| Project/Area Number |
13680841
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Kagawa Medical University |
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Principal Investigator |
WATANABE yasuo Kagawa Medical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (10273228)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUBARA mamoru Nippon Organon, R&D Laboratories, Senior Research Scientist, 医薬研究所, 主任研究員 (90288481)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | calcium / calmodulin / neuronal NO synthase / calmodulin kinase / phosphorylation / dephosphorylation / ischemia / hippocampus / structure biology / リン酸部位特異的認識抗体 / 神経細胞 / リン酸化 / 脱リン酸化反応 / 活性制御 |
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| Research Abstract |
Purpose
Ca2+/calmodulin (CaM) -dependent protein kinase II (CaM-K II) is a broad specificity enzyme with central roles in synaptic plasticity, learning, and memory. Neuronal nitric-oxide synthase (nNOS) is also a Ca2+/CaM-dependent enzyme, which catalyzes the oxidation of L-arginine to generate nitric oxide (NO) and L-citrulline. NO, formed by nNOS, has major signaling functions in the central and peripheral nervous system. It has been established that nNOS is phosphorylated by CaM-K II linking to the decreased catalytic activity. It has been reported recently that constitutively active Cam-K Iiα can phosphorylate nNOS at Ser847, and when transfected into NG108-15 neuronal cells, can attenuate the catalytic activity of nNOS. While nNOS phosphorylation has been investigated in several studies, the reverse reaction is not well documented and the effects in vivo and the physiological consequences are not completely understood. To elucidate the dynamic regulation of nNOS via phosphorylation
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in neuronal cells, the following projects were undertaken: 1. Determination of the dominant protein phosphatase for the dephosphorylation of nNOS at Ser847, being phosphorylated by CaM-Ks in vitro.2. Regulation of nNOS by CaM-Ks in cerebral ischemia. 3. Structure biology of a target-recognition mode of Ca2+/CaM against molecles containing the CaM-binding domain.
Results and discussion
1. We have identified protein phosphates 2A as a major protein phosphatase involved in the dephosphorylation of Nnos at Ser847 using brain extracts as a source of protein phosphatase. 2. We have established that forebrain ischemia causes an increase in the phosphorylation of Nnos at Ser847 in the hippocampus. This Nnos phosphorylation appeared to be catalyzed by CaM-K II. 3. We have determined the structure of MARKS-,a membrane-associated protein essential for the development of the central nerve system, derived peptide in complex with CaM by X-ray crystallography.
Future prospect
Based on our present studies, further analyses are need to elucidate the spatial relationship of nNOS and its binding targets in timers of the regulation of nNOS via its Ser847 phosphorylation by CaM kinases. Less
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