Differential Effects of a Calcineurin Inhibitor on
Glutamate-induced Phosphorylation of
Ca2+/Calmodulin-dependent Protein Kinases in
Cultured Rat Hippocampal Neurons*
Jiro
Kasahara,
Kohji
Fukunaga, and
Eishichi
Miyamoto
From the Department of Pharmacology, Kumamoto University School of
Medicine, Kumamoto 860-0811, Japan
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ABSTRACT |
Calcium/calmodulin-dependent protein
kinases (CaM kinases) are major multifunctional enzymes that play
important roles in calcium-mediated signal transduction. To
characterize their regulatory mechanisms in neurons, we compared
glutamate-induced phosphorylation of CaM kinase IV and CaM kinase II in
cultured rat hippocampal neurons. We observed that dephosphorylation of
these kinases followed different time courses, suggesting different
regulatory mechanisms for each kinase. Okadaic acid, an inhibitor of
protein phosphatase (PP) 1 and PP2A, increased the phosphorylation of
both kinases. In contrast, cyclosporin A, an inhibitor of calcineurin,
showed different effects: the phosphorylation and activity of CaM
kinase IV were significantly increased with this inhibitor, but those of CaM kinase II were not significantly increased. Cyclosporin A
treatment of neurons increased phosphorylation of
Thr196 of CaM kinase IV, the activated form with CaM
kinase kinase, which was recognized with an
anti-phospho-Thr196 antibody. Moreover, recombinant CaM
kinase IV was dephosphorylated and inactivated with calcineurin as well
as with PP1, PP2A, and PP2C in vitro. These results suggest
that CaM kinase IV, but not CaM kinase II, is directly regulated with calcineurin.
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INTRODUCTION |
Calcium (Ca2+)/calmodulin
(CaM)1 transduces increases
in intracellular Ca2+ concentration. These increases
activate Ca2+-dependent molecules involved in
signaling, such as calcium/calmodulin-dependent protein
kinases (CaM kinases). Among the six types of known CaM kinases
(namely, myosin light chain kinase, phosphorylase kinase, and CaM
kinases I, II, III, and IV), CaM kinase II has been the most
extensively characterized. It is abundantly expressed in the brain and
plays an important functional role in hippocampal long-term
potentiations (1-4). After activation by Ca2+/CaM (5), CaM
kinase II shows Ca2+-independent activity, which prolongs
its activity even after intracellular Ca2+ concentrations
return to basal levels. CaM kinase IV is also abundant in the brain,
including the cerebral cortex, the hippocampus, and particularly the
nuclei of cerebellar granule cells (6-10). Because of its expression
in the latter, this enzyme is also called CaM kinase Gr (granule). In
addition, CaM kinase IV is found in the thymus and testis (7),
suggesting functional importance not only in the brain but in the
immune system and reproductive cells. The genomic structure of CaM
kinase IV is quite unique: both CaM kinase IV and calspermin, a male
germ cell-specific CaM-binding protein, are derived from a single CaM
kinase IV gene (7, 11). CaM kinase IV has a broad substrate specificity
and is thought to be multifunctional (8), as is CaM kinase II. Although
less is known about the function of CaM kinase IV than that of CaM kinase II, recent work shows important biochemical and molecular differences between both kinases. CaM kinase IV functions as a monomer
rather than an oligomer (8). CaM kinase IV requires phosphorylation at
Thr196 with another CaM kinase, CaM kinase kinase, for full
activation (12-15), rather than autophosphorylation, as typically
observed in CaM kinase II. CaM kinase IV is localized in the nuclei of neurons (9), possibly associating with chromatin by the polyglutamate sequence in its C-terminal region, whereas the limited isoforms of CaM
kinase II (
B and
B isoforms) exist in the
nuclei. The nuclear localization of CaM kinase IV suggests a role in
the regulation of gene expression. CaM kinase IV can phosphorylate
Ser133, the essential site for activation of CREB (16),
which is essential for the activation of CRE-containing promoters. In
an assay using cultured hippocampal neurons, it was reported that CREB
phosphorylation induced by synaptic stimulations was mediated with CaM
kinase IV (17). Moreover, Ca2+-induced expression of
brain-derived neurotrophic factor that has a CRE motif in its promoter
region (18) was reported to be mediated by CaM kinase IV (19),
suggesting that Ca2+- and CRE-mediated gene expression is
controlled with CaM kinase IV in vivo. Given the functional
differences in these kinases, it is likely that CaM kinase IV and CaM
kinase II are regulated by different mechanisms.
To characterize the differences between CaM kinase IV and CaM kinase II
in the central nervous system and focus on the differences in the
regulatory mechanisms of these kinases, we investigated glutamate-induced phosphorylation and the effects of protein
phosphatase (PP) inhibitors, using primary cultured rat hippocampal
pyramidal neurons. We also examined the susceptibility of recombinant
CaM kinase IV to protein phosphatases.
