Kuo-Ping Huang

Neurogranin/RC3 Enhances Long-Term Potentiation and Learning by Promoting Calcium-Mediated Signaling

In neurons, neurogranin (Ng) binds calmodulin (CaM), and its binding affinity is reduced by increasing Ca2+, phosphorylation by PKC, or oxidation by oxidants. Ng concentration in the hippocampus of adult mice varied broadly (Ng+/+, ∼160-370 and Ng+/-, ∼70-230 pmol/mg); the level in Ng+/+ mice is one of the highest among all neuronal CaM-binding proteins. Among Ng+/- mice, but less apparent in Ng+/+, a significant relationship existed between their hippocampal levels of Ng and performances in the Morris water maze. Ng-/- mice performed poorly in this task; they also displayed deficits in high-frequency-induced long-term potentiation (LTP) in area CA1 of hippocampal slices, whereas low-frequency-induced long-term depression was enhanced. Thus, compared with Ng+/+ mice, the frequency-response curve of Ng-/- shifted to the right. Paired-pulse facilitation and synaptic fatigue during prolonged stimulation at 10 Hz (900 pulses) were unchanged in Ng-/- slices, indicating their normal presynaptic function. Measurements of Ca2+ transients in CA1 pyramidal neurons after weak and strong tetanic stimulations (100 Hz, 400 and 1000 msec, respectively) revealed a significantly greater intracellular Ca2+ ([Ca2+]i) response in Ng+/+ compared with Ng-/- mice, but the decay time constants did not differ. The diminished Ca2+ dynamics in Ng-/- mice are a likely cause of their decreased propensity to undergo LTP. Thus, Ng may promote a high [Ca2+]i by a “mass-action” mechanism; namely, the higher the Ng concentration, the more Ng-CaM complexes will be formed, which effectively raises [Ca2+]i at any given Ca2+ influx. This mechanism provides potent signal amplification in enhancing synaptic plasticity as well as learning and memory.

Environmental Enrichment Enhances Neurogranin Expression and Hippocampal Learning and Memory But Fails to Rescue the Impairments of Neurogranin Null Mutant Mice

Environmental enrichment is known to enhance hippocampal neurogenesis and cognitive functions. Neurogranin (Ng), a specific substrate of protein kinase C (PKC), is abundantly expressed in brain regions important for cognitive functions. Deletion of Ng in mice causes severe deficits in spatial learning and long-term potentiation (LTP) in the hippocampal CA1 region. These Ng−/− mice, as compared with Ng+/+, respond poorly after treatment of their hippocampal slices with agents that activate signaling molecules important for learning and memory, including Ca2+/calmodulin-dependent protein kinase II (αCaMKII), PKC, protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB). In the present study, adult mice were housed in either regular home cages (control group) or more spacious cages with an exercise wheel and change of toys twice per week (enriched group) for at least 3 weeks. Enriched Ng+/+ and Ng+/− mice showed enhanced LTP in the hippocampal CA1 after high-frequency stimulation, but Ng−/− mice were affected only minimally. Behaviorally, the enriched Ng+/+ and Ng+/−, but not Ng−/− mice, performed significantly better than their respective control cohorts in Morris water maze and in step-down fear conditioning. Enriched Ng+/− mice also showed improvement in the radial arm maze. Quantitative immunoblot analyses showed that the enriched groups of all three genotypes exhibited elevated hippocampal levels of αCaMKII and CREB, but not ERK. Interestingly, enrichment caused a significant increase in hippocampal Ng levels both in Ng+/+ and Ng+/− mice that seemed to contribute to their improved LTP and behavioral performances. These results suggest that Ng gates the neuronal signaling reactions involved in learning and memory. During environmental enrichment, these Ng-regulated reactions are also critical for the enhancement of synaptic plasticity and cognitive functions.

Glutathionylation of proteins by glutathione disulfide S-oxide

Aqueous solution of S-nitrosoglutathione (GSNO) underwent spontaneous chemical transformation that generated several glutathione derivatives including glutathione sulfonic acid (GSO3H), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide, and glutathione disulfide. Surprisingly, GS(O)SG (also called glutathione thiosulfinate), which was not identified as a metabolite of GSNO previously, was one of the major products derived from GSNO. This compound was very reactive toward any thiol and the reaction product was a mixed disulfide. The rate of reaction of GS(O)SG with 5-mercapto-2-nitro-benzoate was nearly 20-fold faster than that of GSNO. The mechanism for the formation of GS(O)SG was believed to involve the sulfenic acid (GSOH) and thiosulfinamide (GS(O)NH2) intermediates; the former underwent self-condensation and the latter reacted with GSH to form GS(O)SG. Many reactive oxygen and nitrogen species were also capable of oxidizing GSH or GSSG to form GS(O)SG, which likely played a central role in integrating both the oxidative and nitrosative cellular responses through thionylation of thiols. Treatments of rat brain tissue slices with oxidants resulted in an enhanced thionylation of proteins with a concomitant increase in cellular level of GS(O)SG, suggesting that this compound might play a second messenger role for stimuli that produced a variety of oxidative species.

