| Project/Area Number |
12680748
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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 | Tokyo Metropolitan University |
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Principal Investigator |
ICHIMURA Tohru Graduate School of Science, Associate Professor, 理学研究科, 助教授 (50213012)
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| Co-Investigator(Kenkyū-buntansha) |
ISOBE Toshiaki Graduate School of Science, Professor, 理学研究科, 教授 (70106607)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | 14-3-3 protein / signal transduction / phosphorylation / protein kinase / PC12 cell / proteome / proteomics / タンパク質リン酸化反応 / プロテオーム解析 |
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| Research Abstract |
The 14-3-3 protein is a family of acidic, dimeric proteins distributed widely among eukaryotic cells. This protein family binds to a variety of proteins in a phosphorylation-dependent manner and participants in the regulation of cell proliferation, differentiation and function. In this study, we have attempted to screen 14-3-3-binding proteins in PC12 cells by means of proteomic techniques. We developed a novel multiple affinity tag, called myc-TEV-FLAG (MEF), and transected the MEF-tagged 14-3-3η cDNA into PC12 cells. The proteins associated with the expressed MEF-14-3-3η protein were pulled from the PC12 extracts and were analyzed by SDS-PAGE and mass spectrometry. This procedure allowed us to identify 121 proteins that include 14 proteins already reported as known targets of 14-3-3 and 107 novel interacting partners. These proteins include a series of components in the growth factor signaling pathways such as the growth factor receptor in the plasma membrane and the effectors of small GTP-binding proteins, suggesting that the 14-3-3 protein may act as a coordinator that creates functional signaling complexes in the coupled with the action of protein kinases. It was also observed that one of the identified proteins, kinesin light chain 2 (KLC2), was directly associated with 14-3-3, and their association was dependent on the phosphorylation of KLC2 at Ser575.
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