From the Institute of Interdisciplinary Research, Free University of Brussels, Campus Erasme, Building C, 808 route de Lennik, B-1070 Brussels, Belgium
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ABSTRACT |
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D-myo-Inositol
1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase catalyzes the
production of D-myo-inositol
1,3,4,5-tetrakisphosphate from the second messenger Ins
(1,4,5)P3. Transient and okadaic acid-sensitive activation
of Ins(1,4,5)P3 3-kinase by 8-10-fold is observed in
homogenates prepared from rat cortical astrocytes after incubation with
either carbachol or UTP.
12-O-Tetradecanoylphorbol-13-acetate provokes the
activation of Ins(1,4,5)P3 3-kinase by 2-fold in both cell
systems. The kinase was purified by calmodulin-Sepharose from the two
cell systems. Enzyme activity corresponding to the silver-stained
88-kDa protein could be regenerated after SDS-polyacrylamide gel
electrophoresis. Antibodies to two distinct peptides chosen in the
primary structure of human Ins(1,4,5)P3 3-kinase B
recognized the astrocytic native isoform. In
[32P]orthophosphate-preincubated cells, a major
phosphorylated 88-kDa enzyme could be purified and identified in cells
in response to receptor activation or
12-O-tetradecanoylphorbol-13-acetate treatment. Calmodulin
kinase II inhibitors (i.e. KN-93 and KN-62) and a
protein kinase C inhibitor (i.e. calphostin C) prevented
the phosphorylation of the 88-kDa isoenzyme. In addition to enzyme
activation, a redistribution of Ins(1,4,5)P3 3-kinase from
soluble to particulate fraction of astrocytes was observed. In
vitro phosphorylation of the purified enzyme by calmodulin kinase
II and protein kinase C added together resulted in a maximal
60-70-fold activation.
Hydrolysis of the plasma membrane phosphatidylinositol
4,5-bisphosphate and the formation of
D-myo-inositol 1,4,5-trisphosphate (Ins
(1,4,5)P3)1
result from the activation of a wide variety of cell surface receptors
by neurotransmitters and hormones (1). Ins (1,4,5)P3 3-kinase occupies a crucial position in this signal transduction cascade, inasmuch as it catalyzes a rapid production of
D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins
(1,3,4,5)P4) and generates a metabolic pathway for many
highly phosphorylated inositol phosphates.
D-myo-inositol 3,4,5,6-tetrakisphosphate
displays a second messenger function by selectively blocking epithelial
Ca2+-activated chloride channels (2-4). The production of
Ins (1,3,4,5)P4 in response to receptor activation has been
observed in various cell types, i.e. in rat brain cortical
slices (5, 6), in human astrocytoma 1321N1 cell line (7), and in
primary cultures of rat astrocytes (8). Due in large part to the
efforts of Irvine and colleagues, there is experimental evidence for a
possible second messenger function of Ins (1,3,4,5)P4 in
regulating Ca2+ homeostasis at least in certain cell types
(9, 10). Recently, Ca2+-dependent
K+ and Cl Molecular heterogeneity has been demonstrated for Ins
(1,4,5)P3 3-kinase; cloning of cDNAs encoding rat and
human Ins (1,4,5)P3 3-kinase A (14-16) as well as rat and
human Ins (1,4,5)P3 3-kinase B (17-19) has been reported.
In brain, isoform A is highly expressed in neuronal cells of the
cortex, hippocampus, and cerebellum in both rat and human (20, 21).
Isoform A is mainly expressed in the dendrites of hippocampal CA1
cells, and several reports suggested a second messenger role for Ins
(1,3,4,5)P4 in neuronal cells, e.g. Ins
(1,3,4,5)P4-mediated Ca2+ entry in CA1 cells,
with the suggestion it may contribute to ischemia-induced neuronal
death (22). The distribution of mRNA corresponding to rat Ins
(1,4,5)P3 3-kinase B has been shown to be more general as
compared with isoform A (19). In human brain, in situ
hybridization data indicated that mRNA corresponding to isoenzyme B
was mostly present in astrocytic cells, in contrast to the neuronal
isoenzyme A (21). However, this last point has never been established
at the protein level.
Ins (1,4,5)P3 3-kinase purified from a large number of cell
types appeared to be sensitive to the Ca2+·CaM complex,
i.e. 2-fold in rat and human brain (isoform A) (15, 16, 23)
to 17-fold in human platelets (24). The primary structures of rat and
human Ins (1,4,5)P3 3-kinases A and B reveal the presence
of various potential phosphorylation sites based on consensus
phosphorylation site sequences for
Ca2+·CaM-dependent protein kinase II (CaM
kinase II), protein kinase C (PKC), and cAMP-dependent
protein kinase (PKA) (15-17). In vitro experiments
demonstrated that Ins (1,4,5)P3 3-kinases A (25) and B (26)
are substrates for PKC- and PKA-mediated phosphorylation. Ins
(1,4,5)P3 3-kinase A is the target of a regulatory
mechanism involving CaM kinase II-mediated phosphorylation both
in vitro and in intact cells (27). Treatment of rat brain
cortical slices with carbachol provoked an increase in the
phosphorylation of Ins (1,4,5)P3 3-kinase A and a
corresponding increase in enzymic activity (3-5-fold) and in CaM
affinity (25-fold). An activation of Ins (1,4,5)P3 3-kinase
has also been observed in rat brainstem slices in response to serotonin
through the CaM kinase II pathway. This effect was also sensitive to
okadaic acid (28).
The physiological relevance of the existence of multiple isoforms of
Ins (1,4,5)P3 3-kinase has not yet been pointed out
experimentally in intact cells, especially concerning specific cellular
and subcellular protein distributions and concerning distinct
regulatory mechanisms with variable functional consequences. Data in
the present report revealed for the first time at the protein level
that the astrocytic Ins (1,4,5)P3 3-kinase corresponds to
isoform B. We have shown that the activation mechanism of Ins
(1,4,5)P3 3-kinase A can be generalized to Ins
(1,4,5)P3 3-kinase B in astrocytes. It nevertheless involves other mediators with distinct functional consequences. Activation of this isoform was provoked by PKC and CaM kinase II-mediated phosphorylation in vitro and in intact cells. In
this study, direct evidence is provided for a novel regulatory
mechanism involving phosphorylation by both PKC and CaM kinase II,
activation and redistribution of Ins (1,4,5)P3 3-kinase B
in intact astrocytes.
