Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor by
Cyclic Nucleotide-dependent Kinases in Vitro
and in Rat Cerebellar Slices in Situ*
Lise Sofie
Haug,
Vidar
Jensen
,
Øivind
Hvalby
,
S. Ivar
Walaas, and
Anne Carine
Østvold§
From the Neurochemical Laboratory, P. O. Box 1115 Blindern and the
Department of Neurophysiology, P. O. Box 1104 Blindern,
Department Group of Basic Medical Sciences, University of Oslo,
N-0317 Oslo, Norway
 |
ABSTRACT |
We have examined cyclic nucleotide-regulated
phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate
(IP3) receptor immunopurified from rat cerebellar
membranes in vitro and in rat cerebellar slices in
situ. The isolated IP3 receptor protein was phosphorylated by both cAMP- and cGMP-dependent protein
kinases on two distinct sites as determined by thermolytic
phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and
phosphopeptide 2, representing Ser-1756 in the rat protein (Ferris,
C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. (1991) Biochem. Biophys. Res.
Commun. 175, 192-198). Phosphopeptide maps show that
cAMP-dependent protein kinase (PKA) labeled both sites with
the same time course and same stoichiometry, whereas
cGMP-dependent protein kinase (PKG) phosphorylated Ser-1756
with a higher velocity and a higher stoichiometry than Ser-1589.
Synthetic decapeptides corresponding to the two phosphorylation sites
(peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)) were used to determine kinetic
constants for the phosphorylation by PKG and PKA, and the catalytic
efficiencies were in agreement with the results obtained by in
vitro phosphorylation of the intact protein. In cerebellar slices
prelabeled with [32P]orthophosphate, activation of
endogenous kinases by incubation in the presence of cAMP/cGMP analogues
and specific inhibitors of PKG and PKA induced in both cases a 3-fold
increase in phosphorylation of the IP3 receptor.
Thermolytic phosphopeptide mapping of in situ labeled
IP3 receptor by PKA showed labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the
findings in vitro, PKG preferentially phosphorylated
Ser-1589 in situ. Because both PKG and the IP3
receptor are specifically enriched in cerebellar Purkinje cells, PKG
may be an important IP3 receptor regulator in
vivo.
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INTRODUCTION |
Activation of intracellular signal transduction cascades
frequently involves increased phosphoinositide hydrolysis following stimulation of phospholipase C. Inositol 1,4,5-trisphosphate
(IP3),1 a second
messenger produced by phosphoinositide hydrolysis, mediates Ca2+ release from intracellular stores by binding to
IP3-sensitive Ca2+ channels, thereby increasing
their "open" probability (1). IP3 receptors derive from
at least three different genes, constituting types I, II, and III,
which are approximately 70% identical at the amino acid level but
differ in distribution and regulation (reviewed in Refs. 1-3). An
assembly of four 260-kDa subunits forms the receptor. Each subunit
consists of a cytoplasmic, amino-terminal IP3 binding
domain, a coupling domain, and a Ca2+ channel pore of six
transmembrane segments (2-4). Type I is further diversified by
alternative RNA splicing, resulting in two main forms, of which the
longest (SII+, containing the 40 amino acid residues 1693-1732) is
specifically expressed in neurons (2). One or more IP3
receptor forms have been found in virtually all cell types examined
(reviewed in Ref. 2), but particularly high amounts of type I
IP3 receptor are seen in smooth muscle cells and in
cerebellar Purkinje neurons. Calcium release mediated by
IP3 receptors appears to be an essential step for the
induction of long term depression (LTD) in Purkinje cells (5).
A number of different mechanisms modulates IP3 receptor
function, including binding of ATP, fatty acids, and calcium (reviewed in Ref. 2); a number of neurodegenerative processes (6, 7); and
phosphorylation of the IP3 receptor by specific protein kinases. cAMP-dependent protein kinase (PKA) phosphorylates
the type I IP3 receptor both in vitro and
in vivo (8-11) and has also been reported to phosphorylate
type II and III in intact cells (12).
Ca2+/calmodulin-dependent protein kinase II,
protein kinase C and the tyrosine kinase Fyn have also been reported to
phosphorylate the type I IP3 receptor (13-17). In
addition, the receptor may undergo autophosphorylation (18). Early work
indicating that the neuronal IP3 receptor (SII+) can be
phosphorylated by cGMP-dependent protein kinase (PKG) (8)
was later confirmed by in vitro experiments (19-21).
Likewise, the nonneuronal type I IP3 receptor (SII
) found in smooth muscle cells, also termed the G0 protein (22,
23), is a substrate for phosphorylation by both PKA (10) and PKG (19,
20, 23-25). Recent reports of IP3 receptor phosphorylation by PKA and PKG in hepatocytes (26-28), kidney cells (11), and platelets (29, 30) support these observations.
