2002 Fiscal Year Final Research Report Summary
Molecular mechanism of steroid receptor ubiquitination
| 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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| Keywords | UBE2E2 / yeast two-hybrid / ARA54 / RNF8 / RING-finger / RXR / dominant negative / nuclear localization |
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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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Research Products
(12 results)
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[Publications] Ito, K, Adachi, S., Iwakami, R., Yasuda, H., Muto, Y., Seki, N. and Okano, Y.: "N-terminally extended human ubiquitin-conjugating enzymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8"Eur J Biochem. 268. 2725-2732 (2001)
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「研究成果報告書概要(欧文)」より
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[Publications] Muto, Y., Matsuoka, T., Kida, A., Okano, Y. and Kirino, Y.: "Blepharismins, produced by the protozoan, Blepharisma japonicum, form ion-permeable channels in planar lipid bilayer membranes"FEBS Lett. 508. 423-426 (2001)
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「研究成果報告書概要(欧文)」より
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[Publications] Marumoto, T., Hirota, T., Morisaki, T., Kunitoku, N., Zhang, D., Ichikawa, Y., Sasayama, T., Kuninaka, S., Mimori, T., Tamaki, N., Kimura, M., Okano, Y. and Saya, H.: "Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells"Genes to Cells. 7. 1173-1182 (2002)
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「研究成果報告書概要(欧文)」より
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[Publications] Crosio, C., Fimia, G.M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C.D. and Sassone-Corsi, P.: "Mitotic phosphorylation of histone H3 : Spatio-temporal regulation by mammalian aurora kinases"Mol Cell Biol. 22. 874-885 (2002)
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[Publications] Adachi, S., Okuno, M., Matsushima-Nishiwaki, R., Takano, Y., Kojima, S., Friedman, S.L., Moriwaki, H. and Okano, Y: "Phosphorylation of retinoid X receptor suppresses its ubiquitination in human hepatocellular caricinoma"Hepatology. 35. 332-340 (2002)
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「研究成果報告書概要(欧文)」より