Generation of Antibodies Raised against the C-terminal End of
Human Ins (1,4,5)P3 3-Kinase Isoform B--
An immune
serum recognizing a 20-amino acid peptide (i.e.
SGLNNLVDILTEMSQDAPLA) corresponding to the last 20 amino acids of human
isoenzyme B has been generated. The peptide has been coupled to
hemocyanin in the presence of glutaraldehyde and injected to rabbits.
The antibodies (dilution 1:1000) immunodetected recombinant isoform B
but not isoform A after Western
blotting.2 Another antiserum
(dilution 1:500) raised against a 15-amino acid peptide
(i.e. CPPGDRVGVQPGNSR) in the N-terminal region of human
isoform B has been described before (24). For competition studies, 5 µg of the corresponding peptides were added for 1 h to the
diluted serum before immunodetection.
Preparation of Rat Brain Cortical Astrocytes--
Primary
cultures of rat cerebral cortex astrocytes were established using
dissociated rat cerebral tissue at 2 or 3 days after birth, according
to methods previously described (29). Briefly, dissected cortical
tissue was washed and then dissociated by gentle repeated pipetting in
modified Eagle's medium containing 1 mM sodium pyruvate,
10% fetal calf serum, 2% penicillin/streptomycin, and 1% fungizone.
Cells were decanted by gravity for 5 min, and the supernatant was
saved. The pellet was dissociated again, and the supernatant was added
to the first one. Cells were diluted in complemented cell medium and
plated in 9-cm diameter or 24 × 24-cm2 dishes at
37 °C with 5% CO2. When astrocytes were adherent,
dishes were vigorously agitated overnight and the medium was changed after two washes. Astrocytes reached confluence after 7 days in culture
and could be trypsinized three to four times. Human astrocytoma 1321N1
cells were grown in the same complemented medium. Cell culture medium,
dishes, and antibiotics were from Life Technologies, Inc.
Incubations of Cortical Astrocytes and 1321N1 Cells--
When
the astrocytic cells were ~80% subconfluent (7-8 × 106 cells in 8 ml of culture medium), they were washed
twice with 2 ml of prewarmed KRH medium (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.45 mM CaCl2, 1.25 mM
KH2PO4, 8 mM glucose, 25 mM Hepes/NaOH, pH 7.4). A 2-ml aliquot of the same
prewarmed medium containing the agent(s) was pipetted onto each culture
dish. Cell incubations were terminated by aspirating the incubation
medium prior to rinsing rapidly the cells twice with KRH medium. Cells
were harvested by scraping with a rubber policeman in 200 µl of
ice-cold lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM KCl, 12 mM 2-mercaptoethanol, 0.5% Nonidet
P-40, 100 mM NaF, 50 nM okadaic acid, 1 mM sodium orthovanadate, 0.1 mM Pefabloc, 5 µM leupeptin, and 15 µg/ml calpain inhibitors I and
II). Final cell lysates were obtained by three successive cycles of
freeze/thaw. Ins (1,4,5)P3 3-kinase activity was measured
at 5 µM Ins (1,4,5)P3 (30).
Km values for Ins (1,4,5)P3 were
estimated by measuring initial velocities in the presence of 0-50
µM Ins (1,4,5)P3 and by using a nonlinear least-squares curve fitting of substrate-velocity relationships (Marquardt-Levenberg algorithm). Sensitivity of the enzyme for CaM was
measured by assaying enzymic activity at 5 µM Ins
(1,4,5)P3, 10 µM free Ca2+, and
increasing concentrations of CaM (0-2 µM). Okadaic acid, sodium orthovanadate, NaF, ATP, UTP, carbachol, histamine,
tetradecanoyl phorbol acetate (TPA), Nonidet P-40, and leupeptin were
from Sigma. Pefabloc was from Pentapharm. Calpain inhibitors I and II
were from Roche Molecular Biochemicals. KN-93, KN-62, calphostin C, and
forskolin were from Calbiochem. [3H]Ins
(1,4,5)P3 (15-30 Ci/mmol) was from NEN Life Science
Products. SDS-PAGE, Western blotting and immunodetection were performed as described previously (23).
Cortical Astrocyte or 1321N1 Cell Labeling and Enzyme
Purification by CaM-Sepharose--
When cells were ~80%
subconfluent in 6-cm diameter culture dishes, they were washed two
times and incubated for 2 h in Dulbecco's modified Eagle's
pyrophosphate-free medium supplemented with carrier-free [32P]orthophosphate (0.5 mCi/ml). The cells were
subsequently washed in prewarmed KRH medium, and a 2-ml aliquot of this
medium containing the agent(s) was pipetted onto each culture dish for
an incubation with agonist. Cell extracts were prepared as described
above. Astrocytic Ins (1,4,5)P3 3-kinase was isolated after
each incubation by CaM-Sepharose (Amersham Pharmacia Biotech) (27). The
200-µl cell extract (~1.9 mg of total protein) was applied in the
presence of 0.5 mM CaCl2 to a column containing
200 µl of CaM-Sepharose eluted by gravity at 4 °C. The column was
washed with 5 ml of equilibration buffer (50 mM Tris/HCl pH
7.4, 12 mM 2-mercaptoethanol, 0.1 M NaCl, 0.2 mM CaCl2, 0.1% Triton X-100, 100 mM NaF, 50 nM okadaic acid, 1 mM
sodium vanadate, 0.1 mM Pefabloc, 5 µM
leupeptin, and 15 µg/ml calpain inhibitors I and II) containing no
Triton X-100 (Roche Molecular Biochemicals). The column was washed in the same buffer with 0.5 mM EGTA and no Triton X-100.