Phosphorylation of the IP3 receptor by PKA and PKG
represents a possible mechanism for cross-talk whereby cyclic
nucleotides can modulate IP3-mediated regulation of
Ca2+ levels (20, 31). Because cAMP and cGMP levels in most
cells are regulated by various extracellular signals, identification of
phosphorylation sites labeled by these kinases is of interest. Amino
acid sequencing indicated that PKA phosphorylates the rat neuronal
receptor on Ser-1756 and, less efficiently, on Ser-1589 (9). Similarly,
PKG appears to phosphorylate mainly Ser-1756 in vitro (19).
In contrast, the smooth muscle IP3 receptor is preferentially phosphorylated by PKA on Ser-1589 in vitro
(10), whereas Ser-1756 is more prominently phosphorylated by PKA in kidney cells in vivo (11). Hence, the preferred substrate
sites for PKA-mediated phosphorylation of the IP3 receptor
differ according to receptor isoforms and tissues, and the factors
determining these responses remain unclear. Moreover, IP3
receptor phosphorylation by PKG in neuronal cells has not been
characterized in detail.
In the present study, we have examined in vitro
phosphorylation catalyzed by the cyclic
nucleotide-dependent protein kinases both of the
immunoisolated cerebellar IP3 receptor and of synthetic peptides corresponding to the phosphorylation sites. We have further characterized the in situ phosphorylation of the
IP3 receptor in cerebellar slices stimulated with cyclic
nucleotide analogues. The results confirm earlier reports stating that
the cerebellar IP3 receptor can be efficiently
phosphorylated by PKA and PKG on two distinct sites in
vitro. In addition, we show that both PKA and PKG can
phosphorylate the cerebellar IP3 receptor in
situ on the same two sites, albeit with distinct time courses and kinetics.
Some of these data have been reported in abstract form (21,
32).
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EXPERIMENTAL PROCEDURES |
Materials--
[
-32P]ATP was from ICN (Irvine,
CA). [32P]Orthophosphate, 10 mCi/ml, was from Amersham
Pharmacia Biotech.
8-(4-para-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) and 8-pCPT-cAMP were from BioLog Life Science Institute (Bremen, Germany). KT 5720 and KT 5823 were from
Calbiochem-Novabiochem Corp. Protein A-Sepharose beads were from
Amersham Pharmacia Biotech. Wistar rats were from Møllegaard Breeding
Center (Ejby, Denmark). cGMP-dependent protein kinase, purified from bovine lung, was a gift from Dr. Suzanne Lohmann (University of Würzburg, Würzburg, Germany).
Thermolysin, the catalytic subunit of PKA, the heat-stable inhibitor of
PKA (Walsh inhibitor), and EPPS were from Sigma. Other reagents, of
analytical grade or better, were from standard commercial suppliers.
Preparation of Peptides--
A synthetic peptide comprising the
carboxyl-terminal 18 amino acid residues of the mouse IP3
receptor (33), used to raise antibodies to the IP3 receptor
(6, 7, 34), and two 10-amino acid peptides (peptide 1, with the
sequence AARRDSVLAA, corresponding to residues 1584-1593, and peptide
2, SGRRESLTSF, corresponding to residues 1751-1760) were synthesized
at the Biotechnology Center, University of Oslo. A 30-amino acid
synthetic peptide, termed D32-((Ser-34)8-38), encompassing amino acids
8-38 from DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa) in which the serine residue at position 34 is efficiently
phosphorylated by both PKA and PKG (35, 36), was kindly provided by Dr.
H. C. Hemmings, Jr. (Cornell Medical School, New York, NY).
Immunopurification of the IP3 Receptor for in Vitro
Phosphorylation Assays--
The IP3 receptor was isolated
from rat cerebellum homogenized in 10 volumes of ice-cold buffer
containing 0.32 M sucrose, 20 mM EPPS (pH 8.5),
1 mM EDTA, and the protease inhibitors phenylmethylsulfonyl fluoride (0.1 mM) and leupeptin (10 µg/ml).
Centrifugation and extraction with 1% (w/v) deoxycholate was performed
as described previously (34). The resulting supernatant (containing
approximately 5 mg/ml protein) is referred to as the deoxycholate
extract. Protein A-Sepharose beads were washed and incubated with
anti-IP3 receptor antiserum and subsequently with
deoxycholate extract (diluted 1:3 in TBS-Tween) as described (34), and
the final immunocomplexes were isolated by centrifugation and washing
of the beads.
Phosphorylation of the IP3
Receptor--
Immunopurified IP3 receptor bound to protein
A-Sepharose was phosphorylated in the presence of exogenous protein
kinases. Phosphorylation with the catalytic subunit of PKA was
performed at 30 °C in 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 1 mM EGTA, 10 µM 8-Br-cAMP, the catalytic subunit of PKA (0.5 µM), and phosphatase inhibitors (including 6 mM p-nitrophenylphosphate, 12 mM
-glycerophosphate, and 0.02 mM sodium vanadate).
