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.

通过酵母双杂交筛选，阐明了N端延伸的泛素结合酶UBE2E2的生物学功能，并获得了几个阳性克隆，其中编码RING-finger蛋白的ARA54和RNF8被进一步研究。 - 在体内和体外观察到 UBE2E2 存在时 ARA54/RNF8 的泛素化，表明这些功能与 E3 一样，但 ARA54 却如此。不增强雄激素受体（AR）的泛素化。已证明ARA54定位于细胞核和细胞质，并且RNF8位于细胞核中。我们通过使用各种缺失突变体的双杂交实验进一步证明RNF8与类视黄醇Z受体（RXR）相互作用。研究表明，两种蛋白的 N 末端结构域对于它们的相互作用很重要。体外结合实验和 FRET 测定证实了 RNF8 和 RXR 的关联。 RNF8 的共转染并未增强 RXR 的泛素化，而 UBE2E2 共转染则增强了 RXR 的泛素化。另一方面，RNF8 的存在显着增强了 RXR 的反式激活。突变体（△N）或RING破坏的点突变体（DN），RNF8的△N和DN突变体均未能定位于细胞核。通过免疫荧光研究，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