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EXPERIMENTAL PROCEDURES |
Materials--
The following chemicals and materials were
obtained from the indicated sources: [
-32P]ATP and
125I-protein A, NEN Life Science Products;
[32P]orthophosphate, ICN; calyculin A, okadaic acid, and
cyclosporin A, Wako; CNQX, AP3, and MK801, Tocris Cookson; protein
A-Sepharose CL-4B and glutathione-Sepharose 4B, Pharmacia Biotech;
recombinant human protein phosphatase 1 catalytic subunit, Boehringer
Mannheim; rat brain protein phosphatase 2A holoenzyme, purified
according to the method of Barnes et al. (20) in our
laboratory; bovine purified calcineurin, UBI; and recombinant rat
protein phosphatase 2C, a generous gift from Dr. S. Tamura (Tohoku
University, Institute of Development, Aging and Cancer, Sendai, Japan)
(21).
Preparation of Polyclonal Antibodies--
The following peptides
derived from rat CaM kinase IV were synthesized commercially by Fujiya
and served as the immunogen: (a) the C terminus 10 amino
acids (PQQDAILPEY), (b) Thr196-phosphorylated
peptides that correspond to amino acids 184-197 (LSKIVEHQVLMKT(p)V),
and (c) a peptide corresponding to unphosphorylated amino
acids 184-197. These peptides (except for the latter) were conjugated
to keyhole limpet hemocyanin, mixed with Freund's complete adjuvant,
and injected into Japanese white rabbits. The methods used to prepare
the polyclonal antibody (IgG fraction) were described previously (22).
After repeated injection of the antigen, the anti-serum to each peptide
was prepared and purified as the pellet of an ammonium sulfate cut. The
anti-Thr196-phosphorylated CaM kinase IV antibody
(anti-pT196) was purified using an affinity peptide column using the
SulfoLink kit (Pierce) according to the manufacturer's protocol.
Preparation of the anti-CaM kinase II polyclonal antibody was reported
previously (22).
Cell Culture--
Rat hippocampal pyramidal neurons were
cultured as reported previously (23). Hippocampi were dissected from
Wistar rats at postnatal day 1 and mechanically dispersed. Neurons were
filtered through nylon mesh, plated onto collagen-coated dishes, and
cultured in Eagle's Minimum Essential Medium containing 10% fetal
calf serum. The medium was changed every 3 days.
Cell Stimulation with Glutamate--
Rat hippocampal neurons
cultured for 8 days were stimulated using the following protocol.
Culture medium was aspirated, and the neurons were preincubated with
KRH buffer (23) for 30 min and then stimulated with 10 µM
glutamate and 10 µM glycine for indicated periods. In the
experiments using the phosphatase inhibitors, 5 µM
cyclosporin A (CsA) was added throughout the preincubation and
stimulation or 1 µM okadaic acid was added for 15 min of
the preincubation and throughout the stimulation. Cells were frozen in
liquid nitrogen immediately after stimulation, harvested with homogenizing buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.5% Triton X-100, 10 mM EDTA, 4 mM EGTA, 1 mM Na3VO4,
30 mM Na4P2O7, 50 mM NaF, 0.1% SDS, 0.1 mM leupeptin, 0.075 mM pepstatin A, 0.05 mg/ml trypsin inhibitor, 1 mM phenylmethanesulfonyl fluoride, and 1 mM
dithiothreitol), and homogenized with brief bursts of ultrasonication
using a Branson Sonifier 250. The homogenate was centrifuged at
10,000 × g for 10 min to exclude insoluble materials.
Immunoblot Analysis--
The cell lysate, brain lysate, or
fusion proteins were mixed with Laemmli's SDS-sample buffer (24) and
boiled for 5 min. Samples were subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose
membrane at 70 V for 3.5 h using a Trans Blot Cell (Bio-Rad) in a
cold room (4 °C). After blocking for 1 h at room temperature
with TTBS solution containing 2.5% bovine serum albumin, the membranes
were incubated overnight at 4 °C with the first antibody diluted
with TTBS and bovine serum albumin (1:1000 for the anti-CaM kinase IV
C-terminal antibody and 1:5 for the anti-pT196 antibody). Bound
antibodies were visualized with 125I-protein A (0.1 mCi/ml)
and analyzed by a Bio-Imaging Analyzer BA100 or FLA-2000 (Fuji film).