Phosphorylation by PKG was performed in the same buffer but with 10 µM 8-Br-cGMP instead of 8-Br-cAMP, with the purified
holoenzyme of PKG (0.16 µM) instead of PKA, and with
addition of 10 µM peptide inhibitor of PKA (Walsh
inhibitor). Phosphorylation was initiated by addition of
[
-32P]ATP (final concentration, 8.5 µM,
containing 12 Ci/mmol) and terminated after various incubation times
(1-20 min) by addition of 40 mM EDTA and washing twice in
TBS-Tween. Following boiling of the protein A-Sepharose beads in
SDS-containing stop solution (37), proteins were separated by
SDS-PAGE, the protein content of Coomassie-stained gel bands was
analyzed densitometrically, and phosphoproteins were visualized by
autoradiography. For quantitation, bands of interest were located
with the autoradiograms as guides and excised from the gels.
Radioactivity was measured by Cerenkov counting.
For analysis of sequential addition of the two cyclic
nucleotide-dependent protein kinases, initial
phosphorylation of the immunoisolated IP3 receptor was
performed as described above, but employing nonradioactive ATP (8.5 µM) for 20 min. Following washing of the beads (twice) in
TBS-Tween, the second incubation was initiated by addition of exogenous
kinase and [
-32P]ATP (8.5 µM, 12 Ci/mmol). The reaction was terminated after 5 min by EDTA, and the
samples were washed and analyzed by SDS-PAGE as described above.
Thermolytic Digestion and Phosphopeptide Mapping of
32P-Labeled Protein--
Following SDS-PAGE, the
32P-labeled gel bands containing the IP3
receptor were detected by autoradiography, excised from the gels,
washed, and subjected to proteolysis with thermolysin (1 mg/ml) for
24 h at 37 °C. The thermolytic phosphopeptides were separated
by electrophoresis and ascending chromatography as described (38), and the resulting phosphopeptide maps were visualized by autoradiography.
Phosphorylation of Synthetic IP3 Receptor
Peptides--
The two synthetic decapeptides (peptide 1, residues
1584-1593, and peptide 2, residues 1751-1760) were incubated in a
buffer containing 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.2 mM
[
-32P]ATP (35 mCi/mmol), bovine serum albumin (0.5 mg/ml), and 10-200 µM peptide dissolved in water.
Phosphorylation was initiated by addition either of the catalytic
subunit of PKA (final concentration, 12 nM) or PKG
holoenzyme (final concentration, 4 nM) plus 10 µM 8-Br-cGMP. Incubations were carried out for 5 min at
30 °C, and the reactions were terminated by spotting aliquots onto
P81 phosphocellulose paper (Whatman) and washing in phosphoric acid (75 mM). Phosphorylations were quantified by Cerenkov counting
of the filter papers (39), and kinetic constants were derived from
Hanes-Woolf plots.
Preparation and Incubation of Rat Cerebellar Slices--
Wistar
rats (100-200 g) were sacrificed with halothane, and the cerebellum
was quickly removed and cooled in artificial cerebrospinal fluid (ACSF)
at 0-4 °C of the following composition: NaCl, 124 mM;
KCl, 2 mM; KH2PO4, 1.25 mM; MgSO4, 2 mM;
NaHCO3, 26 mM; glucose, 10 mM;
bubbled with 95% O2/5% CO2 (pH 7.4). The
hemispheres were glued to a mounting block, and sagittal slices (400 µm) were cut with a vibroslice in cold oxygenated ACSF. The slices
were placed in an interface chamber exposed to humidified gas and
maintained in ACSF at a temperature of 25-27 °C for at least 2 h. After equilibration, the slices were carefully transferred to wells
(12-well cell culture cluster, Costar) containing 500 µl of ACSF,
where incubations with different reagents were performed in an
O2-enriched atmosphere at room temperature. The slices were
continuously kept in calcium-free solutions to diminish disturbances
induced by calcium influx, e.g. proteolysis by calpains
(34), protein kinase C or
Ca2+/calmodulin-dependent protein kinase II
phosphorylation (12-15, 30), and protein phosphatase 2B
dephosphorylation (40). Incubations were terminated by removal of the
reaction buffer and addition of 400 µl of ice-cold buffer (containing
0.25 M sucrose, 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and the phosphatase inhibitors
-glycerophosphate (60 mM), sodium vanadate (0.1 mM) and sodium fluoride (0.1 mM)) and
homogenized by sonication. Following centrifugation at 1,000 × g for 15 min at 4 °C, deoxycholate was added to the
supernatant (1% w/v, final concentration), and extraction on ice for
30-45 min was followed by centrifugation for 30 min at 27,000 × g. The resulting supernatant was incubated with protein
A-Sepharose beads preincubated with antiserum to immunoisolate the
IP3 receptor as described above.
In Situ Phosphorylation of the IP3 Receptor in Rat
Cerebellar Slices--
Cerebellar slices were incubated with 625 µCi
of [32P]orthophosphate and cyclic nucleotide analogues
(final concentration, 3.2 mM 8-pCPT-cAMP or 1.6 mM 8-pCPT-cGMP) after equilibration in phosphate-free ACSF.
