| Project/Area Number |
10470031
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | Kobe University |
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Principal Investigator |
KIKKAWA Ushio Kobe University, Biosiganl Research Center, Professor, バイオシグナル研究センター, 教授 (40150354)
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| Co-Investigator(Kenkyū-buntansha) |
TOUHARA Kazushige The University of Tokyo, Graduate School of Frontier Science, Associate Professor, 大学院・新領域創成科学研究科, 助教授 (00280925)
SAKAI Norio Kobe University, Biosiganl Research Center, Associate Professor, バイオシグナル研究センター, 助教授 (70263407)
KONISHI Hiroaki Kobe University, Biosiganl Research Center, Assistant, バイオシグナル研究センター, 助手 (40252811)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥9,700,000 (Direct Cost: ¥9,700,000)
Fiscal Year 1999: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Keywords | PKC / diacylglycerol / protein kinase / tyrosine phosphorylation / hydrogen peroxide / mutant / GFP-fusion protein / membrane translocation |
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| Research Abstract |
It has been shown that PKC, which comprises a family of ten isoforms, is activated by 1, 2-diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On the other hand, the PKC isoforms have been indicated to be tyrosine phosphorylated in response to the treatment of cells with hydrogen peroxide (HィイD22ィエD2OィイD22ィエD2). Among the PKC isoforms, PKCδ is efficiently tyrosine phosphorylated and catalytically activated. In this study, phosphorylation-dependent activation of PKCδ was analyzed. Firstly, the intracellular localization of PKCδ was observed under confocal microscope by monitoring the fluorescence of the GFP-fusion protein of PKCδ. The GFP-fusion protein was translocated to the membrane by the stimuli generating 1, 2-diacylglycerol, whereas the fusion protein did not move in the HィイD22ィエD2OィイD22ィエD2-treated cells. Therefore, PKCδ was concluded to be activated by HィイD22ィエD2OィイD22ィエD2 in a manner independent of the membrane translocation induced by 1, 2-dia
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cyglycerol. It is well known that HィイD22ィエD2OィイD22ィエD2 suppresses the protein-tyrosine phosphatase reaction and thus increase tyrosine phosphorylation of various cellular proteins. Several protein-tyrosine phosphatase inhibitors, however, did not induce tyrosine phosphorylation of PKCδ, indicating that activation of protein-tyrosine kinase rather than inhibition of protein-tyrosine phosphatase is necessary for the covalent modification of PKCδ. Phosphotyrosine residues were determined by the study of a series of mutant protein of PKCδ and by the mass spectrometric analysis of the phosphopetides of PKCδ recovered from the HィイD22ィエD2OィイD22ィエD2-treated cells. Three tyrosine residues were identified, and in vitro phosphorylation of these tyrosine residues by Lck, a non-receptor type protein-tyrosine kinase, activated the enzyme. Activation of PKCδ by tyrosine phosphorylation was detected even three hours after the HィイD22ィエD2OィイD22ィエD2 treatment, and thus the active enzyme may have an important role in the long-term responses of the cells to the extracellular stimuli. Less
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