Immunochemical identification of protein kinase C isozymes as products of discrete genes

Immunocytochemical studies of rat cerebellum using specific antibodies against type I, II, and III PKC revealed the presence of the type I PKC in the Purkinje cells where transcripts of tau cDNA were localized, the type II PKC in the granule cells where transcripts of beta cDNA were detected, and the type III PKC in both the Purkinje and granule cells. Immunoblot analysis revealed that the expressed PKC in COS cells transfected with either alpha, beta, or tau cDNA of PKC were recognized by specific antibodies against the type III, II, and I PKC isozymes, respectively. These results prove that the type I, II, and III PKC are products of PKC genes, tau, beta, and alpha, respectively. With these specific antibodies we have identified the presence of multiple species of PKC in a variety of cell types.

N-Methyl-d-aspartate Induces Neurogranin/RC3 Oxidation in Rat Brain Slices

Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca2+. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter,N-methyl-d-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3–5 min and declined to base line in 30 min. The response was dose-dependent (EC50 ∼100 μm) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitorN G-nitro-l-arginine methyl ester and N G-monomethyl-l-arginine. Ng was oxidized by NO donors, sodium nitroprusside,S-nitroso-N-acetylpenicillamine, andS-nitrosoglutathione, and H2O2 at concentrations less than 0.5 mm. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca2+ and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.

Developmental expression of protein kinase C isozymes in rat cerebellum

Previously we showed that protein kinase C (PKC) isozymes (types I, II, and III) have distinctive neuronal localizations in cerebellum. In the present study, we followed the different appearances of these isozymes during the postnatal development of cerebellum. By immunoblot analysis, type I PKC was found to be low within 2 weeks after birth; an abrupt increase was observed between 2 and 3 weeks and leveled off afterwards. By immunofluorescent staining, the type I PKC-specific antibody recognized the cell bodies and dendrites of Purkinje cells. The increase of this isozyme between 2 and 3 weeks of age correlates with the spreading of Purkinje cell arborization, at which time bulk of synaptogenesis between dendritic spines and axons of granule cells occurs. Both type II and III PKCs were present in granule cells. At birth, the level of type II PKC was relatively high compared to that of type III PKC, and the type II PKC-specific antibody stained the granule cell precursors in the external layer more heavily than did the type III PKC-specific antibody. The level of type II PKC declined slightly after birth and increased again at one week and plateaued after three weeks, whereas that of type III PKC increased gradually until leveling off after three weeks. Throughout the development, the type III PKC-specific antibody also stained the cell bodies of Purkinje cells but not their dendrites. These results demonstrate that the developmental expression of PKC isozymes is under separate control, and their distinct cellular and subcellular localizations suggest their unique functions in the cerebellum.

How is protein kinase C activated in CNS.

Protein kinase C (PKC) enzyme family consists of the Ca(2+)-dependent and -independent subgroups of phospholipid/diacylglycerol (DAG)-stimulated serine/threonine protein kinases. These enzymes exhibit distinct cellular and subcellular localizations in CNS and subtle differences in their biochemical characteristics and substrate specificities. It is believed that each of these isoenzymes respond differently to different input signals. However, detailed mechanism for the functioning of these enzymes in vivo is largely unknown; this is in part due to the absence of specific activator, inhibitor, or substrate for each of these enzymes. Recent advances in biochemical, biophysical, and molecular characterizations have defined certain structural features important to confer the stimulatory responses of these enzymes to Ca2+, DAG or phorbol ester, and Zn2+; other features important for the binding of anionic phospholipids, Ca2+/phospholipid complexes, and cis-unsaturated fatty acids have not yet been characterized. Activation of PKC requires the increase in [Ca2+]i and DAG and/or cis-unsaturated fatty acids. Ca2+ promotes the interactions of the Ca(2+)-dependent subgroup of PKCs with membrane phosphatidylserine (PS) and the enzymes become partially active when simultaneously associated with phosphatidylinositol 4,5-bisphosphate or fully active when DAG is available. Free fatty acids such as arachidonic acid, generated by the activation of phospholipase A2, could synergize with DAG to activate the enzyme maximally. The Ca(2+)-independent subgroup of PKCs also become active when associated with PS at elevated level of DAG. Sustained activation of PKCs leads to the conversion of these enzymes into membrane-inserted and membrane protein-associated forms, which may be responsible for certain long-term neural responses. Activation of PKC results in the phosphorylation of cellular proteins; among them, several calmodulin (CaM)-binding proteins are the prominent substrates of these kinases. Phosphorylation of these proteins by PKC favors the release of CaM, which is required for the Ca2+/CaM-dependent enzymes. Thus, activation of PKCs can lead to diverse cellular responses through such amplification steps. Future studies should be directed at the elucidation of the activation of each PKC isoform in vivo to correlate with the physiological responses.