The specific PKA inhibitor KT 5720 (final concentration, 2.5 µM) or the PKG inhibitor KT 5823 (final concentration,
1.6 µM) was present during the
[32P]orthophosphate incubation of control slices and was
added to the other slices 20 min prior to addition of cyclic
nucleotide analogues (20). Reactions were terminated by
homogenization of the slices as described above, and immunoisolation of
the IP3 receptor was followed by SDS-PAGE. Incorporation of
32P into the IP3 receptor was visualized by
autoradiography and quantified by densitometry and Cerenkov
counting. The phosphoprotein was further characterized by thermolytic
peptide mapping as described above.
For the back-phosphorylation experiments, immunopurified samples from
cerebellar slices that had been incubated with protein kinase
inhibitors and cyclic nucleotide analogues in phosphate-containing ACSF
as described above were in vitro phosphorylated at room
temperature for 20 min using exogenous PKA and
[
-32P]ATP. The reactions were terminated by EDTA and
washing in TBS-Tween, followed by SDS-PAGE, as described above.
Miscellaneous Methods--
SDS-PAGE was performed using the
buffers of Laemmli (37). Protein content was analyzed by the
bicinchoninic acid method (41). When phosphorylation stoichiometry was
examined, the amount of IP3 receptor in the sample was
quantified by densitometry of the gels following staining with
Coomassie Blue, using standard amounts of bovine serum albumin in the
same gels to construct standard curves. Images of autoradiograms were
prepared using a Hewlett-Packard ScanJet IIC/ADF scanner, and Desk Scan
II (version 1.0, Hewlett-Packard) together with Adobe Photoshop 2.5 or
Corel 4.0 software. Statistical analysis and Hanes-Woolf-plots were obtained using Graph-Pad Prism 2.01.
 |
RESULTS |
Phosphorylation of the Cerebellar IP3 Receptor in
Vitro--
Previous work has shown that the ~260 kDa (apparent from
SDS-PAGE) protein band phosphorylated by PKA in cerebellum mostly represents the IP3 receptor (8, 34, 42). When the
IP3 receptor, extracted from rat cerebellar membranes and
immunoprecipitated with rabbit antibodies raised against the
carboxyl-terminal end of the brain type I IP3 receptor, was
used as substrate for PKA and PKG in vitro (Fig.
1A), we found similar
phosphorylation reactions. PKA rapidly phosphorylated the
immunoisolated IP3 receptor during 5-min incubations
(Fig. 1B); longer incubation produced only marginal additional effects (not shown). PKG phosphorylated the
immunoisolated IP3 receptor more slowly and to a lower
maximum level (Fig. 1B).

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Fig. 1.
Phosphorylation of immunoisolated rat
cerebellar IP3 receptor by PKA and PKG. A,
immunopurified IP3 receptor was incubated with
[ -32P]ATP in the presence of 0.5 µM
PKA plus 10 µM 8-Br-cAMP or 0.16 µM PKG
plus 10 µM 8-Br-cGMP for 0 (a), 1 (b), 2 (c), 5 (d), and 10 (e) min. Following termination of the reactions, the
proteins were separated by SDS-PAGE, and the 32P-labeled
proteins were visualized by autoradiography. B, time courses
of the in vitro phosphorylation of immunopurified
IP3 receptor. Phosphorylation was quantified by
densitometry of the autoradiograms shown in A. Left
panel, PKA; right panel, PKG; IOD,
integrated optical density.
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To estimate the extent of phosphorylation, 0.60 µg of IP3
receptor was phosphorylated for 5 min. Employing the predicted
molecular mass of 313 kDa for the immunopurified receptor subunit (33), our results indicated that PKA phosphorylated the immunoisolated IP3 receptor to a stoichiometry of 1.02 mol of phosphate
per mol of protein, whereas PKG phosphorylated the IP3
receptor to a stoichiometry of 0.48 mol of phosphate per mol of protein
under these conditions (see also Fig. 4, a and
b).
Site-specific Phosphorylation of the Immunopurified IP3
Receptor--
The phosphorylated domains of the IP3
receptor were characterized by phosphopeptide mapping. When the
phosphorylated protein was subjected to extensive thermolytic digestion
and peptide separation in two dimensions on silica plates, two major
(phosphopeptides 1 and 2) and two minor phosphopeptides were seen
following incubation with PKA (Fig.
2A). In contrast, incubation
with PKG resulted in one major and one minor phosphopeptide only; the
former comigrated with phosphopeptide 2, and the latter comigrated with
phosphopeptide 1 (Fig. 2B). Quantitation of the
32P-labeled phosphopeptides showed that PKA induced a rapid
phosphorylation of both the major phosphorylation sites and that these
two phosphorylation sites were phosphorylated to the same extent during
the different incubation times (not shown). PKG induced a rapid
phosphorylation of site 2 but a more protracted and less complete
phosphorylation of site 1 as a function of time (Fig.
