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Analysis of G0 phase-specific large complex including p27

G0 期特异性大复合物（包括 p27）的分析

基本信息

批准号：
18570171
负责人：
ISHIDA Noriko
金额：
$ 2.57万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570171/
关键词：
cell cycle affinity chromatography CDK inhibitor GO phase p27 蛋白質細胞周期

项目摘要

We have tried to identify the components of GO-specific large complex including p27 by analytical method for detection of difference between asynchronous and growth arrested in GO phase culture of HEK293T cells.Previously, we reported that the abundance of p27 was decreased in many organs, including brain, thymus. spleen and testis of p27^<Ser10A/Ser10A> knock-in mice. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27^<Ser10A/Ser10A> mice compared with wild-type cells. Then, we analyzed p27 in G0 phase by size fractionation using gel-filtration and obtained that p27 formed large complex specifically in G0 phase inhibitor. By affinity column chromatography using anti-FLAG antiboby and gel-filtration chromatography, we recovered a free-FLAG-p27, and could collect FLAG-p27 interacting proteins. Then we succeeded to identify 26 p27 interacting proteins (including 13 novel and 13 already-known) in total. Whereas we tried to identify the proteins were associated to p27 specifically in G0 phase by comparison of proteins between asynchronous and growth arrested culture cells, we could not succeed. However, we identified PP2A subunit A (Protein Phosphatase 2A) as a novel binding protein of p27 plays an important role for the stability of p27 becomes to be unstable. Based on this hypothesis, we are examining the effects of PP2A on stability of p27 using phosphatase inhibitor or siRNA against PP2A. We also plan to identify the G0-specific binding proteins of p27 using NIH 3T3, like as normal cells.

我们试图通过分析方法鉴定氧化石墨烯特异性大复合体的成分，包括p27，以检测HEK293T细胞氧化石墨烯期培养中异步和生长阻滞的差异。以前，我们报道了p27的丰度在许多器官中减少，包括脑，胸腺。p27^<Ser10A/Ser10A>敲入小鼠的脾脏和睾丸。与野生型细胞相比，p27^<Ser10A/Ser10A>小鼠淋巴细胞G0期p27的稳定性明显降低。然后，我们用凝胶过滤法对p27在G0相中进行粒度分馏分析，得到p27在G0相抑制剂中特异性形成大配合物。通过抗flag抗体亲和柱层析和凝胶过滤层析，我们回收了一个自由的FLAG-p27，并可以收集FLAG-p27相互作用的蛋白。然后我们成功地鉴定了26个p27相互作用蛋白（包括13个新的和13个已知的）。虽然我们试图通过比较异步和生长阻滞培养细胞之间的蛋白质来确定G0期特异性与p27相关的蛋白质，但我们无法成功。然而，我们发现PP2A亚基A（蛋白磷酸酶2A）作为p27的一种新的结合蛋白，在p27的稳定性变得不稳定的过程中起着重要的作用。基于这一假设，我们正在使用磷酸酶抑制剂或siRNA来检测PP2A对p27稳定性的影响。我们还计划像正常细胞一样，使用NIH 3T3鉴定p27的g0特异性结合蛋白。