in its basal state (Fig. 1A). We also tested the ability of PtdIns(3,4,5)P3 to stimulate PLC
activity
measured at pH 5.0, where a pH-dependent conformational
change "uncaps" the active site of the enzyme (30). Consistent with
the model that tyrosine phosphorylation also uncaps the active site,
enzymatic activity of basal state PLC
is elevated at pH 5.0 and
comparable to that of phosphorylated enzyme at either pH 5.0 or pH 6.8. The addition of PtdIns(3,4,5)P3 to the mixed micelle
reaction results in enhanced activity at both pH conditions (Fig.
1B), suggesting that PtdIns(3,4,5)P3 regulates
PLC
2 through a distinct mechanism.
Having established direct effects of PI 3-kinase lipid products on
PLC activity, we next focused on the consequences of PI 3-kinase
activation on overall Ca2+ responses in antigen-stimulated
RBL-2H3 cells. We found that Ca2+ profiles in cells
injected with antibodies to p110
and p110
are remarkably similar
to the response of cells treated with low nanomolar concentrations of
the PI 3-kinase inhibitor wortmannin (10). These data rule out the
possibility that wortmannin inhibits Ca2+ mobilization by
PI 3-kinase-independent means. Both approaches lead to a reduction in
the amplitude of the sustained phase of the Ca2+ response
and to an increase in the lag time to response after antigen addition.
Previous data suggested that the PI 3-kinase D3-phosphatidylinositol
product, Ptd(3,4,5)P3, might independently regulate
Ca2+ influx in RBL-2H3 cells (10). However, our earlier
experiments assessing the state of calcium stores in wortmannin-treated
RBL-2H3 cells were performed after a solution exchange to calcium-free medium, a condition that did not permit the refilling of stores (10).
Here, we compared antigen-stimulated calcium responses in cells that
were injected with either inhibitory PI 3-kinase antibodies (to inhibit
PI 3-kinase contributions to PLC activation), heparin (to block
Ins(1,4,5)P3 receptor binding), or 5-phosphatase I
(to promote Ins(1,4,5)P3 degradation), all in the presence
of extracellular calcium, to facilitate store refilling. It is
particularly revealing that microinjection of 5-phosphatase I markedly
inhibits the extent and duration of store depletion necessary to
support Ca2+ influx, giving results that are very similar
to those seen after inhibition of PI 3-kinase. The best explanation is
that the sustained store-operated calcium influx phase, and not the
initial spike of stores release, is dependent on maintaining high rates
of Ins(1,4,5)P3 synthesis and/or low rates of degradation.
This conclusion agrees with recent work on the properties of the Ca2+ influx current, ICRAC, in RBL cells. First identified as store-operated Ca2+ channels in mast cells by Hoth and Penner (34), ICRAC is highly selective for calcium and is activated in mast cells when intracellular Ca2+ stores are depleted by dialysis with Ca2+ chelators or Ins(1,4,5)P3, by treatment of cells with ionomycin or thapsigargin, or by antigen stimulation (3, 34). Essentially identical ICRAC currents can be recorded in both Jurkat T cells (35, 36) and RBL cells (37). Importantly, ICRAC current induced by Ins(1,4,5)P3 dialysis via the patch clamp requires relatively high concentrations of Ins(1,4,5)P3 (12). The sensitivity of ICRAC current to Ins(1,4,5)P3 levels is not linear but rather resembles an all or nothing phenomenon (12). In the nanomolar to low micromolar range, calcium store release can be measured in the absence of influx through ICRAC. Recent work from the Parekh laboratory suggests that the threshold reflects the state of the Ins(1,4,5)P3-sensitive stores, which can initially be mobilized by submaximal concentrations of Ins(1,4,5)P3 but are subsequently limited by negative feedback regulation or spatial restrictions, curtailing Ca2+ efflux through the Ins(1,4,5)P3-gated channel (11). Recent experiments of Glitsch and Parekh (13) using the nonmetabolizable analogue inositol 2,4,5-triphosphate suggest that dephosphorylation of Ins(1,4,5)P3 by endogenous 5-phosphatase I is likely to be one important negative feedback pathway that limits store-operated ICRAC current.