3). This is in accordance with the time
course for the phosphorylation of the isolated protein, shown in Fig.
1B.

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Fig. 2.
Two-dimensional phosphopeptide maps of
in vitro phosphorylated IP3 receptor.
Immunoisolated IP3 receptor was phosphorylated for 5 min
with [ -32P]ATP and PKA (A),
[ -32P]ATP and PKG (B), or PKG alone for 20 min with nonradioactive ATP followed by PKA with
[ -32P]ATP for 5 min (C). After separation
by SDS-PAGE, the bands corresponding to the IP3 receptor
were excised from the gel and subjected to complete proteolytic
digestion with thermolysin. The phosphopeptides generated were
separated by electrophoresis (pH 3.5) in the first dimension and
chromatography in the second dimension and visualized by
autoradiography. Phosphopeptides (1 and 2) and
application points ( ) are indicated.
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Fig. 3.
Time course of site-specific IP3
receptor phosphorylation by PKG. Immunoisolated IP3
receptor was phosphorylated by PKG plus 8-Br-cGMP and
[ -32P]ATP for 1 (A), 2 (B), and
5 (C) min. After separation by SDS-PAGE, the IP3
receptor was visualized by autoradiography, excised from the gel, and
subjected to proteolytic digestion with thermolysin. The
phosphopeptides generated were separated and visualized as described in
the legend to Fig. 2. Phosphopeptides (1 and 2)
and application points ( ) are indicated.
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Additive Phosphorylation of the Immunopurified IP3
Receptor--
The relation between the serine residues phosphorylated
by PKA and PKG was further examined by phosphorylating the isolated IP3 receptor with sequential addition of the two kinases.
Incubation with nonradioactive ATP in the presence of PKA for 20 min
prevented the protein from subsequent phosphorylation with
[
-32P]ATP in the presence of PKG (Fig.
4). In contrast, incubation of the
IP3 receptor with nonradioactive ATP and PKG for 20 min allowed the protein to become further phosphorylated by subsequent addition of [
-32P]ATP in the presence of PKA, with the
final 32P labeling representing approximately half of that
obtained by PKA alone (Fig. 4). Thermolytic phosphopeptide mapping of
this sample showed that incubation with nonradioactive ATP and PKG led
to a considerable decrease in the subsequent PKA-catalyzed 32P labeling of phosphorylation site 2, but not site 1 (Fig. 2C).

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Fig. 4.
Additive phosphorylation of the
IP3 receptor by PKA and PKG. Immunoisolated
IP3 receptor was phosphorylated for 5 min in the presence
of [ -32P]ATP by the catalytic subunit of PKA
(a) and PKG plus 8-Br-cGMP (b) as described. In
c, the IP3 receptor was phosphorylated for 20 min with nonradioactive ATP in the presence of PKA, followed by
phosphorylation for 5 min with [ -32P]ATP in the
presence of PKG plus 8-Br-cGMP. In d, the IP3
receptor was phosphorylated for 20 min with nonradioactive ATP in the
presence of PKG plus 8-Br-cGMP, followed by phosphorylation for 5 min
with [ -32P]ATP in the presence of PKA. Following
incubations and separation by SDS-PAGE, the amount of IP3
receptor was quantified by densitometry of the Coomassie-stained gels,
and the amount of 32P-labeled IP3 receptor was
measured by Cerenkov counting of the excised bands. The bars represent
mol of incorporated phosphate per mol of IP3 receptor
protein, calculated on basis of a predicted molecular mass of 313 kDa
per subunit. In repeated experiments, essentially identical results
were obtained.
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Taken together, the phosphopeptide mapping of the IP3
receptor indicates that PKA catalyzed in vitro
phosphorylation of at least two residues within 5 min, whereas PKG
catalyzed phosphorylation of one residue within the first minute, with
a subsequent and slower phosphorylation of the second residue (Fig. 3).
Moreover, the residues phosphorylated by PKA and PKG are identical.
