Substrate Phosphorylation in the Protein Kinase Cγ Knockout Mouse

Abstract

The phosphorylation state of three identified neuralspecific 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 wildtype controls by quantitative immunoprecipitation following 32 P i 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.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, calmodulinbinding 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 32 PO 4 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 PROCEDURESThe 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 (Aff...

Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: A study with knockout mice

“…The phosphorylated Ng also stimulates the G-protein-coupled phosphoinositide second messenger pathways that trigger the mobilization of Ca 2ϩ from intracellular stores (8). PKC is the only known Ng kinase in vitro (5), and deletion of PKC ␥ gene in mice negates both the glutamate-and depolarization-mediated phosphorylation of Ng (9). The close functional relationship between Ng and PKC ␥ is further illustrated by their similar developmental expression patterns in the cerebral cortex (10)(11)(12).…”

Neurogranin, a link between calcium/calmodulin and protein kinase C signaling in synaptic plasticity

“…In vivo Ng phosphorylation in response to synaptic activation stimuli has been shown in several preparations such as brain slices (56,57) and cultured hippocampal neurons (58). Using PKCc knockout mice, it could be demonstrated that this PKC isoform is the only kinase involved in Ng phosphorylation upon high K 1 depolarization or glutamate receptor activation (59). Interestingly, Ng and PKCc show similar expression patterns during development and also share a highly coincident localization in the brain, both at the regional and the subcellular levels.…”

“…In a way, this role could be considered as a ''memory effect,'' as occurs with CaMKII autophosphorylation, since it would prolong CaM availability and signaling in the postsynaptic space beyond the time window of the original stimulus. PKCc has been shown to be the isoform involved in the in vivo phosphorylation of Ng (59). Since PKCc needs Ca 21 and DAG for activation, only those stimuli capable to activate ionotropic and metabotropic receptors simultaneously could significantly increase Ng phosphorylation.…”

Pre- and postsynaptic localization of RC3/neurogranin in the adult rat spinal cord: An immunohistochemical study

“…These proteins show sequence homology and have common biochemical characteristics. RC3 plays a role in Ca 2ϩ /calmodulin (Gerendasy et al, 1994a;Prichard et al, 1999) and protein kinase C signal transduction cascades (Baudier et al, 1991;Ramakers et al, 1999), and its phosphorylation is involved in long-term potentiation (Federov et al, 1995;Pasinelli et al, 1995;Chen et al, 1997;Ramakers et al, 1997) and long-term depression (De Graan et al, 1996). The expression level of RC3, but not its anatomical distribution, is under direct control of thyroid hormone at the transcriptional level (Morte et al, 1997;Martínez de Arrieta et al, 1999).…”