Specific elution of Ins (1,4,5)P3 3-kinase was performed in
the presence of 2 mM EGTA, 1% Triton X-100, as well as
phosphatase and protease inhibitors as described before (24, 27). The
two most active fractions (total volume of 400 µl) were concentrated
to 50 µl using Strataclean resin (Stratagene), and proteins were
separated by SDS-PAGE and detected by autoradiography using a
Hyperfilm-MP (Amersham Pharmacia Biotech) exposed for 24 h.
For immunodetection and in vitro phosphorylation
experiments, primary cultures of cortical astrocytes and 1321N1 cell
line were cultured in monolayers in square cell culture dishes (24 cm × 24 cm). The cell crude extracts were applied in the presence of 1 mM CaCl2 onto 40 ml of CaM-Sepharose.
Purified astrocytic Ins (1,4,5)P3 3-kinase was eluted in
the presence of 2 mM EGTA, 1% Triton X-100, together with
phosphatase and protease inhibitors. Fractions presenting the highest
Ins (1,4,5)P3 3-kinase activity (3 fractions, 25 ml) were
concentrated by Amicon Centricon-10 to obtain a final 50-µl sample
(~3 µg of purified enzyme, with a specific activity of 1.4 ± 0.2 µmol/min × mg as determined at 5 µM Ins
(1,4,5)P3 in the absence of the Ca2+·CaM complex).
Separation of Soluble and Particulate Fractions in Astrocytic
Cells--
Homogenates prepared from rat cortical astrocytes or 1321N1
cells (7-8 × 106 cells in 8 ml of culture medium per
incubation condition) were prepared by three successive cycles of
freeze/thaw in the lysis buffer without detergent. After 10 passages in
a 26-gauge needle attached to a 1-ml syringe, cell homogenates were
centrifuged at 4 °C for 1 h at 100,000 × g.
The supernatant (cytosol fraction) was saved; the pellet (particulate
fraction; ~30 µl) was washed three times and recovered in an equal
final volume of the same lysis buffer before assay of enzymic activity.
For Western blot studies, the same procedure was followed to separate
the soluble and particulate fractions from homogenates prepared from
2.3 × 108 1321N1 cells (per incubation condition).
Ins (1,4,5)P3 3-kinase activity was solubilized from the
particulate fraction under strong agitation in the lysis buffer (1 ml)
containing 1% Triton X-100 for 1 h at 4 °C. The solubilized
fraction was recovered after centrifugation at 100,000 × g for 1 h at 4 °C. Enzyme activity assay showed that
solubilization of Ins (1,4,5)P3 3-kinase from the
particulate fraction was efficient at approximately 80-85% as
compared with the total particulate activity (Fig. 7A). The soluble (12 mg of protein) and the solubilized particulate (75 mg of
protein) fractions (1 ml each) were adjusted to 0.1% Triton X-100 and
0.5 mM CaCl2 in a final volume of 50 ml and
purified separately by CaM-Sepharose as described above. The active
fractions were concentrated by Amicon Ultrafree-15 and applied for
SDS-PAGE. Western blotting and immunodetections were performed as
described above.
In Vitro Enzyme Phosphorylation by CaM Kinase II and
PKC--
Phosphorylation of purified astrocytic Ins
(1,4,5)P3 3-kinase (100 ng) by rat brain CaM kinase II (2 ng; Calbiochem) and/or rat brain PKC (2 ng) was performed as described
before (27). Phosphorylation samples were stopped at 4 °C and
directly diluted in ice-cold enzyme dilution buffer (1000-10,000-fold)
before assay of enzymic activity. In case of radioactive
phosphorylation, reactions were carried out in the presence of 50 µM [ Modulation of Ins (1,4,5)P3 3-Kinase Activity following
Receptor Activation in Astrocytes--
Since native Ins
(1,4,5)P3 3-kinase A activity could be regenerated after
SDS-PAGE (see, e.g., Ref. 23), we performed similar experiments with rat astrocytes as starting material. A major 85-90-kDa gel band associated with Ins (1,4,5)P3 3-kinase
activity from rat astrocytes was detected after SDS-PAGE and
regeneration of enzymic activity (Fig.
1A). Similar results were
obtained when using enzyme purified from 1321N1 cells or homogenates of
rat cortical astrocytes and 1321N1 cells (data not shown). Two distinct antibodies were raised against two peptides chosen in the N- and C-terminal sequence regions of human Ins (1,4,5)P3 3-kinase
B. Western blot analysis showed a unique 88-kDa band in purified preparation for both sera (Fig. 1B, lanes 1-4).
Immunodetected signals were greatly reduced when the sera were used in
the presence of the corresponding antigenic peptides (shown for 1321N1
cells) (Fig. 1B, lanes 5 and 6). No
signal could be observed with the corresponding preimmune sera (Fig.
1B, lanes 7-10). No recognition could be seen
with antibodies raised against isoform A (23) (data not shown). Silver
staining of purified astrocytic enzyme from 1321N1 cells revealed a
major band with an approximate molecular weight of 88,000 (Fig.
1C).
Carbachol is a well known agonist for muscarinic cholinoreceptor in rat
brain cortical astrocytes and in 1321N1 cells, where it mediates an
enhancement of PLC activity, the production of Ins
(1,4,5)P3, Ins (1,3,4,5)P4, and intracellular
calcium mobilization (7, 8). The same events were observed in cortical
astrocytes in response to purinoreceptors P2Y and P2U activation with
ATP and UTP (32), but this last response was not observed in 1321N1 cells since this cell line lacks the P2Y receptors (33). Incubation of
rat brain cortical astrocytes or 1321N1 cells with carbachol provoked a
transient increase in Ins (1,4,5)P3 3-kinase activity, i.e. 6-8-fold as compared with basal activity after 15-30
s of incubation with the agonist (Fig.
2). Maximal enzyme activation was
achieved at 10 µM carbachol (Table
I). Activation with UTP provoked in rat
cortical astrocytes the same rapid increase in Ins
(1,4,5)P3 3-kinase activity (Fig. 2A) with
maximal effect at 10 µM agonist (Table I). No effect of
UTP was observed in 1321N1 cells (Fig. 2B and Table I).
Histamine was much less potent (1.3-1.6-fold stimulation) (Fig. 2).