Labeling of Cells and Quantitation of Phosphorylation--
Rat
hippocampal neurons cultured for 8 days were washed once with
phosphate-free and serum-free Dulbecco's modified Eagle's media and
labeled in 1.0 ml of this medium containing carrier-free [32P] orthophosphate (0.25 mCi/ml) for 5 h. The
cells were then preincubated, stimulated, and homogenized as described
above. The homogenate was precleared with 50 µl of protein
A-Sepharose CL-4B (50% v/v). Immunoprecipitation of CaM kinase IV and
CaM kinase II was performed essentially as reported previously (23).
Ten µg of anti-CaM kinase IV C-terminal antibody and 50 µl of
protein A-Sepharose were mixed with the precleared homogenate at
4 °C for 4 h. The immunocomplex was separated by centrifugation
at 10,000 × g for 2 min, washed four times with the
homogenizing buffer, and then prepared as a sample for SDS-PAGE. The
resulting supernatant was mixed with 5 µg of the anti-CaM kinase II
antibody and protein A-Sepharose for another 4 h and then prepared
as a sample for SDS-PAGE. After SDS-PAGE, the gels were dried and
analyzed by a Bio-Imaging Analyzer (FLA-2000; Fuji Film).
Assay for Immunoprecipitated CaM Kinase IV--
Neurons were
stimulated and homogenized, and the immunoprecipitation was performed
with the anti-CaM kinase IV antibody as described above, except for the
32P labeling procedures. After incubation for 4 h with
the anti-CaM kinase IV antibody, the immunocomplex was washed four
times with buffer containing of 50 mM HEPES, pH 7.5, 1 mM EDTA, and 1 mM dithiothreitol.
Immunoprecipitated CaM kinase IV was assayed using peptide-
(8)
(synthesized commercially by Fujiya) in 100 µl of a total volume of
the reaction mixture containing 50 mM HEPES (pH 7.5), 10 mM Mg(OAc)2, 0.5 mM
CaCl2, 0.3 µM CaM, 40 µM
peptide-
, and 0.1 mM [
-32P]ATP
(3000-5000 cpm/pmol). After incubation at 30 °C for 10 min with
gentle tapping, reactions were terminated by centrifugation of the
immunocomplex. Aliquots of the supernatants were spotted onto Whatman
P81 P-cellulose paper. The papers were washed in 75 mM
phosphoric acid buffer, and the radioactivity on the paper was measured
by scintillation spectrometry.
Preparation of GST-Fusion Proteins--
cDNAs encoding rat
CaM kinase IV
(7) and CaM kinase kinase
(15) were amplified
from rat brain total RNA by reverse transcription-polymerase chain
reaction using rTth XL DNA polymerase (Perkin-Elmer) and expressed as
fusion proteins with GST as described below. The primer
sequences used are as follows (5' to 3'): (a) CaM kinase IV
5' primer, GAGTCTCGAGGCGAAGATGCTCAAAGTCACGGT; (b) CaM kinase
IV 3' primer, CCTGGGGCCTCGAGGAAGCTGGGTTAGTACTCTGG; (c) CaM
kinase kinase 5' primer, ACAGGAATTCCCACGGACTGAAGTGATGGAGCG; and
(d) CaM kinase kinase 3' primer, ATTCGAATTCTGTCCCTGGATATGCCTGTGCGC.
All primers contained restriction sites (XhoI for CaM kinase
IV
primers and EcoRI for CaM kinase kinase
primers).
Polymerase chain reaction products were subcloned into plasmid pGEX5X-1
(Pharmacia) using a DNA ligation kit (Takara), sequenced to confirm
that no mutation was introduced, and then transfected into
Escherichia coli BL21 DE3. Expression and purification of
the fusion proteins were performed using GST Purification Modules
(Pharmacia) according to the manufacturer's protocols.
Phosphorylation of Fusion Proteins--
Approximately 250-500
µg of recombinant CaM kinase IV were incubated at 30 °C for 60 min
with approximately 10 µg of recombinant CaM kinase kinase in buffer
containing 50 mM HEPES (pH 7.5), 10 mM
Mg(OAc)2, 0.5 mM CaCl2, 0.3 µM CaM, 0.3 mg of bovine serum albumin, and 0.1 mM [
-32P]ATP (3000-5000 cpm/pmol) in a
total volume of 500 µl. The reactions were terminated by the addition
of EDTA to a final concentration of 20 mM. Phosphorylated
CaM kinase IV was purified as the pellet of an 80% ammonium sulfate
cut. These samples were dialyzed against 5 liters of buffer containing
50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol.
Assay for Protein Phosphatase--
The protein phosphatases we
used had the specific activities listed below, using
microtubule-associated protein 2 phosphorylated with CaM kinase II as a
substrate: (a) protein phosphatase 1 catalytic subunit, 30 nmol/min/mg; (b) purified rat brain protein phosphatase 2A
holoenzyme, 1.5 nmol/min/mg; (c) bovine calcineurin, 2.4 nmol/min/mg; and (d) recombinant protein phosphatase 2C
(21), 9.5 nmol/min/mg. We adjusted sample amounts of each protein
phosphatase to the activities such that a 50% dephosphorylation of
phosphorylated microtubule-associated protein 2 occurred in 15 min.
