| Project/Area Number |
17570156
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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 |
Cell biology
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
KOMADA Masayuki Tokyo Institute of Technology, Dept. Biological Sciences, Associate Professor, 大学院生命理工学研究科, 助教授 (10225568)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | cancer / enzyme / cell-tissue / protein / 受容体ダウンレギュレーション / エンドソーム / 選別輸送 / メンブレントラフィック / ユビチキン / 脱ユビキチン化酵素 |
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| Research Abstract |
1.Regulation of the epidermal growth factor (EGF) receptor downregulation by a deubiquitinating enzyme UBPY
Ubiquitination of ligand-activated EGF receptor serves as a signal that targets the protein for lysosomal trafficking. We found that UBPY deubiquitinates activated EGF receptor on early endosomes and delays its downregulation. These results indicated that by removing the "lysosome-targeting signal" from the receptor and inhibiting its trafficking to lysosomes, UBPY regulates the downregulation of EGF receptor negatively.
2.Regulation of the morphology of early endosomes by a deubiquitinating enzyme UBPY
Inhibition of the UBPY function by expressing a dominant-negative mutant or siRNA resulted in the accumulation of ubiquitinated proteins on early endosomes and the morphological aberration of the organelle. Electromicroscopic study showed that early endosomes are aberrantly aggregated in UBPY-inhibited cells. These results suggested that the regulation of the level of protein ubiquit
… More
ination on early endosomes by UBPY is essential for maintaining the morphology of the organelle.
3.Regulation of the deubiquitinating activity of UBPY by 14-3-3 proteins in the M phase
We found that UBPY binds 14-3-3 proteins via a consensus 14-3-3-binding motif, RSYS^<680>SP. The binding required the phosphorylation of S^<680>. We also found that the binding of 14-3-3 inhibits the catalytic activity of UBPY. Finally, UBPY is dephosphorylated, dissociated from 14-3-3 proteins, and catalytically activated in the M phase, suggesting that elevated activity of UBPY regulates the functions of early endosomes in the M phase.
4.Mechanism of the localization of a deubiquitinating enzyme AMSH to early endosomes
We found that AMSH and its homolog AMSH-like protein (AMSH-LP) bind to the terminal domain of clathrin heavy chain via a novel ~30-amino-acid clathrin-binding motif which is conserved between AMSH and AMSH-LP. Deletion of the clathrin-binding motif from these deubiquitinating enzymes, as well as siRNA-mediated knockdown of cellular clathrin, led to their mislocalization to the cytoplasm. These results suggested that AMSH and AMSH-LP are anchored to early endosomes through the interaction with the clathrin coat on the endosomal membrane. Less
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