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J Biol Chem, Vol. 273, Issue 37, 23750-23757, September 11, 1998
-mediated Calcium Signaling*
,
,, and
From the Divisions of Signal Transduction and
¶ Nephrology, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, Massachusetts 02215, the Schepens Eye
Research Institute, Boston, Massachusetts 02114, and the
§ Laboratory of Cell Signaling, NHLBI, National Institutes
of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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It has been demonstrated that the lipid products
of the phosphoinositide 3-kinase (PI3K) can associate with the Src
homology 2 (SH2) domains of specific signaling molecules and modify
their actions. In the current experiments, phosphatidylinositol
3,4,5-trisphosphate (PtdIns-3,4,5-P3) was found to
bind to the C-terminal SH2 domain of phospholipase C
(PLC) with
an apparent Kd of 2.4 µM and to
displace the C-terminal SH2 domain from the activated platelet-derived
growth factor receptor (PDGFR). To investigate the in vivo
relevance of this observation, intracellular inositol trisphosphate
(IP3) generation and calcium release were examined in HepG2
cells expressing a series of PDGFR mutants that activate PLC with or
without receptor association with PI3K. Coactivation of PLC and PI3K
resulted in an ~40% increase in both intracellular IP3
generation and intracellular calcium release as compared with selective
activation of PLC. Similarly, the addition of wortmannin or LY294002
to cells expressing the wild-type PDGFR inhibited the release of
intracellular calcium. Thus, generation of PtdIns-3,4,5-P3 by receptor-associated PI3K causes an increase in IP3
production and intracellular calcium release, potentially via enhanced
PtdIns-4,5-P2 substrate availability due to
PtdIns-3,4,5-P3-mediated recruitment of PLC to the lipid
bilayer.
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Activation of the receptor-associated phosphoinositide 3-kinase
(PI3K)1 has been shown to
cause mitogenesis and enhanced cell motility, although the exact
mechanism by which PI3K mediates cell signaling during these events has
been difficult to elucidate (1-3). The lipid products of PI3K have now
been found to activate certain calcium-independent protein kinases C
and to bind to a subset of Src homology 2 (SH2) domains (4, 5). In
addition, PtdIns-3,4-P2 and/or PtdIns-3,4,5-P3
has been found to bind to and/or stimulate several pleckstrin homology
(PH) domain-containing proteins, including the Akt/PKB serine/threonine
protein kinase (6, 7), the PDK serine/threonine protein kinase (8), and
the Grp1 exchange factor for Arf1 (9). Falasca et al. (10)
have demonstrated that the PH domain of phospholipase C will bind to
PtdIns-3,4,5-P3, targeting PLC to the membrane.
Recently, Bae et al. (11) have found that the addition of
PtdIns-3,4,5-P3 can enhance phospholipase C-mediated
PtdIns-4,5-P2 hydrolysis in vitro and that
overexpression of a constitutively active form of the p110 catalytic
subunit of PI3K increases intracellular IP3 levels, raising
the possibility that PtdIns-3,4,5-P3 may regulate calcium
signaling as well. This possibility is supported by the observation
that wortmannin, an inhibitor of the catalytic site of PI3K, as well as
several related enzymes, diminishes the intracellular calcium transient
seen in adrenal glomerulosa cells, neutrophils, and rat leukemia cells
following stimulation (12-15).
