COMMUNICATION
Substrate Phosphorylation in the Protein Kinase C
Knockout
Mouse*
Geert M. J.
Ramakers
§,
Dan D.
Gerendasy¶, and
Pierre
N. E.
de Graan
From the
Rudolf Magnus Institute for Neurosciences,
Department of Medical Pharmacology, Unversiteitsweg 100, 3584 CG
Utrecht, The Netherlands and the ¶ Department of Molecular
Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
 |
ABSTRACT |
The phosphorylation state of three identified
neural-specific protein kinase C substrates (RC3, GAP-43/B-50, and
MARCKS) was monitored in hippocampal slices of mice lacking the
-subtype of protein kinase C and wild-type controls by quantitative
immunoprecipitation following 32Pi
labeling. Depolarization with potassium, activation of glutamate receptors with glutamate, or direct stimulation of protein kinase C
with a phorbol ester increased RC3 phosphorylation in wild-type animals
but failed to affect RC3 phosphorylation in mice lacking the
-subtype of protein kinase C. Our results suggests the following biochemical pathway: activation of a postsynaptic (metabotropic) glutamate receptor stimulates the
-subtype of protein kinase C,
which in turn phosphorylates RC3. The inability to increase RC3
phosphorylation in mice lacking the
-subtype of protein kinase C by
membrane depolarization or glutamate receptor activation may contribute
to the spatial learning deficits and impaired hippocampal LTP observed
in these mice.
 |
INTRODUCTION |
Mice lacking the
-subtype of protein kinase C
(PKC
)1 show mild spatial
learning deficits and exhibit impaired hippocampal long term
potentiation (LTP), suggesting that PKC
is a key regulatory component in LTP and spatial memory (1-3). However, the biochemical pathways that are perturbed in these mice have not yet been identified. The acidic, calmodulin-binding PKC substrate RC3 (also called neurogranin), is a likely substrate for PKC
because the two proteins colocalize in the dendrites of excitatory neurons of the cerebral cortex, hippocampus, and striatum and are expressed at the same stages
of development (4-6). Additionally, RC3 is phosphorylated during LTP
and dephosphorylated during LTD, and antibodies to RC3 interfere with
the induction of LTP (7-9). Here, we examine the incorporation of
32PO4 into RC3 and two other major PKC
substrates; GAP-43/B-50 and MARCKS by quantitative immunoprecipitation
(8) before and after treating hippocampal slices from PKC
-deficient
and wild-type mice with potassium, glutamate, or the phorbol ester
4
-phorbol 12,13-dibutyrate (PDB). We show that stimuli that readily
increase RC3 phosphorylation in wild-type mice fail to affect RC3
phosphorylation in PKC
-deficient mice.
 |
EXPERIMENTAL PROCEDURES |
The effects of depolarization, glutamate receptor stimulation,
and direct PKC activation were determined in PKC
knockout and litter
mate control mice (bred into a C57/Bl background for six generations
and kindly provided to us by Dr. J. M. Wehner).
For Western blotting, the forebrain of each mouse was homoginized in 10 ml of buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 2 mM EDTA, 1 mM EGTA, 50 mM dithiothreitol, 0.6 mM phenylmethylsulfonyl fluoride, and 1% SDS. We equalized the protein concentrations of the
homogenates based on three independent protein assays performed in
triplicate prior to adding SDS and dithiothreitol using the BCA method
and then immuno-blotted serial dilutions of each homogenate. The blotts
were probed with polyclonal rabbit anti-RC3 (Affinity Research Products
Ltd., Exeter, UK) at a dilution of 1:1000 and then developed with a
horseradish peroxidase-based, enhanced chemiluminescence protocol.
Hippocampal slices of wild-type (n = 8) and knockout
(n = 8) mice were prepared as described (8) and
collected in carbogenated phosphate-free ACSF containing 124 mM NaCl, 4.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 10 mM glucose, and 20 mM NaHCO3 at
room temperature. After 30 min slices were transferred to reaction
tubes containing 900 µl of carbogenated phosphate-free ACSF at
30 °C, and 45 min later 100 µCi of 32PO4
(specific activity, 40 mCi/ml; ICN Pharmaceuticals) was added. Slices
were labeled for 90 min, and the medium was changed to phosphate-free
ACSF containing 30 mM K+, 1 mM
glutamate (Fluka), or 0.1 µM PDB (Sigma).
32PO4 incorporation into RC3, GAP-43/B-50, and
MARCKS was determined using a quantitative immunoprecipitation as
described (8). Briefly, protein homogenate was incubated overnight at
4 °C with polyclonal rabbit antibodies 8420 (final dilution for RC3,
1:100), 9727 (final dilution for GAP-43/B-50, 1:200), or a polyclonal rat antibody to the MARCKS protein (final dilution, 1:200; gift of Drs.
Lenox and McNamara). Antigen complexes were precipitated with
Pansorbin® (Calbiochem, La Jolla, CA) and solubilized, and immunoprecipitates were separated using 15% (RC3) or 11% (GAP-43/B-50 and MARCKS) SDS-polyacrylamide gel electrophoresis.
32PO4 incorporation into proteins was detected
using a Fuji BAS1000 imaging system (Raytest, Germany) and quantified
using TINA analysis software. The total 32PO4
incorporation into proteins was determined by trichloroacetic acid
precipitation as described previously, and
32PO4 incorporation into RC3, GAP-43/B-50, and
MARCKS was normalized accordingly.
All experiments were performed blind with regard to the genotype of the
animals. Statistical analysis were carried out using a Student's
t test.
 |
RESULTS |
Protein levels of RC3 were not different between wild-type,
heterozygous, and PKC
knockout mice (Fig.
