Molecular analyses of necdin-induced cell growth suppression.

necdin 诱导的细胞生长抑制的分子分析。

• 批准号: 10670123
• 负责人: TANIURA Bideo
• 金额: $ 2.05万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670123/
• 关键词: necdin; p53; pRb; large T antigen; E2F1; postmitotic neuron; growth suppression; transcription factor; E2F; 核マトリックス; 分化; 細胞周期; 転写因子; GI期; G1期

Necdin is a nuclear protein, whose gene is expressed in postmitotic neurons from early embryonal stages. Ectopic expression of necdin suppresses the growth of proliferative NIH3T3 cells. SV40 large T antigen targets both p53 and pRb for cellular transformation. By analogy with the interactions of the large T antigen with these nuclear growth suppressors, we examined the ability of necdin to bind to the large T antigen. Necdin was co-immunoprecipitated with the large T antigen from the nuclear extract of necdin cDNA-transfected COS1 cells. Yeast two-hybrid and in vitro binding analyses revealed that necdin bound to an amino- terminal region of the large T antigen, which encompasses the pRb-binding domain. We then examined the ability of necdin to bind to the transcription factor E2F1, cellular pRb-binding factor involved in cell-cycle progression. Intriguingly, necdin bound to a carboxyl-terminal domain of E2Fl, and repressed E2F-dependent transactivation in vivo. In addition, necdin su … More ppressed the colony formation of Rb-deficient SAOS2 cells. These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to pRb. There is a substantial degree of homology between the E2F1 and p53 activation domains, suggesting a conservation of binding sites for specific proteins. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient SAOS2 cells, necdin markedly suppressed p53-dependent activation of the p21 promoter. Necdin and p53 suppressed cell growth without apoptosis as assessed by the colony formation of SAOS2 cells. On the other hand, necdin inhibited p53-induced apoptosis of U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons. Less

Necdin是一种核蛋白，其基因在胚胎早期的有丝分裂后神经元中表达。necdin的异位表达抑制增殖的NIH 3 T3细胞的生长。SV 40大T抗原靶向p53和pRb用于细胞转化。通过与大T抗原与这些核生长抑制因子的相互作用类比，我们研究了necdin与大T抗原结合的能力。Necdin与来自Necdin cDNA转染的COS 1细胞的核提取物的大T抗原共免疫沉淀。酵母双杂交和体外结合分析显示necdin结合到大T抗原的氨基末端区域，其包含pRb结合结构域。然后，我们研究了necdin与转录因子E2 F1结合的能力，E2 F1是参与细胞周期进程的细胞pRb结合因子。有趣的是，necdin结合到E2 F1的羧基末端结构域，并在体内抑制E2 F依赖的反式激活。此外，necdin su ...更多信息 抑制Rb缺陷SAOS 2细胞的殖民地形成。这些结果表明，necdin是一个有丝分裂后的神经元特异性生长抑制剂，是功能相似的pRb。在E2 F1和p53激活结构域之间存在相当程度的同源性，这表明特定蛋白的结合位点是保守的。酵母双杂交和体外结合分析表明，necdin绑定到一个狭窄的区域之间的MDM 2-结合位点和脯氨酸丰富的区域的氨基末端结构域的p53。电泳迁移率变动分析表明，necdin supershifted p53和它的结合DNA之间的复合物，这意味着p53-necdin复合物是胜任的DNA结合。在p53缺陷的SAOS 2细胞中，necdin显著抑制p53依赖的p21启动子的激活。Necdin和p53抑制细胞生长而不凋亡，如SAOS 2细胞的殖民地形成所评估的。另一方面，necdin抑制p53诱导的U2 OS细胞凋亡。因此，necdin可以是一种生长抑制剂，靶向p53，并调节其在有丝分裂后神经元的生物学功能。少

项目成果

Y.Nakada: "The human chromosomal gene for necdin, a neuronal growthsuppressor, in the Prader-Willi syndrome deletion region."Gene. 213. 65-72 (1998)

Y.Nakada：“神经元生长抑制剂 necdin 的人类染色体基因，位于普瑞德威利综合征缺失区域。”基因。