To determine whether phospholipase C might interact with the lipid
products of PI3K in a manner capable of modifying
ligand-dependent IP3 generation and calcium
signaling, we examined PtdIns-3,4,5-P3 binding to the SH2
domains of PLC and found that PtdIns-3,4,5-P3 associates
with high affinity with the C-terminal SH2 (CT-SH2) domain of PLC
and is capable of displacing the CT-SH2 domain of PLC from the
activated PDGFR. We demonstrate that activation of a PDGFR mutant that
selectively activates PLC (Y1021) results in a substantially
diminished intracellular calcium release when compared with
coactivation of PI3K and PLC by the wild-type PDGFR. In addition,
inhibition of PtdIns-3,4,5-P3 generation by either wortmannin or LY294002 partially inhibits PDGF-mediated calcium release
by the wild-type PDGFR, but has no effect on calcium release initiated
by the Y1021 PDGFR.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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PtdIns-3,4,5-P3 Direct Binding Assay-- Glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli and extracted as described (16). Synthetic water-soluble 3H-labeled dioctanoyl (C8)-PtdIns-3,4,5-P3 (5 × 107 cpm/µmol) (17) was incubated with Sepharose beads containing ~0.3 nmol of GST-CT-SH2 domain fusion protein or GST alone in HNE buffer (30 mM Hepes, pH 7.0, 100 mM NaCl, and 1 mM EDTA) containing 0.02% Nonidet P-40. After 1 h at room temperature, the beads were separated from the supernatant by centrifugation. The supernatant was then collected, mixed with scintillation liquid, and counted on a Beckman counter. The amount of [3H]PtdIns-3,4,5-P3 bound to the CT-SH2 domain was calculated by subtracting the amount of free 3H present in the GST-CT-SH2 domain supernatant from the amount of free 3H present in the control GST supernatant under identical conditions. A nonlinear least squares fit to the data (Kaleidagraph) was determined using the following equation: [bound] = Bmax × [free]/(KD + [free]), where [bound] is the concentration of [3H]C8-PtdIns-3,4,5-P3 bound, [free] is the concentration of [3H]C8-PtdIns-3,4,5-P3 free in solution, Bmax is the saturation binding, and KD is the dissociation constant.
For the competition assay, [3H]C8-PtdIns-3,4,5-P3 (6500 cpm; 0.93 mM) was incubated with unlabeled PtdIns-4,5-P2 (Avanti Polar Lipids) or unlabeled synthetic PtdIns-3,4,5-P3 (17) and 20 µl of Sepharose beads containing ~0.3 nmol of GST-CT-SH2 or GST-N+CT-SH2 domain fusion protein in HNE buffer containing 0.02% Nonidet P-40. Incubation was carried out for 1 h at room temperature to allow binding to reach equilibrium, after which the beads were washed with 1 ml of HNE buffer containing 0.5% Nonidet P-40. The washed beads were mixed with scintillation liquid, and the radioactivity associated with them was measured in a Beckman scintillation counter. The data were plotted as a percentage of the control with no additional competitive lipid added. A nonlinear least squares fit to the data was performed using the following equation: % bound = 100
n × L/(Ki(app) + L), where n
indicates the percent specific binding, L indicates the concentration
of unlabeled lipid added, and Ki(app) is
the apparent competitive dissociation constant for the unlabeled
lipid.
PDGFR Binding Assay-- GST-CT-SH2 domain-containing beads were incubated with the appropriate concentration of PtdIns-4,5-P2 or PtdIns-3,4,5-P3 (sonicated in HNE buffer) for 1 h at room temperature. NIH 3T3 cells were stimulated with PDGF for 5 min and lysed in HNE buffer containing 0.5% Nonidet P-40. Lysates were centrifuged for 10 min at 14,000 × g, and supernatants were added to the GST-CT-SH2 domain/lipids for 10 min. Beads were washed three times with HNE buffer containing 0.5% Nonidet P-40, resuspended in an equal volume of 2× Laemmli buffer, and incubated for 5 min at 100 °C. Proteins were resolved by 7.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, blotted using anti-Tyr(P) antibody (4G10), and visualized using chemiluminescence detection (DuPont). The autoradiograms were quantified using the NIH Image program.
PDGFR Mutants--
These clones are as described previously
(18-21). Briefly, HepG2 cells were transfected with PDGFR constructs
encoding the wild-type PDGFR; a mutant that selectively excludes PI3K
binding (F740/751); a mutant that excludes PI3K, RasGAP, and SHP-2
binding, but associates with PLC (Y1021); a mutant that selectively
associates with PI3K (Y740/751); or a mutant that excludes binding to
all four signaling molecules (F5). Cell lines were
fluorescence-activated cell-sorted to normalize receptor expression and
maintained in Dulbecco's modified Eagle's medium with 10% fetal calf
serum.
