Functional analysis of a novel GTP-binding protein involved in apoptosis.

参与细胞凋亡的新型 GTP 结合蛋白的功能分析。

• 批准号: 12480213
• 负责人: INOUE Jun-ichiro
• 金额: $ 4.86万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480213/
• 关键词: GTP-binding protein; Cell cycle; Apoptosis; RNP K homology domain; RNA-binding; protein, DT40; 細胞増殖; アポートシス; 遺伝子破壊; Gタンパク質; 翻訳後修飾

We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. Thus, H-ERA may play an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain. To elucidate the physiological function of eukaryotic ERA, we have performed a genetic analysis of chicken ERA (GdERA) in DT40 cells. Depletion of GdERA diminished the growth rate of the cells, accompanied by an accumulation of apoptotic cells. The analysis of cell cycle indicates that the elimination of GdERA caused arrest at G1 phase, but not at M phase, which highlights the distinct role of vertebrate ERA in the cell cycle progression compared to prokaryotic ERA. Furthermore, human ERA (HsERA) rescued the phenotype of GdERA-deficient cells, whereas a mutant of HsERA deprived of RNA binding activity did not. These data suggest that vertebrate ERA regulates the G1 phase progression via an as yet unknown molecular mechanism, which involves RNA recognition by ERA.

我们克隆了编码人类ERA同源物(H-ERA)和小鼠ERA同源物(M-ERA)整个编码区的cDNA。ERA在哺乳动物中的同源物由一个典型的GTPase/GTP结合域和一个可能的K同源(KH)结构域组成，被称为RNA结合域。我们用野生型H-ERA或各种H-ERA突变体进行了转染实验。含有GTP酶氨基酸替换突变的H-ERA可诱导HeLa细胞凋亡，其作用可被Bcl2基因表达所阻断。含有部分KH结构域的C末端的缺失减轻了H-ERA突变体的细胞凋亡，表明该结构域在H-ERA功能中的重要性。我们还用固定在珠子或重组H-ERA蛋白上的RNA均聚体进行了下拉实验，证明了H-ERA的RNA结合活性。因此，H-ERA可能通过其GTPase/GTP结合域在调节细胞凋亡信号中发挥重要作用。为了阐明真核细胞ERA的生理功能，我们在DT40细胞中进行了鸡ERA(GdERA)的遗传分析。GdERA的缺失降低了细胞的生长速度，伴随着凋亡细胞的积累。细胞周期分析表明，GdERA的消除导致细胞周期停滞于G1期，而不是M期，这突显了脊椎动物ERA在细胞周期进程中与原核生物ERA不同的作用。此外，人类ERA(HsERA)挽救了GdERA缺陷细胞的表型，而失去RNA结合活性的HsERA突变体则没有。这些数据表明，脊椎动物ERA通过一种未知的分子机制调节G1期的进展，其中涉及ERA对RNA的识别。

项目成果

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Kobayashi,N.: "Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis"EMBO JOURNAL. (in press). (2001)

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Jin Gohda: "Elimination of the vertebrate Escherichia coli Ras-like protein homologue leads to cell cycle arrest at G1 phase and apoptosis"Oncogene. (in press). (2003)

Jin Gohda：“消除脊椎动物大肠杆菌 Ras 样蛋白同源物导致细胞周期停滞在 G1 期并凋亡”癌基因。

Akiyama, T: "Mammalian homologue of E.Ras-like GTPase(ERA) is a possible apoptosis regulator with RNA binding activity"Cenes Cells. 6. 987-1001 (2001)

Akiyama, T：“E.Ras 样 GTP 酶 (ERA) 的哺乳动物同源物是一种可能具有 RNA 结合活性的细胞凋亡调节剂”Cenes Cells。