Phosphorylation of Synthetic IP3 Receptor
Peptides--
PKA has been shown to phosphorylate Ser-1756 and, less
efficiently, Ser-1589, in the neuronal IP3 receptor
in vitro (9). To confirm that the same two serines are
phosphorylated by PKG and to study the kinetics and phosphorylation
efficiency of these in vitro phosphorylation reactions, we
used two synthetic decapeptides as substrates for PKA and PKG: peptide
1, encompassing Ser-1589 (AARRDSVLAA), and peptide 2, encompassing
Ser-1756 (SGRRESLTSF) (10, 19). The results were compared with those
obtained with a 30-amino acid peptide derived from DARPP-32,
D32-((Ser-34)8-38), which is known to represent a good substrate for
both cyclic nucleotide-dependent kinases (36). The
reactions followed Michaelis-Menten kinetics at low substrate
concentrations, but exhibited substrate inhibition at higher substrate
concentrations (not shown). Hanes-Woolf analysis indicated that peptide
1, containing Ser-1589, was phosphorylated with the highest
Vmax by both PKG and PKA (Table
I). In contrast, peptide 2, containing
Ser-1756, was phosphorylated by both PKA and PKG with relatively low
Vmax, but with a lower Km, indicating that both kinases have higher affinity for peptide 2 in vitro. PKA, which phosphorylated sites 1 and 2 of the
immunopurified protein to a similar extent (Fig. 2A),
phosphorylated the synthetic peptides with almost equal catalytic
efficiencies (Kcat/Km; Table
I). PKG, which phosphorylated site 2 of the immunopurified protein more
extensively than site 1 (Figs. 2B and 3), phosphorylated the
synthetic peptide 2 with a higher catalytic efficiency than the
synthetic peptide 1 (Table I). The kinetic data were thus in agreement
with the in vitro phosphorylation of the intact
IP3 receptor. The DARPP-32 peptide showed kinetic
properties similar to those of peptide 2 for both PKA and PKG (Table
I).
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Table I
Kinetic data concerning the phosphorylation, catalyzed by PKA and PKG,
of synthetic peptides 1 and 2 from the IP3 receptor and of the
D32 peptide (D32-((Ser-34)8-38) from DARPP-32
Calculations of the kinetic constants are based on Hanes-Woolf plots.
The results are mean of triplicate determinations. The experiment was
performed three times with essentially identical results.
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Phosphorylation of IP3 Receptor in Situ in Rat
Cerebellar Slices--
It was of interest to ascertain that these
kinases are able to phosphorylate the IP3 receptor in
neurons in situ. Because the IP3 receptor is
specifically abundant in Purkinje neurons of the rat cerebellum (2), we
utilized cerebellar slice preparations to study these neurons. The
slices were incubated for 2-2.5 h in ACSF before stimulation to ensure
equilibration of physiological processes (43). Preparation and
incubation of slices were in accordance with previously published
techniques for electrophysiological slice experiments (44).
Incorporation of [32P]Orthophosphate into Stimulated
Slices from Rat Cerebellum--
Rat cerebellar slices were
preincubated in [32P]orthophosphate, followed by addition
of 8-pCPT-cGMP or 8-pCPT-cAMP together with the specific PKA and PKG
inhibitors, KT 5720 and KT 5823, respectively. Immunopurification of
the IP3 receptor and quantitation of the radioactive gel
bands revealed a considerable increase in phosphate incorporation (Fig.
5a) in stimulated slices
versus control slices. Phosphopeptide mapping showed the
extent to which both the phosphopeptides were phosphorylated (Fig.
5b). In slices treated with PKA inhibitor alone (Fig.
5b, A), the IP3 receptor was weakly
phosphorylated. Stimulation of the slices with 8-pCPT-cAMP resulted in
preferential incorporation of [32P]orthophosphate in
phosphopeptide 2 (Ser-1756; Fig. 5b, B). However, as opposed
to the in vitro observations, stimulation of slices with
8-pCPT-cGMP led to preferential incorporation of 32P in
phosphopeptide 1 (Ser-1589; Fig. 5b, C).

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Fig. 5.
Incorporation of
[32P]orthophosphate into IP3 receptor in
rat cerebellar slices. Rat cerebellar slices preincubated with
[32P]orthophosphate for 90 min and the specific protein
kinase inhibitors KT 5720 (PKA inhibitor) and KT 5823 (PKG inhibitor)
for 20 min were stimulated to induce phosphorylation of the
IP3 receptor. Following stimulation, the slices were
homogenized, the IP3 receptor was immunoisolated, and
SDS-PAGE and phosphopeptide mapping were performed as described under
"Experimental Procedures." The experiment was performed three times
with similar results. a, bar graph representing incorporated
radioactivity per amount of protein in the IP3 receptor gel
band. Each bar represents the mean ± S.D. of two
slices, relative to the control slices. A, control slices,
incubated only with KT 5720 throughout the 32P prelabeling;
B, slices incubated with KT 5823 followed by 8-pCPT-cAMP for
10 min; C, slices incubated with KT 5720 followed by
8-pCPT-cGMP for 10 min. b, phosphopeptide mapping of the gel
bands illlustrated in a. Phosphopeptides (1 and
2) and application points ( ) are indicated.
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Back-phosphorylation of IP3 Receptor from Stimulated
Cerebellar Slices--
Slices of rat cerebellum (equilibrated for
2.5 h in phosphate-containing ACSF) were stimulated for 10 min
with 8-pCPT-cGMP or 8-pCPT-cAMP in the presence of the specific
inhibitors of PKA and PKG, respectively. Following homogenization and
immunopurification, the IP3 receptor was
back-phosphorylated in vitro with PKA and [
-32P]ATP. The decrease in radioactivity seen when
back-phosphorylated receptor is compared with controls (Fig.