Immunoprecipitation and Western Analysis-- Subconfluent cells were washed twice with phosphate-buffered saline and refed with Dulbecco's modified Eagle's medium in the absence of fetal bovine serum (Life Technologies, Inc.). Following 24 h of serum deprivation, cells were treated with either PDGF (100 ng/ml; Upstate Biotechnology, Inc.) or vehicle control for 2 min, washed twice with ice-cold phosphate-buffered saline, and lysed in ice-cold lysis buffer (see above). Cells were vortexed vigorously and centrifuged for 10 min at 12,000 × g. One-mg aliquots were immunoprecipitated overnight at 4 °C with anti-phosphotyrosine antibody (Upstate Biotechnology, Inc.) using protein A-Sepharose beads (Sigma). Beads were washed three times with phosphate-buffered saline, 1% Nonidet P-40, and 2 mM sodium vanadate; two times with 0.1 M Tris, 0.5 M LiCl, and 2 mM sodium vanadate, pH 7.5; and two times with 10 mM Tris, 100 mM NaCl, 1 mM EDTA, and 2 mM sodium vanadate, pH 7.5.
The final pellet was boiled for 5 min in the presence of
-mercaptoethanol and separated by SDS-polyacrylamide gel
electrophoresis. Proteins were electrophoretically transferred to
Immobilon-P membranes (Millipore Corp.). Blots were probed for
anti-phosphotyrosine, anti-p85 (generous gift of Brian Schaffhausen),
or anti-PLC (Santa Cruz Biotechnology) antibody. The blots were
exposed to the appropriate horseradish peroxidase-linked secondary
antibody prior to detection of proteins using a chemiluminescence
system (ECL, Amersham International).
Quantitation of Intracellular IP3 Levels-- Cells were plated at a concentration of 400,000 cells/well on 12-well plates and incubated for 48 h in inositol-free medium supplemented with 5 µCi/ml [3H]inositol (Amersham Pharmacia Biotech). Cells were then washed twice with phosphate-buffered saline and stimulated for 30 s with PDGF (100 ng/ml), and the reaction was stopped by the addition of ice-cold 10% trichloroacetic acid. Cells were lysed by sonication for 30 s, extracted three times with water-saturated ether, and filtered, and [3H]inositol polyphosphates were analyzed by anion-exchange HPLC, using a Partisphere SAX column (Whatman), as described previously (2). All experiments were performed in triplicate.
Intracellular Calcium Determination-- HepG2 cells were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 10% FCS. Cells were loaded with Fura-2/AM (Molecular Probes, Inc.) as follows. Nearly confluent 150-mm2 plates of cells were incubated for 1 min in 5 ml of HBBS/EDTA buffer (HBBS buffer = 118 mM NaCl, 4.6 mM KCl, 10 mM glucose, and 20 mM Hepes, pH 7.2, prepared in calcium-free water; HBBS/EDTA buffer = HBBS + 0.02% EDTA). HBBS/EDTA buffer was then aspirated, and cells were incubated for 1 min at 37 °C, followed by scraping in 25 ml of HBBS/CaCl2 buffer (HBBS buffer + 1 mM CaCl2). Cells were pelleted at 1000 × g for 10 min and resuspended in 5 ml of HBBS/CaCl2 buffer. Fura-2/AM was then added at a final concentration of 1 mM, and cells were incubated in the dark for 40 min at 37 °C. Cells were then washed three times with HBBS/CaCl2 buffer and resuspended in HBBS/CaCl2 buffer at a concentration of 1 × 106 cells/ml.
Intracellular free calcium measurements were performed in a constantly stirred 37 °C cuvette using a dual-beam fluorescence spectrophotometer (Spex AR-CM). For experiments performed in the absence of extracellular calcium, 10 mM EGTA was added at the beginning of the experiment, followed by a 60-s equilibration period. Controls were performed for each condition with addition of vehicle alone. Minimal fluorescence and maximal fluorescence were determined for each experiment by adding digitonin (final concentration = 50 mM) and CaCl2 (final concentration = 11 mM), respectively. Curves shown were generated by determining 5-s [Ca2+]i values using a best fit of the raw data. Results are expressed as mean ± S.E.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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PtdIns-3,4,5-P3 Can Bind to the CT-SH2 Domain of PLC
and Mediate Dissociation from the PDGFR--
To determine whether
PtdIns-3,4,5-P3 might interact with PLC in
vivo, binding of the SH2 domains of PLC to
PtdIns-3,4,5-P3 was examined in vitro.