There was no effect of thrombin (data not shown). Preincubation with
okadaic acid of cortical astrocytes or 1321N1 cells before receptor
activation provided a maximal and more rapid activation of Ins
(1,4,5)P3 3-kinase (i.e. 10-fold) (Fig. 2). TPA
also provoked stimulation of Ins (1,4,5)P3 3-kinase
activity, i.e. 2-2.5-fold, in cortical astrocytes and
1321N1 cells, whereas forskolin did not provoke any change in enzymic
activity (Table I). In both cell systems, activation of Ins
(1,4,5)P3 3-kinase was related to an increase in
Vmax with no change in the apparent
Km value for Ins (1,4,5)P3 (Km = 1.5 ± 0.4 µM) (data not
shown).
Effect of CaM Kinase II and PKC Inhibitors on Ins
(1,4,5)P3 3-Kinase Activation--
We investigated the
effect of specific membrane-permeable PKC and CaM kinase II inhibitors
on the activation of astrocytic Ins (1,4,5)P3 3-kinase in
intact cells. Preincubation in the presence of increasing
concentrations (up to 500 nM) of calphostin C (potent inhibitor of PKC) before agonist stimulation partially prevented (up to
40%) activation induced by cell stimulation by carbachol (Fig.
3). Additionally, preincubation of both
cell types with increasing concentrations (up to 2 µM) of
two potent CaM kinase II inhibitors, i.e. KN-93 and KN-62,
prevented agonist-mediated activation of Ins (1,4,5)P3
3-kinase up to 85% (Fig. 3). The same results were obtained in UTP (10 µM)-stimulated rat cortical astrocytes (data not shown).
Direct enzyme phosphorylation was evidenced in rat cortical astrocytes
prelabeled with [32P] orthophosphate and incubated with
an agonist (carbachol or UTP) to stimulate Ins (1,4,5)P3
3-kinase activity (Fig. 4). Ins (1,4,5)P3 3-kinase was purified by CaM-Sepharose
specifically eluted in the presence of Triton X-100 and EGTA, and
analyzed by SDS-PAGE. Enzyme activation coincided with phosphate
incorporation into a 88-kDa protein band (Fig. 4). Maximal
32P incorporation occurred after incubation of astrocytes
with 10 µM carbachol for 30 s or with 10 µM UTP for 30 s (Fig. 4). Similar results were
observed after stimulation of 1321N1 cells by carbachol but UTP did not
present any effect (data not shown). Preincubation with okadaic acid
(75 nM) before receptor activation potentiated the
phosphate incorporation into the 88-kDa enzyme in both cell systems
(Fig. 4). 32P incorporation was also observed after
incubation of both cell types with TPA for at least 3 min (Fig.
5). On the other hand, preincubation with
KN-93 or KN-62 before receptor activation prevented 32P
incorporation into the enzyme in a dose-dependent manner.
Calphostin C also prevented phosphate incorporation (Fig. 5). No enzyme
phosphorylation could be seen in response to forskolin (data not
shown).
Distribution of Ins (1,4,5)P3 3-Kinase Activity and
Immunodetection after Receptor Activation--
Intracellular
distribution of astrocytic Ins (1,4,5)P3 3-kinase activity
was studied after in vivo phosphorylation and activation of
the enzyme in intact astrocytes. Ins (1,4,5)P3 3-kinase
activity was assayed in stimulated cortical astrocytes or 1321N1 cells after separation of cytosolic and particulate fractions by
centrifugation at 100,000 × g for 1 h. In
quiescent rat cortex astrocytes, enzymic activity was soluble at
approximately 65% and particulate at 35% (Fig.
6A). This was also observed in
1321N1 cells (Fig. 6B). Under conditions where the enzyme
was activated in response to carbachol or UTP, enzymic activity was
mainly measured in the particulate fraction and only a residual
activity (approximately 10% of total cell activity) was detected in
the soluble fraction after maximal activation at 30 s (Fig. 6).
This was not observed in 1321N1 cells in response to UTP (Fig.
6B). Western blot analysis of purified Ins
(1,4,5)P3 3-kinase B from 1321N1 cells permitted to observe a relocation of the enzyme to the particulate fraction in response to
carbachol (Fig. 7B). In
quiescent cells, approximately the same amounts of enzyme were detected
in soluble and particulate fractions, whereas the enzyme was relatively
more abundant in the particulate fraction after cell stimulation by
carbachol (10 µM) for 30 s (Fig. 7B).
Relocation occurred when the enzyme was maximally phosphorylated and
activated (see Figs. 2 and 4).
Sensitivity to the Ca2+·CaM Complex--
When
enzymic activity was determined in the presence of increasing
concentrations of CaM (0-2 µM), in vivo
phosphorylation of Ins (1,4,5)P3 3-kinase in rat cortex
astrocytes stimulated by carbachol (as well as in stimulated 1321N1
cells; data not shown) did not provoke any change in CaM sensitivity as
compared with the enzyme in quiescent cells (Fig.
8). Half-maximal stimulation of
astrocytic Ins (1,4,5)P3 3-kinase activity was reached at
approximately 17 nM CaM for non-phosphorylated enzyme as
well as for phosphorylated enzyme (Fig. 8). In the first case, the
maximal stimulation factor by the Ca2+·CaM complex was
14-fold, whereas in the second case, this factor was only 8-fold,
indicating that the maximal enzyme activation (due to enzyme
phosphorylation and CaM binding) was about 70-fold (Fig. 8).
In Vitro Phosphorylation of Astrocytic Ins (1,4,5)P3
3-Kinase B by CaM Kinase II and PKC--
Since in vivo
phosphorylation of Ins (1,4,5)P3 3-kinase isolated from
astrocytes was inhibited by potent CaM kinase II and PKC inhibitors, we
investigated in vitro phosphorylation of astrocytic Ins
(1,4,5)P3 3-kinase by CaM kinase II and PKC. Basal activity was stimulated 14-fold by the Ca2+·CaM complex when added
in the activity assay (Fig.
9A, black column). CaM kinase II-catalyzed phosphorylation and direct
CaM binding resulted in an increase in Ins (1,4,5)P3
3-kinase activity in the presence of 10 µM free
Ca2+ and 2 µM CaM, i.e.