6) represents the increase in in
situ phosphorylation induced by the pharmacological agents applied. Stimulation with 8-pCPT-cGMP reduced back-phosphorylation to
75.5 ± 15.5% of control (p = 0.029, Mann-Whitney
U test),2 and
stimulation with 8-pCPT-cAMP reduced the back-phosphorylation to
75.3 ± 8.1% of control (p = 0.012, Student's
t test).

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|
Fig. 6.
Back-phosphorylation of IP3
receptor from cerebellar slices stimulated in
situ. Rat cerebellar slices were preincubated in
nonradioactive, phosphate-containing ACSF for 2.5 h, followed by
incubation with the PKG inhibitor KT 5823 (control) (a), KT
5823 for 20 min and 8-pCPT-cAMP for 10 min (b), the PKA
inhibitor KT 5720 (control) (c), KT 5720 for 20 min and
8-pCPT-cGMP for 10 min (d). Homogenization of the slices and
immunopurification of the IP3 receptor was followed by
in vitro phosphorylation with exogenous PKA and
[ -32P]ATP for 20 min. Termination of the reaction,
SDS-PAGE, and quantitation of the IP3 receptor was
performed as described. The bars represent mean ± S.D.
of four slices; the experiment was performed three times with similar
results. *, significantly different from control, p < 0.05 (Student's t test); , p < 0.05 (Mann-Whitney U test).
|
|
 |
DISCUSSION |
Phosphorylation of the IP3 receptor has been observed
in different cell types, involving different signaling systems and
protein kinases. In the present work, we show that both PKA and PKG
mediate in vitro phosphorylation of at least two sites in
the neuronal IP3 receptor immunopurified from rat
cerebellum. When the intact neuronal IP3 receptor is
phosphorylated in vitro, low concentrations of PKA label
Ser-1756, whereas high levels of PKA label both Ser-1756 and Ser-1589
(9). In vitro phosphorylation by PKG appears to mimic the
effect of low PKA, because a strong preference for Ser-1756 is observed
(20). In contrast, the smooth muscle IP3 receptor is
phosphorylated by PKA in vitro only on Ser-1589 (10). The smooth muscle G0 protein, later reported to represent an
IP3 receptor (23), is preferentially phosphorylated by
endogenous PKG in vitro on a phosphopeptide comigrating on
phosphopeptide maps with the IP3 receptor phosphopeptide
containing Ser-1589 (25). A proposed cause for the differential
phosphorylation between neuronal and smooth muscle IP3
receptors is the 40-amino acid stretch comprising the alternatively
spliced residues 1693-1732, which are not found in the nonneuronal
IP3 receptor subtype from smooth muscle (SII
).
Under the experimental conditions used in this work, we have found that
the two phosphorylation sites showed distinct kinetic features when
their in vitro phosphorylation was studied by the use of
synthetic peptide substrates. Phosphorylation of synthetic decapeptides
containing the putative phosphorylation sites for PKA and PKG showed
that both enzymes display higher values for Vmax
and Kcat for the Ser-1589-containing peptide 1 than for the Ser-1756-containing peptide 2 (Table I). Despite the lower
maximal rate of phosphorylation, PKG had approximately 4-fold higher
affinity for peptide 2 than peptide 1, whereas PKA exhibited 1.5-fold
higher affinity for peptide 2 compared with peptide 1. Hence, the
catalytic efficiency
(Kcat/Km) of PKG was higher
for peptide 2, indicating a preference for Ser-1756 by this kinase
(Table I), which is in agreement with earlier results (19). However,
the differences in catalytic efficiency were small, and both
IP3 receptor-derived peptides appeared to be phosphorylated
by the two kinases with a higher catalytic efficiency than
D32-((Ser-34)8-38) (Table I). The latter peptide represents the
phosphorylation domain of DARPP-32, a protein that is a substrate for
both PKA (35) and PKG (45) in situ. Thus, from these data we
cannot exclude any of these IP3 receptor peptides as
possible targets for cyclic nucleotide-activated protein kinases
in vivo.
Our in situ data appear to contradict the report from
Komalavilas and Lincoln (19), in which Ser-1589 was proposed not to represent a physiological substrate site for PKG. Our experiments employing [32P]orthophosphate-incorporated cerebellar
slices, which were stimulated with a highly specific cGMP analogue,
showed that the IP3 receptor was phosphorylated mostly on
Ser-1589 in situ (Fig. 5), whereas a cAMP analogue mainly
produced the Ser-1756 phosphopeptide. This difference in the
phosphorylation pattern, which apparently is not caused by distinct
IP3 receptor subtypes, may be due to the properties of the
protein kinases and their localization in the Purkinje cells (46).
These results emphasize the care needed when extrapolation from
in vitro experiments is used to identify physiologically
important processes.