Phosphorylated CaM kinase IV (1 µg) was incubated at 30 °C for the
indicated periods with protein phosphatases under the following buffer
conditions (total, 50 µl): (a) protein phosphatases 1 and
2A, 50 mM HEPES (pH 7.5) and 1 mM
MnCl2; (b) calcineurin, 50 mM HEPES
(pH 7.5), 1 mM MnCl2, 1 mM
CaCl2, and 0.3 µM CaM; and (c)
protein phosphatase 2C, 50 mM HEPES (pH 7.5) and 10 mM Mg(OAc)2. Reactions were terminated by
(a) the addition of 5 µl of SDS-sample buffer in the case
of SDS-PAGE, (b) the addition of 20 µl of 50%
trichloroacetic acid and 150 µg of bovine serum albumin, followed by
centrifugation at 10,000 × g for 10 min and
measurement of the radioactivity of the released phosphate by
scintillation spectrometry, or (c) the addition of
termination buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% Triton X-100, 10 mM EDTA, and 1 mM dithiothreitol), followed by purification of GST-CaM
kinase IV with glutathione-Sepharose 4B. To determine changes in CaM
kinase IV activity by phosphorylation and dephosphorylation, GST-CaM
kinase IV was previously phosphorylated with 0.1 mM cold
ATP and then assayed for activity using [
-32P]ATP and
peptide-
as a substrate.
 |
RESULTS |
Characterization of Anti-CaM Kinase IV Polyclonal
Antibodies--
We prepared a rabbit anti-rat CaM kinase IV polyclonal
antibody. This antibody recognized a 63-kDa protein (CaM kinase IV
) found in a rat hippocampal lysate and a 90-kDa GST-fusion protein of
rat CaM kinase IV (Fig. 1A).
Using the antibody, we performed immunoprecipitation of the cell
lysates obtained from 32P-labeled hippocampal neurons (Fig.
1B). In our previous study (23), we demonstrated that the
activation of CaM kinase II by glutamate stimulation was mediated by a
NMDA-type glutamate receptor. We examined whether this observation was
also applicable to the activation of CaM kinase IV. The incorporation
of phosphate into the 63-kDa protein was significantly increased by
stimulation with glutamate for 3 min (182.6 ± 10%;
n = 4) compared with a control and was reduced by the
application of 20 µM MK801 (a noncompetitive inhibitor of
NMDA-type glutamate receptor; 92.7 ± 2% of control) (Fig.
1B), indicating that phosphorylation of CaM kinase IV
depended on NMDA-type glutamate receptors. The application of MK801
without glutamate did not show significant changes in the
phosphorylation levels of CaM kinase II and CaM kinase IV compared with
controls (89% and 87%, respectively). Other inhibitors of glutamate
receptors (20 µM CNQX (a specific inhibitor of AMPA-type
glutamate receptors; 212.7 ± 11%) and 1 mM AP3 (an
inhibitor of metabotropic glutamate receptors; 191.5 ± 7%) did
not show inhibitory effects on CaM kinase IV phosphorylation.

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Fig. 1.
Characterization of polyclonal anti-CaM
kinase IV antibodies. A, immunoblot analysis with polyclonal
anti-CaM kinase IV antibody. Rat hippocampal lysate (50 µg;
lanes 1 and 5), cultured rat hippocampal
pyramidal neuron lysate (50 µg; lanes 2 and 6),
GST-fusion CaM kinase IV (0.1 µg; lanes 3 and
7), and GST (1 µg; lane 4) were subjected to
SDS-PAGE. Immunoblot was performed using the anti-CaM kinase IV
antibody (lanes 1-4) and the anti-CaM kinase IV antibody
preabsorbed with antigen (lanes 5-7). B,
immunoprecipitated CaM kinase IV with the anti-CaM kinase IV antibody
from an extract of 32P-labeled neurons. Radiolabeled
neurons were stimulated with control (none), 10 µM
glutamate, or glutamate plus 20 µM MK801 in KRH buffer
for 3 min. The medium was then aspirated, and cells were frozen in
liquid N2. Cells were homogenized, and the Triton
X-100-soluble fraction was immunoprecipitated with the anti-CaM kinase
IV antibody. The immunocomplex was subjected to SDS-PAGE as duplicate
samples, followed by autoradiography. The application of MK801 without
glutamate did not show significant differences compared with control
(data not shown). C, immunoblot was performed using the
anti-CaM kinase IV antibody (lanes 1 and 2), the
anti-pT196 antibody (lanes 3-8), or the anti-pT196 antibody
preabsorbed with the antigen (lanes 9 and 10).