35-40-fold, whereas PKC alone induced an inhibitory effect (50%) in
the presence of its cofactors (as compared with basal activity measured
after preincubation in the absence of any protein kinase). When the
experiment was performed in the presence of CaM kinase II and PKC
together and corresponding effectors, a maximal stimulation of Ins
(1,4,5)P3 3-kinase activity was observed, i.e.
approximately 60-70-fold as compared with the control (Fig. 9A). This last stimulation effect was not affected when the
preincubated enzyme was assayed in the presence of the
Ca2+·CaM complex (data not shown). We checked that
in vitro modulation of Ins (1,4,5)P3 3-kinase
activity by PKC and/or CaM kinase II was associated with
32P incorporation at the level of an 88-kDa protein after
SDS-PAGE and autoradiography (data not shown). Similar results were
obtained for in vitro phosphorylation of purified Ins
(1,4,5)P3 3-kinase from 1321N1 cells (data not shown).
Moreover, stoichiometry of Ins (1,4,5)P3 3-kinase B
phosporylation by CaM kinase II and/or PKC was estimated in
vitro. CaM kinase II and PKC provoked an incorporation of
32P to the enzyme with a molar ratio of 1.1 after 2 min and
of 2.3 after 5 min, respectively. When used together, 1 mol of enzyme was labeled with 3.3 mol of 32P after 5 min, indicating
that distinct sites were phosphorylated independently by these two
protein kinases (Fig. 9B).
On Western blots, two distinct antibodies raised against two
distinct peptides chosen in the primary structure of human Ins (1,4,5)P3 3-kinase B recognized the purified 88-kDa Ins
(1,4,5)P3 3-kinase isoform in rat astrocytes and human
astrocytoma cells. From this, we concluded that the isoform present in
astrocytic cells is isoform B. Regulation of Ins (1,4,5)P3
3-kinase could be driven by two types of mechanisms: direct binding of
the Ca2+·CaM complex and enzyme phosphorylation by
protein kinases. In response to a Ca2+-raising agent, PKC
and CaM kinase II provoke an okadaic acid-sensitive phosphorylation and
activation of Ins (1,4,5)P3 3-kinase in intact astrocytes.
The Ca2+·CaM complex contributes to maximal stimulation
of Ins (1,4,5)P3 3-kinase activity. This coincided with a
redistribution of the enzyme to the particulate fraction and no change
in sensitivity to the Ca2+·CaM complex. In the present
study, we provided evidence that Ins (1,4,5)P3 3-kinase
isoform B expressed in astrocytes is activated by both PKC and CaM
kinase II. This was shown in primary cultures of rat cortical
astrocytes stimulated through the muscarinic and purinergic pathways.
Similar results were obtained in 1321N1 cells stimulated through the
muscarinic pathway. Phosphorylation of the enzyme in astrocytic cells
was potentiated and more rapid in the presence of okadaic acid, as
shown for activation and 32P labeling of the enzyme. This
could be explained by the sensitivity of Ins (1,4,5)P3
3-kinase B or of the protein kinases mediating its activation to an
okadaic-sensitive protein phosphatase. The control by dephosphorylation
and deactivation of CaM kinase II by specific okadaic acid-sensitive
protein phosphatases has been reported (34, 35). In rat brain, CaM
kinase II was an excellent substrate for brain protein phosphatase 2A
and the decrease of the CaM kinase II phosphorylation level observed in
depolarized hippocampal synaptosomes was blocked in the presence of
okadaic acid (35). The presence of Ins (1,4,5)P3 3-kinase A
in rat brain, especially in neurons, as the major expressed isoform has
been previously reported (15, 20). A pre- and post-embedding
immunoelectron microscopic study made it possible to show that rat Ins
(1,4,5)P3 3-kinase A was present at highest levels in the
dendritic spines of cerebellar Purkinje cells and hippocampal CA1
pyramidal cells at the level of the cytoplasmic matrix (36, 37).
Expression of messenger RNA corresponding to isoenzyme B has been
reported in rat astrocytic cells (21), which is consistent with our
data obtained in this study.
The regulation of Ins (1,4,5)P3 3-kinase by phosphorylation
is thus quite general, although the present study reveals that the
functional consequences of this novel regulation of astrocytic Ins
(1,4,5)P3 3-kinase by CaM kinase II and PKC are distinct
from those observed for isoform A in rat brain cortical slices
(summarized in Table II): (a)
the degree of activation was higher (up to 10-fold in comparison with
5-fold in homogenates, respectively); (b) there was no
change in sensitivity to the Ca2+·CaM complex, whereas
isoform A presented a 25-fold decrease in Ka for
this complex; and, finally, (c) isoenzyme B was redistributed to the particulate fraction when phosphorylated and
activated (shown in this study), whereas isoform A remained soluble in
the same separation conditions in rat brain cortical slices (data not
shown). The higher degree of activation could explain the differences
in the stimulation of the phosphoinositide cycle by amine
neurotransmitters (e.g. carbachol) that have been revealed
in cultured rat forebrain neurons and astrocytes (8). Measurements of
[3H]inositol phosphates in the two last systems suggested
that the metabolic pathway involving Ins (1,4,5)P3 3-kinase
prevails in astrocytes and the one involving Ins (1,4,5)P3
5-phosphatase prevails in neurons. Recent theoretical work simulated
the effects of Ins (1,4,5)P3 3-kinase and 5-phosphatase
activities on the temporal pattern of Ca2+ oscillations
(38). It was predicted, on the basis of the Ins (1,4,5)P3
3-kinase stimulation by the Ca2+·CaM complex and the
experimentally determined kinetic characteristics of kinase isoform A
and phosphatase type I, that Ins (1,4,5)P3 5-phosphatase
primarily controls the Ins (1,4,5)P3 levels and so the
occurrence and frequency of Ca2+ oscillations. However,
this model predicted that high activation (more than 20-fold) of Ins
(1,4,5)P3 3-kinase, as is the case for the astrocytic