Calcium responses are either abolished or consist of only a brief
calcium spike in cells microinjected with heparin (Fig. 6). These data
suggest that Ins(1,4,5)P3 may be solely responsible for
FcRI-mediated release of internal Ca2+ stores.
Thapsigargin challenge demonstrated that calcium influx, per
se, is not inhibited by heparin injection because
thapsigargin-mediated responses are indistinguishable from controls. A
potential explanation for the Ins(1,4,5)P3 dependence of
Ca2+ influx is provided by recent work on the transient
receptor potential (trp) family of Ca2+ channels. Six
mammalian trp isoforms have been identified as potential store-operated
channels. Although the electrophysiological properties of
overexpressed, recombinant trp channels do not resemble those of
ICRAC (38), it remains possible that heterotetramers of
different family members, expressed at a low level, may form the
ICRAC channel. An early report detected trp2 and trp5
expression in RBL-2H3 cells by reverse transcription-polymerase chain
reaction analysis (39). Recent studies have demonstrated that trp1 and trp3 couple to Ins(1,4,5)P3 receptors (40, 41) and that
this coupling is required for activation of trp3 (40). Others have observed that Ins(1,4,5)P3 receptors may detect the state
of calcium stores in a manner that regulates the formation of this
complex (42). We demonstrate here that heparin microinjection does not inhibit thapsigargin-mediated Ca2+ influx. This suggests
that if the Ins(1,4,5)P3 receptor acts as the sensor of
store depletion, it does so independently of Ins(1,4,5)P3 binding.
We showed previously that the type II Ins(1,4,5)P3 receptors of RBL cells cluster in the endoplasmic reticulum within minutes of calcium mobilization induced by antigen, inonomycin, or thapsigargin treatment (43). We speculate that this dramatic redistribution results in the formation of Ca2+ pools within the ER whose efflux is limited by distance from the Ins(1,4,5)P3 receptor clusters. Although it does not preclude the influence of other feedback mechanisms operating on Ins(1,4,5)P3 receptors (44), it offers one potential explanation for increased dependence on high Ins(1,4,5)P3 levels during the latter, but not the early, phase of the calcium response.
Based on antigen-stimulated increases in tyrosine-phosphorylated
proteins found in immune complexes prepared using each of the three
class IA PI 3-kinase-specific antibodies, all of the p110/p85
heterodimers are likely to function in some aspect of FcRI
signaling. Nevertheless, only antibodies to the p110
and p110
catalytic subunits blocked Ca2+ responses and ruffling
after antigen stimulation. Absence of inhibition with antibodies to
p110
may have several explanations. Based on semiquantitative
analysis by immunoprecipitation and immunoblotting comparisons (Fig.
2), p110
appears to be the least abundant class IA isoform in
RBL-2H3 cells. Thus, the low expression levels may be a simple
explanation for the lack of inhibition using blocking p110
antibodies. Alternatively, distinct functions of the class IA PI
3-kinases may reflect intrinsic differences in the isoforms that
mediate differential coupling to specific receptors and/or downstream
effectors. An important component of coupling specificity may be the
p85 isoform present in the PI 3-kinase heterodimer, as suggested the
recent report that cKit signaling, but not that of Fc
RI, is blocked
in p85
-deficient bone marrow-derived mast cells (45).
In conclusion, our results demonstrate that class IA PI 3-kinase
isoforms p110 and p110
act synergistically to regulate Fc
RI-induced Ca2+ signaling and plasma membrane
ruffling. The principal role for these PI 3-kinases in mast cell
Ca2+ responses is likely to be the enhancement of PLC
activity by PtdIns(3,4,5)P3-mediated translocation and
phosphorylation of PLC
1 (8) and the direct activation of both PLC
isoforms (10).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Janet Oliver for critical reading of the manuscript. Use of the Fluorescence Microscopy Facility at the University of New Mexico Cancer Research Facility is gratefully acknowledged.
![]() |
FOOTNOTES |
---|
* This work was supported by American Cancer Society Grant RPG-99-233-01-CIM (to B. S. W.).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: University of New
Mexico Cancer Research Facility, Rm. 205, Albuquerque, NM 87131. Tel.:
505-272-8852; Fax: 505-272-1435; E-mail: bwilson@thor.unm.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M100417200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PLC, phospholipase C; PI 3-kinase, phosphatidylinositol 3-kinase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; PtdIns(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; trp, transient receptor potential; DNP, dinitrophenol.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Park, D. J.,
Min, H. K.,
and Rhee, S. G.