Phosphorylation of HMG-I by Protein Kinase C Attenuates Its Binding Affinity to the Promoter Regions of Protein Kinase C γ and Neurogranin/RC3 Genes

Abstract: A 20‐kDa DNA‐binding protein that binds the AT‐rich sequences within the promoters of the brain‐specific protein kinase C (PKC) γ and neurogranin/RC3 genes has been characterized as chromosomal nonhistone high‐mobility‐group protein (HMG)‐I. This protein is a substrate of PKC α, β, γ, and δ but is poorly phosphorylated by PKC ε and ζ. Two major (Ser44 and Ser64) and four minor phosphorylation sites have been identified. The extents of phosphorylation of Ser44 and Ser64 were 1:1, whereas those of the four minor sites all together were <30% of the major one. These PKC phosphorylation sites are distinct from those phosphorylated by cdc2 kinase, which phosphorylates Thr53 and Thr78. Phosphorylation of HMG‐I by PKC resulted in a reduction of DNA‐binding affinity by 28‐fold as compared with 12‐fold caused by the phosphorylation with cdc2 kinase. HMG‐I could be additively phosphorylated by cdc2 kinase and PKC, and the resulting doubly phosphorylated protein exhibited a > 100‐fold reduction in binding affinity. The two cdc2 kinase phosphorylation sites of HMG‐I are adjacent to the N terminus of two of the three predicted DNA‐binding domains. In comparison, one of the major PKC phosphorylation sites, Ser64, is adjacent to the C terminus of the second DNA‐binding domain, whereas Ser44 is located within the spanning region between the first and second DNA‐binding domains. The current results suggest that phosphorylation of the mammalian HMG‐I by PKC alone or in combination with cdc2 kinase provides an effective mechanism for the regulation of HMG‐I function.

Conversion of protein kinase C from a Ca2+-dependent to an independent form of phorbol ester-binding protein by digestion with trypsim

Tryptic fragments of protein kinase C containing the kinase (45 KDa) and phorbol ester-binding activity (38 KDa) were separated by Mono O column chromatography. The purified phorbol ester-binding fragment exhibits a higher affinity for phosphatidylserine than the native enzyme but comparable Kd for [3H]phorbol 12,13-dibutyrate as the native enzyme. This proteolytic fragment binds phorbol ester equally efficient either in the presence or absence of Ca2+ and the addition of the kinase fragment did not restore the Ca2+-requirement for the binding. These results indicate that protein kinase C is composed of two functionally distinct units which can be expressed independently after limited proteolysis with trypsin.

Nitric Oxide Modification of Rat Brain Neurogranin IDENTIFICATION OF THE CYSTEINE RESIDUES INVOLVED IN INTRAMOLECULAR DISULFIDE BRIDGE FORMATION USING SITE-DIRECTED MUTAGENESIS

Neurogranin (Ng) is a neuron-specific protein kinase C-selective substrate, which binds calmodulin (CaM) in the dephosphorylated form at low levels of Ca2+. This protein contains redox active Cys residues that are readily oxidized by several nitric oxide (NO) donors and other oxidants to form intramolecular disulfide. Identification of the Cys residues of rat brain Ng, Cys3, Cys4, Cys9, and Cys51, involved in NO-mediated intramolecular disulfide bridge formation was examined by site-directed mutagenesis. Mutation of all four Cys residues or single mutation of Cys51 blocked the oxidant-mediated intramolecular disulfide formation as monitored by the downward mobility shift under nonreducing SDS-polyacrylamide gel electrophoresis. Single mutation of Cys3, Cys4, or Cys9 or double mutation of any pair of these three Cys residues did not block such intramolecular disulfide formation, although the rates of oxidation of these mutant proteins were different. Thus, Cys51 is an essential pairing partner in NO-mediated intramolecular disulfide formation in Ng. Cys3, Cys4, and Cys9 individually could pair with Cys51, and the order of reactivity was Cys9 > Cys4 > Cys3, suggesting that Cys9 and Cys51 form the preferential disulfide bridge. In all cases tested, the intramolecularly disulfide bridged Ng proteins displayed dramatically attenuated CaM-binding affinity and ∼2-3-fold weaker protein kinase C substrate phosphorylation activity. The data indicate that the N-terminal Cys3, Cys4, and Cys9 are in close proximity to the C-terminal Cys51 in solution. The disulfide bridge between the N- and C-terminal domains of Ng renders the central CaM-binding and phosphorylation site domain in a fixed conformation unfavorable for binding to CaM and as a substrate of protein kinase C.