enzyme, may provoke a large contribution of Ins (1,4,5)P3
3-kinase in the control of Ins (1,4,5)P3 and Ins (1,3,4,5)P4 levels as well as in the Ca2+
oscillations. Concerning its subcellular distribution, previously reported immunodetection analysis have indicated that rat Ins (1,4,5)P3 3-kinase B exists as a cytosolic protein and was
also associated with peripheral membranes of the cytosolic face of the
extended endoplasmic reticulum network in transfected cells (39). Our
data obtained in quiescent astrocytes are consistent with a particulate
and cytosolic distribution (35% and 65%, respectively) of Ins
(1,4,5)P3 3-kinase B activity. We showed by activity assay and Western blot analysis that the distribution of this isoform is
modified, i.e. relocation to the membrane fraction, in
response to the stimulation of astrocytic cells by
Ca2+-raising agents. This could be a mechanism of
concentrating the enzyme at specific membranes directly involved in
Ca2+ homeostasis, e.g. endoplasmic reticulum
membranes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channels have been shown to be
activated by the non-metabolizable D-myo-inositol 2,4,5-trisphosphate and Ins
(1,3,4,5)P4 in primary cultures of mouse lacrimal acinar
cells (11). Its high affinity and high isomeric specificity of binding
to a GTPase-activating protein (GAP) (Ras-GAP1IP4BP
isolated from pig platelets) have been reported (12). An increase in
D-myo-inositol 2,4,5-trisphosphate-mediated
Ca2+ mobilization in the presence of Ins
(1,3,4,5)P4 has been reported in permeabilized L-1210
cells. This effect probably involves the participation of
GAP1IP4BP acting together with an activated monomeric
G-protein (13).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (final activity ~50
µCi/ml) instead of 1 mM ATP and stopped in SDS sample
buffer before SDS-PAGE and autoradiography. In order to measure CaM
kinase II and/or PKC-mediated 32P incorporation into
astrocytic Ins (1,4,5)P3 3-kinase, enzyme (0.5 µg) was
phosphorylated in the presence of 40 µM
[
-32P]ATP (final activity ~500 µCi/ml) for various
times (0-10 min) in the presence of CaM kinase II (10 ng), 2 µM CaM, and 10 µM free Ca2+ or
in the presence of PKC (10 ng), 0.25 mg/ml phosphatidylserine, and 20 µg/ml diacylglycerol. After incubation, each sample was spotted onto
P81 phosphocellulose (Whatman), precipitated, and washed in the
presence of 75 mM phosphoric acid before counting the
radioactivity (31).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Fig. 1.
Regeneration and immunodetection by specific
polyclonal anti-(human Ins(1,4,5)P3 3-kinase B) antibodies
of purified astrocytic Ins(1,4,5)P3 3-kinase.
A, Purified rat astrocytic enzyme (0.5 µg, 0.65 nmol/min)
was separated by SDS-PAGE before cutting the gel and assaying enzymic
activity at 5 µM Ins(1,4,5)P3. The major
active gel band at 85-90 kDa is indicated by an arrow.
B, Enzyme purified from 1321N1 cells (lanes
1, 3, 5-7, and 9) or from
rat cortex astrocytes (lanes 2, 4,
8, and 10) were separated by SDS-PAGE,
electroblotted, and immunodetected with antibodies raised against a
C-terminal peptide (lanes 1, 2, and
5) or a N-terminal peptide (lanes 3,
4, and 6) derived from the human
Ins(1,4,5)P3 3-kinase B primary structure. Competition
experiments were performed by immunodetecting enzyme from 1321N1 cells
in the presence of 5 µg of the corresponding peptides
(lanes 5 and 6). Immunodetections were
also performed with the corresponding preimmune sera (lanes
7-10). C, Enzyme purified from 1321N1 cells (40 ng, 50 pmol/min) was electrophoretically separated and silver-stained.
The 88-kDa band is indicated by an arrow.
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Fig. 2.
Effect of Ca2+-raising agonists
and okadaic acid on Ins(1,4,5)P3 3-kinase activity in rat
brain cortical astrocytes (A) and in human 1321N1
astrocytoma cells (B). The two cell types (two
dishes per condition) were incubated at 37 °C at the indicated times
with 10 µM carbachol ( ), UTP (
), or histamine (×).
Okadaic acid (75 nM) (
) was added for 30 min before
agonist stimulation, i.e. 10 µM carbachol.
Astrocytic cells were then lysed in the presence of protease and
phosphatase inhibitors, and enzyme activity was measured at 5 µM Ins(1,4,5)P3. Results are means of
triplicates ± S.D of one representative experiment out of
four.
Effect of Ca2+-raising agonists, TPA and forskolin on
Ins(1,4,5)P3 3-kinase activity in rat brain cortex astrocytes
and 1321N1 cells
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Fig. 3.
Effect of PKC inhibitor calphostin C and two
CaM kinase II inhibitors KN-93 and KN-62 on Ins(1,4,5)P3
3-kinase activation in stimulated rat brain cortical astrocytes
(A) and 1321N1 cells (B).
Calphostin C ( ), KN-62 (
), and KN-93 (×) inhibitors were added
for 30 min at the indicated concentrations before stimulation of both
cell types with 10 µM carbachol for 30 s. Enzyme activity
was assayed at 5 µM Ins(1,4,5)P3. Each value
is the mean of duplicates ± S.D. The results are from one
representative experiment out of five.
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Fig. 4.
Okadaic acid-sensitive phosphorylation of
Ins(1,4,5)P3 3-kinase in rat brain cortical
astrocytes. Cells were preincubated with
[32P]orthophosphate for 2 h before incubation at the
indicated times in the presence of various agents, i.e.
carbachol (Cchol), UTP, or okadaic acid (O.A.).
Cells were lysed in the presence of protease and phosphatase inhibitors
and enzyme was purified by CaM-Sepharose in each condition. The 88-kDa
enzyme was detected by autoradiography following SDS-PAGE. Carbachol
and UTP were at 10 µM and okadaic acid at 75 nM. Okadaic acid was added for 30 min before
Ca2+-raising agonist stimulation. Entire autoradiographies
are shown in the two first stimulation conditions with an
arrow indicating the 88-kDa protein.
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Fig. 5.
