| Project/Area Number |
13670135
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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 |
Pathological medical chemistry
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| Research Institution | Gifu University |
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Principal Investigator |
OKANO Yukio Gifu University, School of Medicine, Professor, 医学部, 教授 (10177066)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Masashi Gifu University, School of Medicine, Research associate, 医学部, 助手 (40260575)
YOSHIOKA Takashi Gifu University, School of Medicine, Research associate, 医学部, 助手 (20311699)
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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 |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | UBE2E2 / yeast two-hybrid / ARA54 / RNF8 / RING-finger / RXR / dominant negative / nuclear localization / FRET / RING-finger蛋白 / ユビキチン化 / リン酸化 / レチノイド誘導体 |
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| Research Abstract |
Yeast two-hybrid screening was performed to clarify the biological functions of an N-terminally extended ubiquitin-conjugating enzyme, UBE2E2, and several positive clone were obtained. Among those, ARA54 and RNF8, encoding RING-finger proteins, were further studied. Poly-ubiquitination of ARA54/RNF8 in the presence of UBE2E2 was observed in vivo and in vitro, suggesting that these function as E3s. However, ARA54 did not enhance ubiquitination of androgen receptor (AR). It was demonstrated that ARA54 localizes both nuclear and cytoplasm and RNF8 in the nucleus.
We further demonstrated that RNF8 interacts with retinoid Z receptor (RXR) by two-hybrid assay. Experiments with various deletion mutants revealed that the N-terminal domains of both proteins are important for their interaction. The association of RNF8 and RXR was confirmed with in vitro binding experiment and FRET assay. Although ubiquitination of RXR was not enhanced by co-transfection of RNF8, it was enhanced with UBE2E2 co-transfection. On the other hand, transactivation of RXR was significantly enhanced by RNF8. This activation was abolished by the presence of either an N-terminal deletion mutant (△N) or a RING-disrupted point mutant (DN). Both △N and DN mutant of RNF8 failed to localize in the nucleus as revealed by immunofluorescence study, indicating that nuclear localization of RNF8 is important for the transactivation activity.
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