Covalent modifications of protein by phosphorylation and oxidation are important mechanisms for the modulation of a plethora of cellular responses. Protein phosphorylation catalyzed by a variety of protein kinases and oxidation by many endogenously produced oxidants have been linked to the regulation of cellular processes as diverse as ion channels, cellular metabolism, synaptic plasticity, and growth and differentiation. This laboratory investigates the roles of protein phosphorylation and oxidation involved in the synaptic transmission and plasticity. Studies of these neural processes are essential to the understanding of the complex problem related to learning and memory. Our approach is to generate genetically modified mice by deletion or expression of a gene specifically expressed in the brain. These efforts have led us to generate a strain of mice devoid of a neural-specific protein, neurogranin (Ng). This protein is normally expressed at high level in the neurons within cerebral cortex, hippocampus, and amygdala and has been implicated in the modulation of synaptic plasticity. Ng binds calmodulin (CaM) in an inverse Ca2+-sensitive manner; namely, its binding affinity for CaM decreases with increasing Ca2+ concentration. It is believed that at basal levels of Ca2+ all CaM is sequestered by Ng, whose cellular concentration is at least twice as much as that of CaM. Upon synaptic stimulation, the influxed Ca2+ will displace Ng from the Ng/CaM complex to form Ca2+/CaM. The buffering of CaM by Ng serves as a mechanism to regulate neuronal free Ca2+ and Ca2+/CaM concentrations. Furthermore, Ng is readily phosphorylated by protein kinase C (PKC) and oxidized by nitric oxide (NO) and other oxidants. Both the phosphorylated and oxidized Ng exhibit lower affinities for CaM than the unmodified protein; thus, these modifications of Ng extend the availability of CaM even after the intracellular level of Ca2+ is reduced to basal levels. Synaptic responses triggering long-term potentiation (LTP) or long-term depression (LTD) depend on the amplitude of Ca2+ influx and the sensitivity of the transduction machinery to amplify the signal. Based on our previous finding that the autophosphorylation of Ca2+/CaM-dependent protein kinase II was enhanced by Ng, we speculate that other Ca2+- and Ca2+/CaM-regulated pathways are also upregulated by neurogranin. The multifarious effect of Ng could account for the positive impact of this protein on the behavior of mice. Investigation of the regulatory mechanism of synaptic transmission by Ng will help us to design novel therapeutic approaches with the potential to improve memory in human and alleviate symptoms related to dementia. The concentration of hippocampal Ng in adult wild type mice was estimated to be one of the highest of the known neuronal CaM-binding proteins. Induction of LTP caused a rapid phosphorylation of this protein in the hippocampal CA1 region. Testing with the Morris water maze showed significant relationships between the levels of hippocampal Ng among heterozygous mice and their performances; however, such relationships were less significant among the wild type mice. These findings suggest that the wild type mice contain supra-threshold levels of Ng for proficient performance of these tasks. Ng knockout mice performed poorly in all spatial tasks tested and exhibited deficits in LTP and concurrent CaMKII autophosphorylation. The tetanus-frequency response curve of the knockout mice is shifted to the right compared to that of the wild type mice; low frequency stimulation (5-10 Hz) induced LTD in the former and modest LTP in the latter. Thus, Ng might regulate neuronal function by increasing the efficiency of Ca2+- and Ca2+/CaM-mediated signaling and thus improve the performance in behavioral tests. The neuronal signaling mediated by activation of NMDA receptor is one of the critical steps leading to the enhancement of synaptic plasticity that underlie learning and memory. Calcium influx induced by the activation of this receptor triggers the stimulation of PKC, Ca2+/CaM-dependent protein kinases, adenylyl cyclases, and nitric oxide synthase. It is the activations of these Ca2+- and Ca2+/CaM-dependent enzymes and their downstream targets that contribute to NMDA receptor-dependent LTP. Stimulation of PKC causes phosphorylation of Ng, which promotes the release of Ca2+ from intracellular stores through G protein-coupled phosphoinositide second messenger pathways. In addition, Ng is susceptible to oxidant-mediated modification, which causes glutathionylation and/or formation of intramolecular disulfide bonds. Using mouse hippocampal slices, we showed that NMDA induced a rapid and transient phosphorylation and oxidation of Ng. NMDA also caused activation of various PKC isozymes as evidenced by their phosphorylation, notably at the carboxyl terminal autophosphorylation sites; such activations were much reduced in the Ng knockout mice. A high degree of phosphorylation of Ca2+/CaM-dependent kinase II and activation of cAMP-dependent protein kinase were also evident in the wild type compared to those of the knockout mice. These findings demonstrate the functional role of Ng in Ca2+- and Ca2+/CaM-dependent signaling pathways subsequent to stimulation of the NMDA receptor, suggesting that both phosphorylation and oxidation of Ng may regulate neuronal signaling. Redox regulation through modifications of proteins has emerged as one of the major cellular responses to oxidative and nitrosative stresses. In the CNS, neurotransmission under normal or pathological condition generates a variety of oxidants. Thionylation of protein is one of the mechanisms that can serve as a protection against oxidative insults, as well as for cell signaling. Protein S-glutathionylation introduces the gamma-glutamyl tri-peptide into a protein with additional ionic charges resembling the well-characterized mechanism of protein phosphorylation in cellular regulation. The potential target proteins for thionylation are likely to be as abundant as those for phosphorylation. However, unlike protein phosphorylation, where numerous protein kinases have been identified, there is no evidence for the involvement of specific thionylating enzyme for each target protein. Thus, the specificity for the thionylation may be endowed within each protein depending on its affinity for the modifiers, namely, thionylating agents, and the accessibility of the sulfhydryl group. The thionylating agent has to be highly reactive and preferably with a short half-life, so that the reaction will be localized nearby the origin of the oxidant. Recently, we have identified a highly reactive glutathionylating agent, glutathione disulfide S-oxide (GS(O)SG), from the aqueous solution of S-nitrosoglutathione (GSNO), which fulfills some of these features. This compound, also called glutathione thiosulfinate, is the anhydride of glutathione sulfenic acid (GSOH). Introduction of an oxygen atom into a disulfide bond significantly decreases the bond energy and transforms it into a highly reactive agent toward thiol to form mixed disulfides. Many reactive oxygen and nitrogen species are also capable of oxidizing GSH or GSSG to form GS(O)SG, which probably plays a central role in integrating the oxidative and nitrosative cellular responses through thionylation of thiols.
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