Effects of TPA, calphostin C, KN-93, and
KN-62 on phosphorylation of Ins(1,4,5)P3 3-kinase in rat
brain cortical astrocytes (A) and in human 1321N1
astrocytoma cells (B). Cells were preincubated
with [32P]orthophosphate for 2 h before incubation
in the presence of the various agents. The procedures for lysis and
enzyme purification are the same as described in the legend of Fig. 4.
The 88-kDa enzyme was detected by autoradiography. TPA was added at 400 nM for 0-5 min. Calphostin C, KN-93, and KN-62 were added
at the indicated concentrations for a 30-min preincubation before cell
stimulation with carbachol at 10 µM for 30 s.
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Fig. 6.
Subcellular distribution of
Ins(1,4,5)P3 3-kinase activity before and after enzyme
activation in intact astrocytes. Soluble (S) and
particulate (P) fractions were separated by centrifuging at
100,000 × g for 1 h starting from homogenates
prepared from rat cortex astrocytes (A) or from 1321N1 cells
(B). Enzyme activity was assayed at 5 µM
Ins(1,4,5)P3. Homogenates were prepared from
quiescent cells (Ctrl) and from the cells stimulated by 10 µM carbachol (Cchol) at the indicated times or
by UTP for 30 s.
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Fig. 7.
Relocation of Ins(1,4,5)P3
3-kinase before and after enzyme activation in intact astrocytes.
A, homogenates were prepared from 1321N1 quiescent cells and
from the cells stimulated by 10 µM carbachol for 30 s. Soluble and particulate fractions were separated by centrifuging at
100,000 × g for 1 h. The particulate fractions
were solubilized in 1% Triton X-100 for 1 h at 4 °C. The
soluble and the solubilized particulate fractions (1 ml) were purified
by CaM-Sepharose, and the active fractions eluted in the presence of 2 mM EGTA/1% Triton X-100 were concentrated. Enzyme activity
was assayed at 5 µM Ins(1,4,5)P3.
B, concentrated fractions issued from CaM-Sepharose
purification of soluble (1, 3) and particulate
(2, 4) fractions of quiescent (1,
2) and stimulated (3, 4) 1321N1 cells
were separated by SDS-PAGE and immunodetected in the presence of the
anti-(human Ins(1,4,5)P3 3-kinase B) antibodies dressed
against the C-terminal peptide (dilution 1/750). The band with an
apparent molecular weight of 88,000 is indicated by an
arrow.
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Fig. 8.
CaM dependence of astrocytic
Ins(1,4,5)P3 3-kinase activity before and after
phosphorylation in intact cells. Rat cortical astrocytes were
incubated in the presence ( ) or absence (
) of 10 µM
carbachol for 30 s at 37 °C. Enzymic activity of homogenates
was assayed at 5 µM Ins(1,4,5)P3 and 10 µM free Ca2+ with increasing concentrations
of CaM (0-2 µM). Results are means of triplicates ± S.D.
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Fig. 9.
Effect on enzymic activity and stoichiometry
of phosphate incorporation related to in vitro
phosphorylation of astrocytic Ins(1,4,5)P3 3-kinase
by CaM kinase II and PKC. A, purified rat astrocytic
Ins(1,4,5)P3 3-kinase (100 ng) was preincubated during 5 min at 37 °C and pH 7.4 in the presence or absence of 2 ng of
protein kinases (CaM kinase II or PKC). Free Ca2+ and CaM
were at 10 and 2 µM, respectively. Phosphatidylserine and
1,2-diacylglycerol were at 0.25 mg/ml and 20 µg/ml, respectively.
After incubation, samples were diluted 1000-10,000-fold before assay
of enzymic activity at 5 µM Ins(1,4,5)P3. In
the case of preincubation in the absence of any agent, enzymic activity
was also assayed in the presence of 10 µM
Ca2+ and 0.1 µM CaM (black column).
PS, DAG, and CaMKII are for
phosphatidylserine, 1,2-diacylglycerol, and CaM kinase II,
respectively. Results are means of triplicates ± S.D.
B, CaM kinase II and/or PKC-catalyzed 32P
incorporation into astrocytic Ins(1,4,5)P3 3-kinase has
been measured by phosphorylating enzyme (0.5 µg) with CaM kinase II
(10 ng) (×) or PKC (10 ng) ( ) and 40 µM
[
-32P]ATP (final activity ~500 µCi/ml) for various
times (0-10 min) in the presence of 2 µM CaM and 10 µM free Ca2+ or 0.20 mg/ml phosphatidylserine
and 20 µg/ml 1,2-diacylglycerol, respectively. The phosphorylation
reaction was also performed with both protein kinases and related
cofactors together (
). The autophosphorylation state of CaM kinase
II (
) and PKC (
) in the presence of their cofactors was estimated
in the absence of 3-kinase. After each incubation time, the enzyme was
precipitated onto P81 phosphocellulose with 75 mM
phosphoric acid before counting radioactivity. Results are means of
triplicates ± S.D.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Regulatory properties of Ins(1,4,5)P3 3-kinase isoforms A and B
As shown in the present study, regulation mechanisms of astrocytic Ins (1,4,5)P3 3-kinase B are more complex than for neuronal Ins (1,4,5)P3 3-kinase A, since it requires at least a dual phosphorylation by two distinct protein kinases, i.e. CaM kinase II and PKC, and so phosphorylation at multiple sites. CaM kinase II and PKC are multifunctional enzymes catalyzing phosphorylation of many proteins, presenting a wide tissue distribution and being particularly abundant in brain, e.g. in astrocytic cells (40-42). CaM kinase II is a major mediator of dynamically organized Ca2+ oscillations and actions. Intracellular distributions of the signaling by CaM kinase II and Ca2+ were studied in the same astrocytes (43). These results indicated that local spatio-temporal Ca2+ signals induced protein phosphorylation (e.g. vimentin) by CaM kinase II in the same cellular compartment in astrocytes and that Ca2+ waves induced global protein phosphorylation by CaM kinase II. The abundance of total PKC isoforms has been reported in rat astrocytes and phorbol esters cause a rapid translocation of PKC activity from cytosol to membrane compartments in this cell system (44, 45). Interestingly, Ras-GAP1IP4BP presents a specific mRNA distribution in rat brain, since it is highly expressed in the hippocampus and cerebellum, specially in neurons and oligodendrocytes, but not in astrocytes (46). This is compatible with cell type-specific roles of the second messenger Ins (1,3,4,5)P4, for which our data suggest its levels are controlled by distinct isoenzymes of Ins (1,4,5)P3 3-kinase presenting a specific cellular and subcellular distribution, as well as specific regulation mechanisms by phosphorylation.