Further evidence for in situ phosphorylation of the
IP3 receptor in Purkinje cells came from
back-phosphorylation experiments. Following both cAMP and cGMP analogue
stimulation, such experiments decreased the subsequent in
vitro phosphorylation induced by PKA by 25%. Because the
IP3 receptor was extracted and isolated in the presence of
phosphatase inhibitors, these results indicate that the various
treatments had decreased the amount of dephospho-IP3 receptor. Hence, although phosphorylation/dephosphorylation reactions are rapid and dynamic processes, and the stimulation with cyclic nucleotide analogues took place without phosphatase inhibitors present,
these results demonstrate notable changes in IP3 receptor phosphorylation caused by in situ stimulation.
Recent work has provided increasing knowledge about the functional
consequences of IP3 receptor phosphorylation. Cyclic
nucleotides mediate regulation of intracellular Ca2+
levels, e.g. by phosphorylation of Ca2+
channels, Ca2+ transporters, and regulatory proteins (47,
48). Phosphorylation of the IP3 receptor by PKA regulates
the sensitivity of the IP3 receptor to IP3
(49), although the effect of phosphorylation remains uncertain: in
microsomal membranes from rat brain and human platelets,
cAMP-dependent phosphorylation has been reported to inhibit
Ca2+ release (29, 30, 40, 50, 51), whereas phosphorylation of purified rat brain IP3 receptor increases
Ca2+ flux in reconstituted lipid vesicles (52). Increased
Ca2+ flux is seen when IP3 receptor is
phosphorylated by PKA in permeabilized SH-SY5Y human neuroblastoma
cells (12), and Ca2+ release and oscillations are
stimulated by cAMP and IP3 in rat hepatocytes (28).
Furthermore, Ca2+/calmodulin-dependent protein
kinase II and protein kinase C phosphorylation of the IP3
receptor are reported to increase Ca2+ mobilization in rat
brain, liver nuclei, and fibroblasts (15, 40, 53), whereas endogenous
kinases increase Ca2+ release through IP3
receptors in platelets (30), and the tyrosine kinase Fyn increases the
open probability of IP3 receptors in T-lymphocytes
(17).
Multiple mechanisms may also mediate the effects of cGMP on
intracellular Ca2+ (54), including cGMP-mediated regulation
of smooth muscle plasma membrane Ca2+-ATPase (23) and
PKG-induced decreases in agonist-induced IP3 generation
(55). The consequences of PKG-phosphorylation of the IP3
receptor are not well defined, although in rat and guinea pig
hepatocytes, cGMP potentiates Ca2+ release and induces
oscillations (26, 27). PKG also appears to inhibit IP3
receptor activity in platelets (29), similar to the effects of PKA.
However, functional effects of IP3 receptor phosphorylation
in brain remain uncertain, because both decreased and increased
Ca2+ release have been observed (12, 50, 52).
In the cerebellum, the Purkinje cells contain large amounts of both
IP3 receptor and PKG (8, 46). The effect of conjunction of
parallel fiber activation and climbing fiber-induced depolarization in
Purkinje cells is a long lasting synaptic depression (LTD). Recent
studies have proposed that climbing fiber activity may induce calcium
release from intracellular stores via IP3 receptors, which
in turn may sensitize IP3 receptors to the IP3
released as a consequence of parallel fiber activation (56, 57).
Induction of LTD is prevented by a specific, blocking antibody to the
IP3 receptor, and LTD fails to develop in mice with a
disrupted IP3 receptor type I gene (5). One underlying
molecular mechanism whereby climbing fiber-induced nitric oxide release
(58), the resulting generation of cGMP, and the activation of PKG may
regulate LTD in these synapses is the phosphorylation of the
IP3 receptor.
 |
ACKNOWLEDGEMENTS |
We thank Iren Sefland for technical
assistance, Dr. Ingeborg Torgner and Prof. Elling Kvamme for advice
concerning enzyme kinetics, Dr. Suzanne Lohmann (University of
Würzburg, Germany) for purified PKG, and Dr. H.C. Hemmings,
Jr. (Cornell Medical School, New York) for the D32-((Ser-34)8-38) peptide.
 |
FOOTNOTES |
*
This study was supported by the Norwegian Research Council
(to L. S. H.), by grants from Nordic Insulin Foundation (Gentofte, Denmark), and by the Jahre Foundation (Oslo, Norway).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.: 47-22-85-10-97;
Fax: 47-22-85-14-36; E-mail: annecari{at}basalmed.uio.no.
2
Student's t test may not be used
when variances in the two groups are significantly different, as they
were for 8-pCPT-cGMP-stimulation. Student's t test with
Welch's approximation: p = 0.057.
 |
ABBREVIATIONS |
The abbreviations used are:
IP3, inositol 1,4,5-trisphosphate;
PAGE, polyacrylamide gel electrophoresis;
PKA, cyclic AMP-dependent protein kinase;
PKG, cyclic
GMP-dependent protein kinase;
ACSF, artificial
cerebrospinal fluid;
8-pCPT, 8-(4-para-chlorophenylthio);
DARPP-32, dopamine- and cAMP-regulated phosphoprotein of 32 kDa;
EPPS, N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid;
LTD, long term depression.
 |
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