The amount of the protein run in each lane was as follows: 50 µg of
rat hippocampal lysate (lane 1); 0.1 µg of GST-CaM kinase
IV (lanes 2 and 7); 50 µg of rat hippocampal
lysate treated with 1 mM EGTA for 5 min (lane
3); 50 µg of rat hippocampal lysate treated with
Ca2+/CaM for 1 min (lane 4), 3 min (lane
5), and 10 min (lanes 6 and 9); and 0.1 µg
of GST-CaM kinase IV phosphorylated with GST-CaM kinase kinase
(lanes 8 and 10). The minor bands below CaM
kinase IV may be degradation products because these bands disappeared
when the anti-pT196 antibody was preabsorbed with antigen (lane
9). Left, markers (MW) indicate a molecular
mass of 116, 97.4, 66, and 45 kDa (from top to
bottom).
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We also raised an antibody against CaM kinase IV phosphorylated at
Thr196 with CaM kinase kinase designated anti-pT196. This
antibody recognized the 63-kDa protein in rat hippocampal lysate (Fig.
1C, lane 5) when it was phosphorylated with
Ca2+/CaM and recognized 90-kDa GST-CaM kinase IV (Fig.
1C, lane 8) when it was phosphorylated with GST-CaM kinase
kinase, indicating that the antibody was specific for CaM kinase IV
phosphorylated at Thr196 (Fig. 1C).
Glutamate-induced Phosphorylation of CaM Kinase IV and CaM Kinase
II--
We next examined the time courses of glutamate-induced
phosphorylation of CaM kinases IV and II in cultured neurons, using a
labeling assay technique. As shown in Fig.
2, A and B, inset, glutamate-induced phosphorylation of CaM kinase IV was transient without CsA (Fig. 2A and inset). The maximal peak
was obtained at 3 min and declined afterward. In contrast, the
glutamate-induced phosphorylation of CaM kinase II was relatively
sustained without CsA after phosphorylation (Fig. 2B and
inset). The difference in the phosphorylation level of CaM
kinase IV between 3 and 10 min was significant (p < 0.01, Student's t test), whereas that of CaM kinase II was
not significant. These results suggest the existence of different
dephosphorylation mechanisms for CaM kinase IV and CaM kinase II.

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Fig. 2.
Effects of protein phosphatase inhibitors on
the time course of phosphorylation of CaM kinases. A and
B, effects of 5 µM CsA, 10 µM
glutamate, or 5 µM CsA plus 10 µM glutamate
on the phosphorylation of CaM kinase IV (A) and CaM kinase
II (B). 32P-labeled hippocampal neurons were
stimulated with control (none), 5 µM CsA ( ), 10 µM glutamate ( ), or 5 µM CsA plus 10 µM glutamate ( ) in KRH buffer for the indicated times
(n = 4). CsA was added to the medium 30 min before
stimulation. After stimulation, samples were subjected to
immunoprecipitation, SDS-PAGE, and autoradiography as described under
"Experimental Procedures." Quantitation of phosphorylation was
performed by a Bio-Imaging analyzer. Values were expressed as a
percentage of the control at each stimulation time. Lanes
1-4 indicate the autoradiographs of duplicate samples stimulated
with control (none), glutamate, CsA plus glutamate, and CsA,
respectively, for 3- and 10-min stimulation periods. Insets
indicate the phosphorylation of CaM kinase IV (A) or CaM
kinase II (B) stimulated with glutamate alone in the time
course. C and D, effects of 1 µM
okadaic acid, 10 µM glutamate, or 1 µM
okadaic acid plus 10 µM glutamate on the phosphorylation
of CaM kinase IV (C) and CaM kinase II (D).
32P-labeled hippocampal neurons were stimulated with
control (none), 1 µM okadaic acid ( ), 10 µM glutamate ( ), or 1 µM okadaic acid
plus 10 µM glutamate ( ) in KRH buffer for the
indicated times (n = 4). Okadaic acid was added to the
medium 15 min before stimulation. Immunoprecipitation, SDS-PAGE, and
quantitation of phosphorylation were performed as described above and
under "Experimental Procedures." Lanes 1-4 indicate the
autoradiographs of duplicate samples stimulated with control (none),
glutamate, okadaic acid plus glutamate, and okadaic acid alone,
respectively, for 3- and 10-min stimulation periods.
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The Effects of CsA on Phosphorylation--
To test the
abovementioned idea, we examined the effects of protein phosphatase
inhibitors using these points of the time in the following experiments.