Water-soluble C8-PtdIns-3,4,5-P3 bound to the
recombinant C-terminal SH2 domain of PLC1 with a
Kd of 2.4 µM and a stoichiometry of
~1 mol/mol (Fig. 1). In contrast, the
N-terminal SH2 domain did not significantly associate with C8-PtdIns-3,4,5-P3 under these conditions,
whereas a construct including both SH2 domains had an affinity for
PtdIns-3,4,5-P3 that was similar to that of the C-terminal
SH2 domain alone (data not shown).
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1
C-terminal SH2 domain, competition assays were performed. When
incubated with
[3H]C8-PtdIns-3,4,5-P3, unlabeled
dipalmitoyl (C16)-PtdIns-3,4,5-P3 competed for
association with the PLC1 C-terminal SH2 domain with a
Ki(app) of 1.3 µM, whereas
PtdIns-4,5-P2 had an ~3-fold lower affinity (Fig.
2). Inositol 1,3,4,5-tetrakisphosphate and inositol 1,4,5-trisphosphate, at concentrations up to 100 µM, competed minimally with the
C8-PtdIns-3,4,5-P3 binding. The combined
N-terminal + C-terminal SH2 domain construct displayed a slightly lower
affinity than did the isolated C-terminal domain (Ki(app) = 3.7 µM for
PtdIns-3,4,5-P3 and 11 µM for
PtdIns-4,5-P2), consistent with this construct containing
both a high and low affinity binding site.
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with
phosphotyrosine residues, we postulated that
PtdIns-3,4,5-P3 might compete for PLC binding to the
phosphorylated PDGFR. To examine this, the CT-SH2 domain of PLC1,
immobilized on beads, was preincubated with increasing concentrations
of either PtdIns-3,4,5-P3 or PtdIns-4,5-P2. PDGF-stimulated NIH 3T3 cell lysates were added to the beads, and the
ability of the SH2 domain to associate with the PDGFR was determined.
PtdIns-3,4,5-P3 blocked the association of PLC with the
phosphorylated PDGFR in a dose-dependent fashion with a
Ki(app) of ~9 µM (Fig.
3). The higher
Ki(app) for PtdIns-3,4,5-P3
binding in this circumstance as compared with competition with
[3H]dioctanoyl-PtdIns-3,4,5-P3 (Fig. 2) is
likely due to the high affinity of the phosphorylated PDGFR for the
CT-SH2 domain of PLC. In agreement with the relative affinities
observed in Fig. 2, PtdIns-4,5-P2 competed with the PDGFR
for SH2 domain binding with a 3.6 times lower affinity than did
PtdIns-3,4,5-P3.
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Activation of Receptor Mutants with PDGF--
To examine the
in vivo effect of the lipid products of PI3K on PLC
signaling, we utilized PDGFR mutants stably expressed in HepG2 cells.
To ensure that the PDGFR mutants were similarly expressed and
recruiting the appropriate downstream signaling proteins,
anti-phosphotyrosine immunoprecipitates of PDGF-stimulated HepG2 cells
were examined for co-immunoprecipitation of phospholipase C and PI3K
(Fig. 4). Phosphorylation of the PDGFR in
response to added PDGF was nearly equivalent for all cell lines
(top panel). As expected from previous studies,
PLC associated with the F740/751, Y1021, and wild-type receptors in
a ligand-dependent fashion, but did not associate with the
F5 receptor (middle panel). Interestingly, the
association of PLC with the Y1021 mutant was increased as compared
with the wild-type receptor (see "Discussion"). The p85 subunit of
PI3K associated only with the wild-type receptor (bottom panel) and with the Y740/751 mutant (data not shown). Thus,
activation of only the wild-type receptor resulted in recruitment of
both signaling molecules.
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Intracellular IP3 Generation in Response to PDGF Is
Enhanced by Coactivation of PLC and PI3K--
HepG2 cells
expressing either the wild-type PDGFR or the F740/751 receptor were
loaded with [3H]inositol for 48 h, and total
IP3 generation in response to PDGF was determined by HPLC
analysis. A time course of IP3 generation in cells
expressing the wild-type PDGFR revealed that total IP3 began increasing 15 s following PDGF stimulation and peaked at 30 s (Fig. 5A). The
addition of PDGF to cells expressing the F740/751 mutant receptor
resulted in a lesser increase in intracellular total IP3 as
compared with the wild-type receptor (Fig. 5B, 1762 ± 201 dpm for the F740/751 mutant versus 3028 ± 148 dpm
for the wild-type receptor at 30 s following stimulation;
p < 0.01), with the increase in IP3
diminished by ~40%.