1A), showing that there was no
up- or down-regulation of RC3 levels induced by the PKC
knockout.
Basal in situ phosphorylation of RC3 and MARCKS in
hippocampal slices from mice lacking PKC
did not differ
significantly from those observed in wild-type littermate controls
(102.3 ± 8.1% (mean ± S.E.) and 99.3 ± 9.2% of
basal phosphorylation in controls for RC3 and MARCKS respectively,
p > 0.1, n = 8, and n = 4). However, increased basal phosphorylation of GAP-43/B-50 was
observed in the null mutant (148.9 ± 17.8% (mean ± S.E.)
of basal phosphorylation in controls, p < 0.05, n = 8). As expected, depolarization with potassium,
excitation with glutamate, or activation of PKC with PDB induced
phosphorylation of RC3 in wild-type controls (Fig. 1, B and
C). Strikingly, none of the treatments induced an increase in RC3 phosphorylation in slices from PKC
-deficient mice. Thus, RC3
is a highly specific substrate for PKC
during excitation. PDB
increased the phosphorylation of MARCKS and GAP-43/B-50 in slices
derived from knockout (147.4 ± 6.6% (n = 4) and
162.7 ± 14.2% (n = 8)), as well as the wild-type
(173.7 ± 14.5% (n = 4) and 288.2 ± 35.2%
(n = 8)) mice (Fig. 1B). However,
GAP-43/B-50 phosphorylation was significantly attenuated in the former
compared with the latter (43.5 ± 4.4% of wild type,
p < 0.01, n = 8). Decreased incorporation of 32PO4 into GAP-43/B-50 in
knockout mice upon direct stimulation of PKC was probably due to higher
initial levels of phospho-GAP-43/B-50 because the two appear to offset
each other, although the possibility that GAP-43/B-50 could be a
substrate for PKC
cannot be ruled out.

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|
Fig. 1.
Quantification of RC3 protein levels and
in situ phosphorylation following different physiological
relevant stimuli in PKC knockout mice. A, Western
blots showing RC3 protein levels from wild-type (+/+),
heterozygous (+/ ), and PKC knockout ( / )
mice. Different amounts of brain homogenates (20, 10, 5, and 2.5 µg
from left to right) were loaded. B,
typical immunoprecipitations for GAP-43/B-50, MARCKS, and RC3 from
wild-type (+/+) and PKC knockout ( / ) mice.
Phorbol ester treatment induces a clear increase in GAP-43/B-50 and
MARCKS phosphorylation in both wild-type and PKCg knockout mice. High
potassium, glutamate, and phorbol ester treatment does not affect RC3
phosphorylation in PKC knockout mice. C, RC3
phosphorylation in hippocampal slices from PKC knockout mice
(open bars) and control mice (filled bars) under
control conditions (Con), after depolarization
(K+), glutamate receptor stimulation
(Glu), or PKC activation (PDB). On average,
depolarization with potassium, excitation with glutamate, and
activation of PKC with PDB increased RC3 phosphorylation in wild-type
controls (124.8 ± 6.9, 149.2 ± 15.0, and 192.4 ± 19.5% of basal levels, respectively, n = 8). In PKC
knockout mice, none of these treatments induced a change in RC3
phosphorylation (103.9 ± 8.0, 98.4 ± 9.1, and 94.7 ± 7.7% of basal levels respectively, n = 8). *,
p < 0.05, Student's t test.
|
|
 |
DISCUSSION |
The experiments described here demonstrate that depolarization
with potassium, activation with glutamate, or direct stimulation of PKC
with a phorbol ester leads to phosphorylation of RC3 solely by PKC
.
Thus, the results unequivocally delineate the following biochemical
pathway: activation of a postsynaptic (metabotropic) glutamate receptor
stimulates PKC
, which in turn phosphorylates RC3. Basal levels of
phospho-RC3 appear to be dictated by a calcium and
diacylglycerol-independent atypical isoform of PKC, possibly
or
. Basal levels of phospho-GAP-43/B-50 are higher in the PKC
knockout mouse, and this might be a presynaptic mechanism to compensate
for the inability to phosphorylate RC3 in dendrites, perhaps by
increasing neurotransmitter release in response to decreased
postsynaptic gain (6, 10-13). Inability to phosphorylate RC3 in the
PKC
knockout mouse by either membrane depolarization or by
activation of postsynaptic glutamate receptors may contribute to the
electrophysiological and behavioral phenotypes of the PKC
knockout mouse.
 |
ACKNOWLEDGEMENTS |
We thank Dr. J. Wehner for providing two
breeding pairs of heterozygous PKC
knockout mice, Dr. S. Tonegawa
for permitting their use, and Drs. R. Lenox and R. McNamara for the
gift of the MARCKS antibody.
 |
FOOTNOTES |
*
This work was supported by Netherlands Organization for
Scientific Research Grant 910-20-901 (to G. M. J. R.),
NIGMS, National Institutes of Health Grant GM-32355 (to J. Gregor
Sutcliffe), NINDS, National Institutes of Health Grant NS-35831 (to
D. D. G.), European Science Foundation Grant ENP 16/3, and
BIOMED II Grant BMH4-CT96-0228 (to P. N. E. de G.).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: Inst. of Basal Medical
Sciences, Dept. of Physiology, University of Oslo, PB 1103 Blindern,
0317 Oslo, Norway. Tel.: 47-22851407; Fax: 47-22851249; E-mail:
geert.ramakers{at}basalmed.uio.no.
The abbreviations used are:
PKC, protein kinase
C; ACSF, artificial cerebrospinal fluid; LTP, long term potentiation; PDB, 4-
-phorbol 12,13-dibutyrate.
 |
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