Molecular mechanism of steroid receptor ubiquitination

类固醇受体泛素化的分子机制

• 批准号: 13670135
• 负责人: OKANO Yukio
• 金额: $ 2.24万
• 依托单位: Gifu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670135/
• 关键词: 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

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.

进行酵母两杂化筛选，以阐明N末端扩展的泛素偶联酶，UBE2E2的生物学功能，并获得了几个阳性克隆。其中，编码无名指蛋白的ARA54和RNF8进一步研究了。在体内和体外观察到在UBE2E2存在下ARA54/RNF8的多泛素化，这表明这些功能作为E3S。但是，ARA54并未增强雄激素受体（AR）的泛素化。已证明ARA54在核中将核和细胞质和RNF8定位。我们进一步证明RNF8通过两个杂交测定法与类维生素类Z受体（RXR）相互作用。具有各种缺失突变体的实验表明，两种蛋白质的N末端结构域对于它们的相互作用都很重要。通过体外结合实验和FRET分析，确认了RNF8和RXR的关联。尽管RNF8的共转染并未增强RXR的泛素化，但使用UBE2E2共转染增强了RXR。另一方面，RNF8显着增强了RXR的反式激活。存在N末端缺失突变体（△N）或环中破坏点突变体（DN），从而消除了这种激活。如免疫荧光研究所揭示的那样，RNF8的△n和dn突变体都无法定位在核中，这表明RNF8的核定位对于反式激活活性很重要。

项目成果

Kimura M.: "Identification and assignment of the human NIMA-related protein kinase 7 gene(NEK7) to human chromosome 1q31.3."Cytogenet.Cell Genet.. 94. 33-38 (2001)

Kimura M.：“人类 NIMA 相关蛋白激酶 7 基因 (NEK7) 到人类染色体 1q31.3 的鉴定和分配。”Cytogenet.Cell Genet.. 94. 33-38 (2001)

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)

Crosio, C.、Fimia, G.M.、Loury, R.、Kimura, M.、Okano, Y.、Zhou, H.、Sen, S.、Allis, C.D.

Marumoto T.: "Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells"Genes to Cells. 7. 1173-1182 (2002)

Marumoto T.：“极光 A 激酶在哺乳动物细胞有丝分裂进入和 G2 检查点中的作用”《基因到细胞》。

Kimura, M. and Okano, Y: "Identification and assignment of the human NIMA-related protein kinase 7 gene (NEK7) to human chromosome 1q31.3."Cytogenet Cell Genet. 94. 33-38 (2001)

Kimura, M. 和 Okano, Y：“人类 NIMA 相关蛋白激酶 7 基因 (NEK7) 到人类染色体 1q31.3 的识别和分配。”Cytogenet 细胞基因。

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)

Adachi, S.、Okuno, M.、Matsushima-Nishiwaki, R.、Takano, Y.、Kojima, S.、Friedman, S.L.、Moriwaki, H. 和 Okano, Y：“类视黄醇 X 受体的磷酸化抑制其泛素化