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Intracellular Calcium Release in Response to PDGF Is Enhanced by
Coactivation of PLC and PI3K--
HepG2 cells were loaded with
Fura-2 as described, and extracellular calcium was removed by the
addition of excess extracellular EGTA. Resting intracellular calcium in
all clones in the absence of extracellular calcium was ~15
nM (Fig. 6) and remained
stable for 
5 min. The addition of PDGF to HepG2 cells expressing the wild-type PDGFR resulted in a peak intracellular free calcium concentration of 93 ± 6 nM ~75 s following
stimulation (Fig. 6, closed circles). In cells
expressing either the Y1021 mutant (which selectively activates PLC)
or the F740/751 mutant (which activates all of the signaling pathways
except PI3K), the peak intracellular calcium response to PDGF was
significantly diminished (51 ± 7 and 58 ± 7 nM,
respectively) and delayed (105 and 90 s, respectively). Cells
expressing the Y740/751 mutant (which selectively binds PI3K, but not
PLC, RasGAP, or SH-PTP2) or the F5 mutant (which binds none of these
signaling enzymes) demonstrated no release of intracellular calcium in
response to PDGF. Thus, coactivation of PLC and PI3K resulted in a
1.5-2 fold increase in intracellular free calcium release as compared
with selective activation of PLC.
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Lipid Products of PI3K Mediate Enhanced Calcium Signaling--
The
in vivo effect of the interaction between
PtdIns-3,4,5-P3 and PLC was examined by inhibition of
PI3K-dependent PtdIns-3,4,5-P3 generation by
the HepG2 mutant receptor cell line. The addition of 100 nM
wortmannin to HepG2 cells (which results in inhibition of 80% of the
immunoprecipitable PI3K activity in these cells) expressing the
wild-type PDGFR resulted in a 24% inhibition of the calcium transient
(Fig. 7), whereas wortmannin had no
effect on the calcium transient in either the Y1021 or F740/751 mutant. The specificity of this effect of wortmannin for tyrosine kinase receptor-mediated coactivation of PI3K and PLC was confirmed by
comparison with the calcium transient induced following fetal calf
serum addition (Fig. 8). Fetal calf serum
contains lysophosphatidic acid, which causes a rapid calcium transient
(peak [Ca2+]i at ~10 s) secondary to release of
intracellular calcium stores following GTP-dependent
phosphoinositide hydrolysis (22, 23). Wortmannin inhibited the
intracellular calcium release following PDGF addition while having no
effect on calcium release in response to FCS.
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[Ca2+]i = 39.0 ± 1.9 nM following PDGF, 31.0 ± 4.7 nM
following PDGF + wortmannin, and 18.7 ± 5.0 nM
following PDGF + LY294002). Thus, inhibition of
PtdIns-3,4,5-P3 generation using PI3K inhibitors prevented
the enhanced release of intracellular calcium following PDGF
stimulation of cells that coactivate PI3K and PLC and had no effect
on the lesser calcium response in the Y1021 and/or F740/751 cells,
which activate PLC without coactivation of PI3K.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present data demonstrate that the PDGF-dependent
coactivation of phospholipase C and the phosphoinositide 3-kinase
results in enhanced intracellular generation of IP3 and an
augmented intracellular calcium transient as compared with those
observed with selective activation of PLC, suggesting that
recruitment of active PI3K to the PDGFR results in increased hydrolytic
activity of PLC. The importance of PtdIns-3,4,5-P3 in
this PI3K-dependent increase in intracellular calcium
release is demonstrated by two observations. First, the inhibition of
intracellular calcium release following the addition of wortmannin or
LY294002 (Figs. 7-9) (10, 12-15) argues that
PtdIns-3,4,5-P3 is upstream of PLC activation since wortmannin and LY294002 inhibit PtdIns-3,4,5-P3 production
without preventing PI3K association with the
receptor.2 Second, the
observation by Bae et al. (11)that constitutive expression
of the p110 catalytic subunit of PI3K can cause sustained increases in
cellular IP3 levels, without the necessity of receptor activation, also supports the importance of local generation of PtdIns-3,4,5-P3 in enhancing PLC activity.