(1991)
J. Biol. Chem.
266,
24237-24240 |
2. | Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1983) Nature 306, 67-69[Medline] [Order article via Infotrieve] |
3. | Zhang, L., and McCloskey, M. A. (1995) J. Physiol. (Lond.) 483, 59-66[Abstract] |
4. | Siraganian, R. P., Kulczycki, A. J., Mendoza, G., and Metzger, H. (1975) J. Immunol. 115, 1599-1602[Abstract] |
5. |
Yano, H.,
Nakanishi, S.,
Kimura, K.,
Hanai, N.,
Saitoh, Y.,
Fukui, Y.,
Nonomura, Y.,
and Matsuda, Y.
(1993)
J. Biol. Chem.
268,
25846-25856 |
6. | Barker, S. A., Caldwell, K. K., Hall, A., Martinez, A. M., Pfeiffer, J. R., Oliver, J. M., and Wilson, B. S. (1995) Mol. Biol. Cell 6, 1145-1158[Abstract] |
7. |
Fluckiger, A. C.,
Li, Z.,
Kato, R. M.,
Wahl, M. I.,
Ochs, H. D.,
Longnecker, R.,
Kinet, J. P.,
Witte, O. N.,
Scharenberg, A. M.,
and Rawlings, D. J.
(1998)
EMBO J.
17,
1973-1985 |
8. |
Barker, S. A.,
Caldwell, K. K.,
Pfeiffer, J. R.,
and Wilson, B. S.
(1998)
Mol. Biol. Cell
9,
483-496 |
9. |
Bae, Y. S.,
Cantley, L. G.,
Chen, C. S.,
Kim, S. R.,
Kwon, K. S.,
and Rhee, S. G.
(1998)
J. Biol. Chem.
273,
4465-4469 |
10. | Barker, S. A., Lujan, D., and Wilson, B. S. (1999) J. Leukocyte Biol. 65, 321-329[Abstract] |
11. |
Fierro, L.,
and Parekh, A. B.
(2000)
J. Physiol. (Lond.)
522,
247-257 |
12. | Parekh, A. B., Fleig, A., and Penner, R. (1997) Cell 89, 973-980[Medline] [Order article via Infotrieve] |
13. |
Glitsch, M. D.,
and Parekh, A. B.
(2000)
J. Physiol. (Lond.)
523,
283-290 |
14. |
Hsu, A. L.,
Ching, T. T.,
Sen, G.,
Wang, D. S.,
Bondada, S.,
Authi, K. S.,
and Chen, C. S.
(2000)
J. Biol. Chem.
275,
16242-16250 |
15. |
Pasquet, J. M.,
Quek, L.,
Stevens, C.,
Bobe, R.,
Huber, M.,
Duronio, V.,
Krystal, G.,
and Watson, S. P.
(2000)
EMBO J.
19,
2793-2802 |
16. | Choi, O. H., Kim, J. H., and Kinet, J. P. (1996) Nature 380, 634-636[CrossRef][Medline] [Order article via Infotrieve] |
17. | Downing, G. J., Kim, S., Nakanishi, S., Catt, K. J., and Balla, T. (1996) Biochemistry 35, 3587-3594[CrossRef][Medline] [Order article via Infotrieve] |
18. | Majerus, P. W. (1996) Genes Dev. 10, 1051-1053[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Malbec, O.,
Fong, D. C.,
Turner, M.,
Tybulewicz, V. L.,
Cambier, J. C.,
Fridman, W. H.,
and Daeron, M.
(1998)
J. Immunol.
160,
1647-1658 |
20. |
Huber, M.,
Helgason, C. D.,
Damen, J. E.,
Liu, L.,
Humphries, R. K.,
and Krystal, G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11330-11335 |
21. |
De Smedt, F.,
Missiaen, L.,
Parys, J. B.,
Vanweyenberg, V.,
De Smedt, H.,
and Erneux, C.
(1997)
J. Biol. Chem.
272,
17367-17375 |
22. |
Siddhanta, U.,
McIlroy, J.,
Shah, A.,
Zhang, Y.,
and Backer, J. M.