After a 30-min pretreatment of cells with 5 µM CsA, an
inhibitor of calcineurin, glutamate-induced phosphorylation of CaM
kinase IV increased significantly (Fig. 2A), whereas CaM
kinase II phosphorylation was not significantly affected (Fig.
2B). The addition of CsA alone had no effect on the
phosphorylation of either CaM kinase IV (Fig. 2A) or CaM
kinase II (Fig. 2B). These results suggest that calcineurin
is involved in CaM kinase IV dephosphorylation but not in CaM kinase II dephosphorylation.
The Effects of Okadaic Acid on Phosphorylation--
Unlike the
results obtained using CsA, a 15-min treatment of neurons with 1 µM okadaic acid inhibits both protein phosphatases 1 and
2A and significantly increases the phosphorylation of both CaM kinase
IV and CaM kinase II (Fig. 2, C and D), showing
that both enzymes are regulated with protein phosphatase 1 and/or 2A. Protein phosphatase 2A has been reported to regulate both CaM kinase II
(25) and CaM kinase IV (13, 26) and to be associated with CaM kinase IV
(27). The increased level of CaM kinase IV phosphorylation after
treatment with okadaic acid was about 2-fold higher than that seen
after treatment with CsA alone. It seems that CaM kinase IV is more
strictly regulated with okadaic acid-sensitive protein phosphatases
than with CsA-sensitive protein phosphatases.
Effects of CsA on the Activity of CaM Kinase IV--
Although the
regulation of CaM kinase IV with an okadaic acid-sensitive protein
phosphatase (e.g. PP2A) has been reported previously (26),
the effect of CsA on CaM kinase IV activity has not been reported
previously. We examined whether increased phosphorylation of CaM kinase
IV by CsA treatment resulted in an increase in CaM kinase IV activity.
As shown in Fig. 3, a 3-min application
of glutamate to neurons significantly increased
Ca2+/CaM-dependent CaM kinase IV activity. In
comparison, cells stimulated for 10 min showed decreased activity.
Moreover, CaM kinase IV activities after stimulation with glutamate
plus CsA for both 3 and 10 min were significantly increased compared
with those of cells stimulated for the same periods with glutamate or
CsA alone. Because the phosphorylation of Thr196 of CaM
kinase IV with CaM kinase kinase significantly increased both
Ca2+/CaM-dependent and -independent activities
in vitro (15, 28), the results indicate that phosphorylation
of Thr196 of CaM kinase IV and subsequent
autophosphorylation in the N terminus may occur. Thus, calcineurin is
involved in the dephosphorylation of at least Thr196.

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Fig. 3.
Effects of CsA and glutamate on the activity
of CaM kinase IV. Cultured hippocampal neurons were stimulated
with control (none), CsA, glutamate, and glutamate plus CsA in KRH
buffer for the indicated times. After stimulation, CaM kinase IV was
immunoprecipitated with the anti-CaM kinase IV antibody. The
immunocomplexes were assayed for
Ca2+/CaM-dependent activity of CaM kinase IV,
as described under "Experimental Procedures." The
Ca2+/CaM-independent activities were less than 10% of the
Ca2+/CaM-dependent activities of the samples.
Statistical analysis was performed using Student's t
test.
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Examination of the Phosphorylation of Thr196 of CaM
Kinase IV--
Phosphorylation of CaM kinase IV by treatment with
glutamate alone and glutamate plus CsA increased the kinase activity,
as described above (Fig. 3). Because this finding indicates that Thr196 of CaM kinase IV is phosphorylated, we asked whether
the phosphorylation level of Thr196 of CaM kinase IV
increases after treatment with glutamate and/or CsA using the
anti-pT196 antibody. CsA alone had no effect on the phosphorylation of
Thr196 of CaM kinase IV. Treating neurons with glutamate
significantly increased phosphorylation, and an additional increase was
observed when neurons were treated with glutamate plus CsA (Fig.
4). The total protein levels of CaM
kinase IV were not altered by stimulation with glutamate and/or CsA.
These results show that Thr196 of CaM kinase IV is
phosphorylated with CaM kinase kinase in response to glutamate and/or
CsA.

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Fig. 4.
Immunoblot analysis of stimulated neurons
using the anti-pT196 antibody. A, cultured hippocampal
neurons were stimulated with control (none; lanes 1 and
5), 10 µM glutamate (lanes 2 and
6), 10 µM glutamate plus 5 µM
CsA (lanes 3 and 7), and 5 µM CsA
(lanes 4 and 8) for 3 min. After stimulation,
immunoblot analysis was performed as described under "Experimental
Procedures," using the anti-pT196 antibody (lanes 1-4)
and the anti-CaM kinase IV antibody (lanes 5-8).
B, immunoreactivities observed in A were
quantified by a Bio-Imaging analyzer and analyzed statistically. *,
p < 0.05; **, p < 0.01 (Student's
t test). n = 3.