Recent data demonstrating that PtdIns-3,4,5-P3 can directly
associate with SH2 domain-containing proteins and regulate their activity therefore led us to examine PtdIns-3,4,5-P3
association with the SH2 domains of PLC1. The in vitro
data presented demonstrate that PtdIns-3,4,5-P3 binds to
the C-terminal SH2 domain of PLC1 with an affinity similar to that
which we have observed with PtdIns-3,4,5-P3 binding to the
C-terminal SH2 domain of p85PI3K and ~3-fold weaker than
its binding to the PH domain of BTK (24) and Grp1 (25).
In a related series of experiments, Bae et al. (11) have
found that PtdIns-3,4,5-P3 can directly activate PLC
hydrolytic activity in vitro and that this activation can be
blocked by the addition of isolated SH2 domains of PLC. In addition,
the PH domain of PLC may be involved in the association of
PtdIns-3,4,5-P3 with the enzyme. Data from the laboratory
of Dr. Joseph Schlessinger (10) demonstrate that the N-terminal PH
domain of PLC can bind PtdIns-3,4,5-P3 and that
mutations of this domain diminish recruitment of PLC to the cell
membrane, raising the possibility that simultaneous binding of the PH
and SH2 domains to PtdIns-3,4,5-P3 may be involved in
enzyme activation. In contrast, the split PH domain of PLC does not
appear to bind PtdIns-3,4,5-P3 (11).
We postulate that the mechanism by which the PI3K mediates enhanced
activation of PLC may directly relate to the SH2
domain/PtdIns-3,4,5-P3 interaction. As seen in Fig. 3, the
association of PtdIns-3,4,5-P3 with the C-terminal SH2
domain of PLC prevents association of the SH2 domain with the
tyrosine-phosphorylated PDGFR. Dissociation of PLC from the receptor
due to local production of PtdIns-3,4,5-P3 could thus allow
translocation of the enzyme to the lipid bilayer, increasing its
substrate availability. Since PtdIns-3,4,5-P3 itself is not
hydrolyzed by PLC, its presence in the lipid bilayer would also
serve to stabilize PLC at that site, via association with the SH2
domain, the amino-terminal PH domain, or both domains simultaneously.
Coactivation of PLC and PI3K by the wild-type PDGFR, with subsequent
dissociation of PLC from the activated receptor due to local
production of PtdIns-3,4,5-P3, might also explain the
observation of greater association of PLC with the Y1021 PDGFR
mutant as compared with the wild-type receptor (Fig. 4).
In our experiments, PtdIns-3,4,5-P3 prevented binding of
the PLC CT-SH2 domain to the phosphorylated PDGFR with only a
3.6-fold higher affinity than did PtdIns-4,5-P2. The low
total cellular concentration of PtdIns-3,4,5-P3 as compared
with PtdIns-4,5-P2 thus raises the question of the
physiologic relevance of this observation. It is likely, we believe,
that recruitment of active PI3K to the receptor results in the local
conversion of much of the PtdIns-4,5-P2 into
PtdIns-3,4,5-P3, resulting in a reversal of these
concentrations in the immediate vicinity of the receptor. We postulate
that the generation of PtdIns-3,4,5-P3 as a high affinity
"anchor" for PLC at the membrane is therefore important for
stabilization of the enzyme in the vicinity of its substrate.
It is conceivable that PtdIns-3,4,5-P3 production at the
membrane could recruit non-phosphorylated PLC to this site (via association with the PLC SH2 and/or PH domain) and thereby trigger PtdIns-4,5-P2 hydrolysis in the absence of PLC
recruitment to receptors. This is not the case in all circumstances,
however, since activation of neither the Y740/751 PDGFR mutant (Fig. 6) nor the insulin receptor (26), which activate PI3K but not PLC, will
cause an intracellular calcium transient.
