COMMUNICATION
Substrate Phosphorylation in the Protein Kinase Cgamma Knockout Mouse*

Geert M. J. RamakersDagger §, Dan D. Gerendasy, and Pierre N. E. de GraanDagger

From the Dagger  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
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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 gamma -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 gamma -subtype of protein kinase C. Our results suggests the following biochemical pathway: activation of a postsynaptic (metabotropic) glutamate receptor stimulates the gamma -subtype of protein kinase C, which in turn phosphorylates RC3. The inability to increase RC3 phosphorylation in mice lacking the gamma -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
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Abstract
Introduction
References

Mice lacking the gamma -subtype of protein kinase C (PKCgamma )1 show mild spatial learning deficits and exhibit impaired hippocampal long term potentiation (LTP), suggesting that PKCgamma 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 PKCgamma 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 PKCgamma -deficient and wild-type mice with potassium, glutamate, or the phorbol ester 4alpha -phorbol 12,13-dibutyrate (PDB). We show that stimuli that readily increase RC3 phosphorylation in wild-type mice fail to affect RC3 phosphorylation in PKCgamma -deficient mice.

    EXPERIMENTAL PROCEDURES

The effects of depolarization, glutamate receptor stimulation, and direct PKC activation were determined in PKCgamma 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 PKCgamma knockout mice (Fig. 1A), showing that there was no up- or down-regulation of RC3 levels induced by the PKCgamma knockout. Basal in situ phosphorylation of RC3 and MARCKS in hippocampal slices from mice lacking PKCgamma 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 PKCgamma -deficient mice. Thus, RC3 is a highly specific substrate for PKCgamma 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 PKCgamma 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 PKCgamma knockout mice. A, Western blots showing RC3 protein levels from wild-type (+/+), heterozygous (+/-), and PKCgamma 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 PKCgamma 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 PKCgamma knockout mice. C, RC3 phosphorylation in hippocampal slices from PKCgamma 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 PKCgamma 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 PKCgamma . Thus, the results unequivocally delineate the following biochemical pathway: activation of a postsynaptic (metabotropic) glutamate receptor stimulates PKCgamma , 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 lambda  or zeta . Basal levels of phospho-GAP-43/B-50 are higher in the PKCgamma 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 PKCgamma knockout mouse by either membrane depolarization or by activation of postsynaptic glutamate receptors may contribute to the electrophysiological and behavioral phenotypes of the PKCgamma knockout mouse.

    ACKNOWLEDGEMENTS

We thank Dr. J. Wehner for providing two breeding pairs of heterozygous PKCgamma 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-alpha -phorbol 12,13-dibutyrate.
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