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In Vitro Dephosphorylation of Recombinant CaM Kinase IV with
Protein Phosphatases--
To further examine whether CaM kinase IV is
phosphorylated with CaM kinase kinase and dephosphorylated with protein
phosphatases, recombinant GST fusions of CaM kinase kinase and CaM
kinase IV were used (Fig. 5). GST-CaM
kinase IV phosphorylated with CaM kinase kinase with an incorporation
of 43 pmol phosphate/µg CaM kinase IV was dephosphorylated with
protein phosphatase 1, protein phosphatase 2A, calcineurin, and protein
phosphatase 2C in a time-dependent manner (Figs. 5,
A and B). To quantitate the relative potency of
each protein phosphatase activity, we adjusted the protein levels of
protein phosphatases to that which can dephosphorylate an equivalent
amount of phosphate from microtubule-associated protein 2. Among the
protein phosphatases tested, protein phosphatase 1 was the most active
in dephosphorylating CaM kinase IV phosphorylated with CaM kinase
kinase, followed by calcineurin and protein phosphatases 2A and 2C
(Fig. 5B).

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Fig. 5.
In vitro dephosphorylation of GST-CaM
kinase IV and correlation of activity and dephosphorylation.
A, 1 µg of GST-CaM kinase IV phosphorylated with
[32P]ATP by GST-CaM kinase kinase was dephosphorylated
with protein phosphatases as indicated for 0, 3, 10, and 30 min.
CaMKIV, CaM kinase IV. After incubation, the reaction
mixtures were subjected to SDS-PAGE as duplicate samples, followed by
autoradiography. Minor bands with molecular masses lower than that of
GST-CaM kinase IV may be degradation products. B, protein
phosphatase activities were assayed for 32P-labeled
phosphorylated GST-CaM kinase IV with protein phosphatase 1 ( ),
protein phosphatase 2A ( ), calcineurin ( ), and protein
phosphatase 2C ( ) during a time course. , a sample incubated
without protein phosphatases. Values represent the means of triplicate
determinations. Similar experiments were performed at least three
times, and a representative experiment is shown. C, effects
of dephosphorylation on phosphorylated GST-CaM kinase IV activity. One
µg of GST-CaM kinase IV phosphorylated with GST-CaM kinase kinase
using cold ATP was dephosphorylated with control (none), PP1, PP2A,
calcineurin, and PP2C for 30 min. After incubation, GST-CaM kinase IV
was immunoprecipitated with the anti-CaM kinase IV antibody, and the
immunocomplexes were assayed for CaM kinase IV activity as described
under "Experimental Procedures." n = 4, *,
p < 0.01 (Student's t test).
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The dephosphorylation of CaM kinase IV with protein phosphatases was
correlated with decreases in the activities of CaM kinase IV, which
were determined using peptide-
as a substrate (Fig. 5C).
Because CaM kinase IV is activated by the phosphorylation of
Thr196 with CaM kinase kinase, these results show that
protein phosphatase 1, protein phosphatase 2A, calcineurin, and protein
phosphatase 2C can each dephosphorylate this site and regulate kinase activity.
 |
DISCUSSION |
In previous reports, we have investigated the activation of CaM
kinase II by autophosphorylation in response to neurotransmitters, growth factors, and hormones in various cells (rat embryo fibroblasts (29), hippocampal neurons (23), cerebellar granule cells (5), NG 108-15 cells (30), hippocampal slices (1), rat cortical astrocytes (31), MIN 6 cells (32), and bovine adrenal chromaffin cells (33)). This study
describes the activation of CaM kinase IV in hippocampal neurons by
stimulation with an excitatory neurotransmitter, glutamate. In this
connection, Bito et al. (17) reported that CREB
phosphorylation caused by synaptic stimulation or by high potassium-induced depolarization was mediated by CaM kinase IV activation in hippocampal neurons. Furthermore, Park and Soderling (26)
reported activation of CaM kinase IV in Jurkat T lymphocytes by
stimulation of the antigen receptor CD3. Thus, CaM kinase IV as well as
CaM kinase II in living cells is activated in response to
neurotransmitters. This is the first report to show directly that CaM
kinase IV is activated in neurons by glutamate stimulation, an
observation lending support to the idea that CaM kinase IV is involved
in Ca2+-mediated gene expression.