The present results, along with the recent observations that
PtdIns-3,4-P2 and/or PtdIns-3,4,5-P3 can
activate the serine/threonine protein kinases Akt/PKB and PDK1 (3) and
that PtdIns-3,4,5-P3 can stimulate the Arf1 nucleotide
exchange protein, Grp1 (25), suggest that PI3K may regulate multiple
signaling pathways via PtdIns-3,4-P2- and
PtdIns-3,4,5-P3-dependent interactions with the
SH2 and/or PH domains of proteins within those pathways. We have
previously shown that either selective activation of PI3K or the direct
addition of PtdIns-3,4,5-P3 to epithelial cells is capable
of initiating cell motility, at least in part by activation of
calcium-independent protein kinases C (4, 27). Our present data suggest
that receptor-mediated activation of PI3K could result in enhanced
activation of calcium-dependent protein kinase C isoforms by enhancing PLC substrate hydrolysis and therefore IP3
production and intracellular calcium release. The possibility that the
magnitude and/or duration of intracellular calcium transients can
moderate cell fate has recently been confirmed by the work of Dolmetsch et al. (28), in which it was shown that activation of
NF-
B and c-Jun N-terminal kinase occurred with rapid high calcium
transients, whereas NFAT was preferentially activated by a low, more
sustained calcium release.
The present data demonstrate that coactivation of PI3K and PLC
results in an increase in IP3 generation and intracellular calcium release. The ability of PtdIns-3,4,5-P3 to bind to
the CT-SH2 domain of PLC and to compete for its association with the
phosphorylated PDGFR suggests that the mechanism of the increased calcium signaling involves PtdIns-3,4,5-P3-mediated
recruitment and/or stabilization of PLC at the lipid bilayer.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK48871 (to L. G. C.), GM48339 (to A. K.), and GM41890 (to L. C. C.) and by the Medical Foundation of Massachusetts (to L. E. R.).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. Tel.: 617-667-2147; Fax: 617-667-5276.
The abbreviations used are:
PI3K, phosphoinositide 3-kinase; PtdIns-3, 4-P2,
phosphatidylinositol 3,4-bisphosphatePtdIns-4, 5-P2,
phosphatidylinositol 4,5-bisphosphatePtdIns-3, 4,5-P3,
phosphatidylinositol 3,4,5-trisphosphatePLC, phospholipase CSH2 domain, Src homology 2 domainCT-SH2 domain, C-terminal SH2
domainPH domain, pleckstrin homology domainIP3, inositol trisphosphatePDGF, platelet-derived growth factorPDGFR, PDGF receptorGST, glutathione S-transferaseHPLC, high
pressure liquid chromatographyFCS, fetal calf serum.
2
The degree of inhibition of calcium release
and/or IP3 generation by wortmannin appears to be cell
type-dependent, with marked inhibition of calcium release
in COS-1 cells expressing the PDGFR, granulosa cells, and leukemia
cells (10, 12, 15) and essentially no inhibition of A31 cells (L. G. Cantley, personal observation). This could be due to differences in
the importance of PtdIns-3,4,5-P3 for PLC activation by
different receptors or to different cell sensitivities to wortmannin
inhibition.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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 S. Dewitt, W. Tian, and M. B. Hallett Localised PtdIns(3,4,5)P3 or PtdIns(3,4)P2 at the phagocytic cup is required for both phagosome closure and Ca2+ signalling in HL60 neutrophils J. Cell Sci., February 1, 2006; 119(3): 443 - 451. [Abstract] [Full Text] [PDF]
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 J. C. Qi, J. Wang, S. Mandadi, K. Tanaka, B. D. Roufogalis, M. C. Madigan, K. Lai, F. Yan, B. H. Chong, R. L. Stevens, et al. Human and mouse mast cells use the tetraspanin CD9 as an alternate interleukin-16 receptor Blood, January 1, 2006; 107(1): 135 - 142. [Abstract] [Full Text] [PDF]
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 Y. D. Barac, N. Zeevi-Levin, G. Yaniv, I. Reiter, F. Milman, M. Shilkrut, R. Coleman, Z. Abassi, and O. Binah The 1,4,5-inositol trisphosphate pathway is a key component in Fas-mediated hypertrophy in neonatal rat ventricular myocytes Cardiovasc Res, October 1, 2005; 68(1): 75 - 86. [Abstract] [Full Text] [PDF]