Ins (1,4,5)P3 3-kinase A was phosphorylated in vitro and in vivo by CaM kinase II at the level of one unique residue, i.e. Thr-311 (in the primary structure of the human isoenzyme). This residue is one of the two predicted phosphorylation consensus sites for CaM kinase II previously identified in the sequence of Ins (1,4,5)P3 3-kinase A (i.e. Arg-Ala-Val-Thr311 for consensus site Arg/Lys-X-X-Ser/Thr; Refs. 15 and 47) (27). Interestingly, this residue is conserved in the primary structure of human and rat Ins (1,4,5)P3 3-kinase B (17-19), suggesting the novel phosphorylation mechanism presented here to involve at least this common residue as target for CaM kinase II-mediated phosphorylation and enzyme activation. Many phosphorylation consensus sites for PKC are found in the primary structures of rat and human Ins (1,4,5)P3 3-kinase B: 10 sites in the human sequence (i.e. Ser-8, Ser-71, Ser-184, Ser-216, Ser-278, Ser-332, Ser-391, Ser-407, Ser-423, and Ser-508) and 8 sites in the rat sequence (i.e. Ser-8, Ser-172, Ser-315, Ser-374, Ser-390, Ser-406, Ser-491, and Thr-261). Six of them are conserved between both species (in italic) and only one is conserved in human sequence of isoform A (in bold). We showed here that Ins (1,4,5)P3 3-kinase B is not only a target of CaM kinase II, it is also a substrate of PKC to observe maximal enzyme activation in intact astrocytic cells. Recombinant rat Ins (1,4,5)P3 3-kinase B has been shown to be a substrate of PKC and PKA in vitro; both protein kinases provoked a decrease in enzymic activity in the presence of the Ca2+·CaM complex (26). Our data in intact astrocytes with TPA and calphostin C revealed that the PKC pathway did not provoke any enzyme inhibition. Instead, maximal phosphorylation and activation of Ins (1,4,5)P3 3-kinase B was observed in the presence of PKC and CaM kinase II. Our results indicated also that the effect of PKC may require prior phosphorylation by CaM kinase II to mediate maximal activation of Ins (1,4,5)P3 3-kinase B in astrocytes. This is supported by the inhibitory effect of KN-62 and KN-93 inhibitors on the TPA-induced phosphorylation and activation of astrocytic Ins (1,4,5)P3 3-kinase, as well as the in vitro PKC-induced activation of the enzyme, which is observed only in the presence of CaM kinase II. In vitro experiments indicated that CaM kinase II and PKC provoke the phosphorylation of one and two phosphorylation sites on astrocytic Ins (1,4,5)P3 3-kinase, respectively. Concerning PKC phosphorylation of Ins (1,4,5)P3 3-kinase B, this result is in agreement with previously reported in vitro experiments where at least two sites were labeled (26). Forskolin did not induce any change in Ins (1,4,5)P3 3-kinase activity or 32P incorporation in cortical astrocytes or 1321N1 cells, indicating that PKA does not seem to be a physiological effector of isoenzyme B.
As for Ins (1,4,5)P3 3-kinase isoenzymes in this study,
specific cell distribution has also been demonstrated for Ins
(1,4,5)P3 receptor isoforms in brain. Immunohistochemical
and biochemical studies in rat central nervous system indicated that
Ins (1,4,5)P3-induced Ca2+ release is mediated
by receptor isoform type 1 in neuronal cells, whereas it is mainly
directed by receptor isoforms 2 and/or 3 in astrocytes (48, 49).
Molecular heterogeneity generating specific regulatory mechanisms and
subcellular distributions of PLC, Ins (1,4,5)P3 receptor
and Ins (1,4,5)P3 3-kinase isoforms may be considered as
critical parameters involved in the complex mechanisms that underlie
Ca2+ wave formation and propagation in brain cells,
e.g. astrocytes. Since no regulatory mechanism involving
Ca2+ has been demonstrated for Ins (1,4,5)P3
5-phosphatase type I, the metabolic pathway through highly regulated
Ins (1,4,5)P3 3-kinase may be the critical route for
Ca2+-controlled Ins (1,4,5)P3 and Ins
(1,3,4,5)P4 levels and actions.
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ACKNOWLEDGEMENTS |
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We thank Dr. P. Gourlet (Laboratoire de Chimie Biologique et Nutrition, , Free University of Brussels, Brussels, Belgium) for peptide synthesis. We are grateful to Dr. Serge Schiffman for helpful discussions. We thank Roberte Menu for experimental help in first preparations of cortical astrocytes. Rat brain PKC was a generous gift from Dr. Mark H. Rider (Institut de Pathologie Cellulaire, Université Catholique de Louvain, Brussels, Belgium).
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FOOTNOTES |
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* This work was supported in part by grants from Actions de Recherche Concertées, Fonds de la Recherche Scientifique Médicale, EU Biomed 2 Program BMH4-CT-972609 and by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Federal Service for Science, Technology and Culture.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.
Chargé de Recherches at the Fonds National pour la Recherche
Scientifique. To whom correspondence should be addressed. Tel.: 32-2-555-41-64; Fax: 32-2-555-46-55; E-mail: dcommuni{at}ulb.ac.be.
§ Fellow of the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture.
2 V. Dewaste, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Ins (1, 4,5)P3, D-myo-inositol 1,4,5-trisphosphate; Ins (1, 3,4,5)P4, D-myo-inositol 1,3,4,5-tetrakisphosphate; GAP, GTPase-activating protein; PKC, protein kinase C: PKA, protein kinase A; CaM, calmodulin; CaM kinase, Ca2+·CaM-dependent protein kinase II; PAGE, polyacrylamide gel electrophoresis; TPA, 12-O- tetradecanoylphorbol-13-acetate.
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