Although CsA alone had no effect on the phosphorylation of CaM kinase
IV in cultured hippocampal neurons, the combination of glutamate and
CsA greatly increased its phosphorylation compared with the increases
observed with glutamate alone (Fig. 2A). In contrast, no
difference in phosphorylation of CaM kinase II was observed when
stimulation with glutamate alone was compared with stimulation with
glutamate plus CsA. This finding suggested that calcineurin could
dephosphorylate CaM kinase IV but not CaM kinase II. The CaM kinase II
results were consistent with in vitro observations made in a
previous report (34). Recombinant GST-CaM kinase IV phosphorylated with
GST-CaM kinase kinase could be dephosphorylated with calcineurin
purified from rat brain. In addition, FK506, a calcineurin inhibitor,
was reported to delay the time course of dephosphorylation of CREB
phosphorylated with CaM kinase IV (17), suggesting the regulation of
CaM kinase IV or CaM kinase kinase with calcineurin. On the other hand,
okadaic acid combined with glutamate stimulated the phosphorylation of
both CaM kinases IV and II (Figs. 1, C and D).
This suggests that okadaic acid-sensitive protein phosphatases are
involved in the dephosphorylation of CaM kinases IV and II. CaM kinase
II could be dephosphorylated with protein phosphatases 1 (35), 2A (36),
and 2C (21). As predicted, GST-CaM kinase IV could also be
dephosphorylated with purified protein phosphatases 1, 2A, and 2C in
addition to calcineurin. In contrast to this study, Park and Soderling
(26) reported that the catalytic subunit of protein phosphatase 1 could not dephosphorylate CaM kinase IV. The difference between our results
and their data has not yet been clarified. We suppose that it may be
due to the difference in the isoforms of protein phosphatase 1; they
used an
-isoform, whereas we used a human
-isoform that was
purchased commercially. Because both of the catalytic subunits of
protein phosphatase 1 are recombinant, the preparations have no
contamination by other protein phosphatases.
CaM kinase IV has multiple autophosphorylation sites in the N-terminal
region (37) and in a calmodulin-binding domain in the C-terminal region
(38) in addition to the Thr196 phosphorylated with CaM
kinase kinase. Although the relationship between kinase activity and
N-terminal autophosphorylation is not yet clearly understood,
C-terminal autophosphorylation was reported to cause inactivation of
CaM kinase IV (38). Because the physiological significance of this
phosphorylation is still unclear, it is important to determine the
protein phosphatases responsible for the dephosphorylation of the
autophosphorylated C-terminal because dephosphorylation of this site
may reactivate CaM kinase IV.
Because it was shown in this study that CaM kinase IV was
dephosphorylated with calcineurin (Fig. 5), the interaction between CaM
kinase IV and calcineurin is predicted to occur in neurons. CaM kinase
IV, which is primarily localized to the nuclei of neurons (17), has
also been seen in the axons of neurons (9). Although calcineurin is
thought to be localized to the cytoplasm, it has been reported that
calcineurin may translocate to the nucleus with NF-AT after ionomycin
treatment (39), which shows Ca2+-mediated translocation of
calcineurin to the nucleus. It is unclear how the phosphorylation state
of nuclear CaM kinase IV compares with that of the putative cytoplasmic
form and whether translocation of calcineurin with glutamate
stimulation occurs in neurons. How these enzymes interact should be
elucidated in a further study.
This study focused on the dephosphorylation of CaM kinase IV in
cultured hippocampal neurons. Because CaM kinase IV is activated with
CaM kinase kinase in a Ca2+/CaM-dependent
manner, CaM kinase kinase is also activated in response to glutamate.
Because it was reported that CaM kinase kinase showed strong
autophosphorylation in the presence of Ca2+/CaM (40), we do
not exclude the possibility that activation of CaM kinase IV may be
controlled by the regulation of CaM kinase kinase with protein phosphatases.
 |
ACKNOWLEDGEMENTS |
We thank Prof. S. Tamura for providing PP2C,
Drs. H. Yamamoto, H. Kanasaki, and M. Ohmitsu for useful comments, and
K. Ohta for technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-Aid for
Scientific Research and for Scientific Research on Priority from the Ministry of Education, Science, Sports and Culture of Japan and by a
research grant from the Human Frontier Science Program (to E. M. and K. F.).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: Dept. of Pharmacology,
Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan. Tel.: 81-96-373-5076; Fax: 81-96-373-5078; E-mail: emiyamot{at}gpo.kumamoto-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
CaM, calmodulin;
CaM
kinase, Ca2+/calmodulin-dependent protein
kinase;
MK801, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine;
CsA, cyclosporin A;
PP, protein phosphatase;
KRH, Krebs-Ringer HEPES;
PAGE, polyacrylamide gel electrophoresis;
TTBS, 50
mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1%
Tween 20;
GST, glutathione S-transferase;
AMPA, (S)-
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate;
AP3, DL-2-amino-3-phosphonopropionate;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
CRE, cyclic AMP-responsive
element;
CREB, cyclic AMP-responsive element-binding protein;
NF-AT, nuclear factor of activated T cells;
NMDA, N-methyl-D-aspartate.
 |
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