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 A. A. Beharka, J. E. Crowther, F. X. McCormack, G. M. Denning, J. Lees, E. Tibesar, and L. S. Schlesinger Pulmonary Surfactant Protein A Activates a Phosphatidylinositol 3-Kinase/Calcium Signal Transduction Pathway in Human Macrophages: Participation in the Up-Regulation of Mannose Receptor Activity J. Immunol., August 15, 2005; 175(4): 2227 - 2236. [Abstract] [Full Text] [PDF]
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 Z. Xie, P. A. Singleton, L. Y.W. Bourguignon, and D. D. Bikle Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-{gamma}1 Mol. Biol. Cell, July 1, 2005; 16(7): 3236 - 3246. [Abstract] [Full Text] [PDF]
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 M. Van Stry, A. Kazlauskas, S. L. Schreiber, and K. Symes Distinct effectors of platelet-derived growth factor receptor-{alpha} signaling are required for cell survival during embryogenesis PNAS, June 7, 2005; 102(23): 8233 - 8238. [Abstract] [Full Text] [PDF]
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 B.-G. Kerfant, D. Gidrewicz, H. Sun, G. Y. Oudit, J. M. Penninger, and P. H. Backx Cardiac Sarcoplasmic Reticulum Calcium Release and Load Are Enhanced by Subcellular cAMP Elevations in PI3K{gamma}-Deficient Mice Circ. Res., May 27, 2005; 96(10): 1079 - 1086. [Abstract] [Full Text] [PDF]
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 A. Sasamoto, M. Nagino, S. Kobayashi, K. Naruse, Y. Nimura, and M. Sokabe Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch Am J Physiol Cell Physiol, May 1, 2005; 288(5): C1012 - C1022. [Abstract] [Full Text] [PDF]
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 M. F. Ethier, E. Cappelluti, and J. M. Madison Mechanisms of Interleukin-4 Effects on Calcium Signaling in Airway Smooth Muscle Cells J. Pharmacol. Exp. Ther., April 1, 2005; 313(1): 127 - 133. [Abstract] [Full Text] [PDF]
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S. D. Barbee and J. Alberola-Ila Phosphatidylinositol 3-Kinase Regulates Thymic Exit J. Immunol., February 1, 2005; 174(3): 1230 - 1238. [Abstract] [Full Text] [PDF]
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 L. Fischer, A. S. Gukovskaya, S. H. Young, I. Gukovsky, A. Lugea, P. Buechler, J. M. Penninger, H. Friess, and S. J. Pandol Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase Am J Physiol Gastrointest Liver Physiol, December 1, 2004; 287(6): G1200 - G1212. [Abstract] [Full Text] [PDF]
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B. Tolloczko, P. Turkewitsch, M. Al-Chalabi, and J. G. Martin LY-294002 [2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] Affects Calcium Signaling in Airway Smooth Muscle Cells Independently of Phosphoinositide 3-Kinase Inhibition J. Pharmacol. Exp. Ther., November 1, 2004; 311(2): 787 - 793. [Abstract] [Full Text] [PDF]
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S. P. Lee, C. H. So, A. J. Rashid, G. Varghese, R. Cheng, A. J. Lanca, B. F. O'Dowd, and S. R. George Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal J. Biol. Chem., August 20, 2004; 279(34): 35671 - 35678. [Abstract] [Full Text] [PDF]
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F. Sekiya, B. Poulin, Y. J. Kim, and S. G. Rhee Mechanism of Tyrosine Phosphorylation and Activation of Phospholipase C-{gamma}1: TYROSINE 783 PHOSPHORYLATION IS NOT SUFFICIENT FOR LIPASE ACTIVATION J. Biol. Chem., July 30, 2004; 279(31): 32181 - 32190. [Abstract] [Full Text] [PDF]
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 V. Nauc, E. De Lamirande, P. Leclerc, and C. Gagnon Inhibitors of Phosphoinositide 3-Kinase, LY294002 and Wortmannin, Affect Sperm Capacitation and Associated Phosphorylation of Proteins Differently: Ca2+-Dependent Divergences J Androl, |
, base line
(+EGTA); 
, PDGF; 
, PDGF + wortmannin.