(1998)
J. Cell Biol.
143,
1647-1659 |
23. | Johanson, S. O., Naccache, P. A., and Crouch, M. F. (1999) Exp. Cell Res. 248, 223-233[CrossRef][Medline] [Order article via Infotrieve] |
24. | Pfeiffer, J. R., Seagrave, J. C., Davis, B. H., Deanin, G. G., and Oliver, J. M. (1985) J. Cell Biol. 101, 2145-2155[Abstract] |
25. |
Hooshmand-Rad, R.,
Hajkova, L.,
Klint, P.,
Karlsson, R.,
Vanhaesebroeck, B.,
Claesson-Welsh, L.,
and Heldin, C. H.
(2000)
J. Cell Sci.
113,
207-214 |
26. |
Hill, K.,
Welti, S., Yu, J.,
Murray, J. T.,
Yip, S. C.,
Condeelis, J. S.,
Segall, J. E.,
and Backer, J. M.
(2000)
J. Biol. Chem.
275,
3741-3744 |
27. | Hill, T. D., Berggren, P. O., and Boynton, A. L. (1987) Biochem. Biophys. Res. Commun. 149, 897-901[Medline] [Order article via Infotrieve] |
28. | Ono, M., Bolland, S., Tempst, P., and Ravetch, J. V. (1996) Nature 383, 263-266[CrossRef][Medline] [Order article via Infotrieve] |
29. | Lee, R. J., and Oliver, J. M. (1995) Mol. Biol. Cell 6, 825-839[Abstract] |
30. |
Zhou, C.,
Horstman, D.,
Carpenter, G.,
and Roberts, M. F.
(1999)
J. Biol. Chem.
274,
2786-2793 |
31. | Vanhaesebroeck, B., Jones, G. E., Allen, W. E., Zicha, D., Hooshmand-Rad, R., Sawyer, C., Wells, C., Waterfield, M. D., and Ridley, A. J. (1999) Nat. Cell Biol. 1, 69-71[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Wahl, M. I.,
Jones, G. A.,
Nishibe, S.,
Rhee, S. G.,
and Carpenter, G.
(1992)
J. Biol. Chem.
267,
10447-10456 |
33. | Li, W., Deanin, G. G., Margolis, B., Schlessinger, J., and Oliver, J. M. (1992) Mol. Cell. Biol. 12, 3176-3182[Abstract] |
34. | Hoth, M., and Penner, R. (1992) Nature 355, 353-356[CrossRef][Medline] [Order article via Infotrieve] |
35. | Lewis, R. S., and Cahalan, M. D. (1989) Cell Regul. 1, 99-112[Medline] [Order article via Infotrieve] |
36. |
Kerschbaum, H. H.,
and Cahalan, M. D.
(1999)
Science
283,
836-839 |
37. |
Fasolato, C.,
Hoth, M.,
and Penner, R.
(1993)
J. Biol. Chem.
268,
20737-20740 |
38. | Hofmann, T., Schaefer, M., Schultz, G., and Gudermann, T. (2000) J. Mol. Med. 78, 14-25[CrossRef][Medline] [Order article via Infotrieve] |
39. | Garcia, R. L., and Schilling, W. P. (1997) Biochem. Biophys. Res. Commun. 239, 279-283[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Ma, H. T.,
Patterson, R. L.,
van Rossum, D. B.,
Birnbaumer, L.,
Mikoshiba, K.,
and Gill, D. L.
(2000)
Science
287,
1647-1651 |
41. | Rosado, J. A., and Sage, S. O. (2000) Biochem. J. 350, 631-635[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Boulay, G.,
Brown, D. M.,
Qin, N.,
Jiang, M.,
Dietrich, A.,
Zhu, M. X.,
Chen, Z.,
Birnbaumer, M.,
Mikoshiba, K.,
and Birnbaumer, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14955-14960 |
43. |
Wilson, B. S.,
Pfeiffer, J. R.,
Smith, A. J.,
Oliver, J. M.,
Oberdorf, J. A.,
and Wojcikiewicz, R. J.
(1998)
Mol. Biol. Cell
9,
1465-1478 |
44. | Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Lu-Kuo, J. M.,
Fruman, D. A.,
Joyal, D. M.,
Cantley, L. C.,
and Katz, H. R.
(2000)
J. Biol. Chem.
275,
6022-6029 |