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Application of ubiquitin ligase against tumor suppressor gene products for cancer therapy

泛素连接酶针对抑癌基因产物在癌症治疗中的应用

基本信息

批准号：
12480192
负责人：
KITAGAWA Masatoshi
金额：
$ 9.09万
依托单位：
Hamamatsu University School of Medicine
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

项目摘要

In this study, we analyzed the molecular mechanisms of proteolytic degradation of two major tumor suppressor gene products such as p27^<Kip1> and RB protein. Fast of all, we tried to identify the ubiquitin ligase for RB protein and analyze its function in malignant transformation process (2). The other project is identification of the down-regulation mechanisms of p27^<Kip1> (1).(1) Down-regulation mechanisms of p27^<Kip1>We as well as the other groups reported that p27^<Kip1> is efficiently degraded in human tumors with poor prognosis. Target disruption of Skp2, a ubiquitin ligase for p27^<Kip1>, resulted to decrease degradation of p27^<Kip1> in vivo. However, another mechanism of p27Kip1 degradation was stronglt suggested. We have reported that are down regulated by two mechanisms, one is ubiquitin dependent pathway and the other is site specific cleavage. We should identify the cleavage enzyme. In this study, we identified Ser10 as a major phosphorylation site for p27^<Kip1>. The phosphorylation at Ser10 stabilized p27^<Kip1> in cultured cells.(2) Identification of ubiquitin ligase for RB proteinWe established in vitro ubiquitination assay of RB protein and successfully obtain the candidate protein for ubiquitin ligase for RB protein using biochemical approach. In this study, it was confirmed by in vivo ubiquitination analysis and pulse chase experiment. It may be a novel mechanisms in malignant transformation via proteolytic degradation of RB protein.

在本研究中，我们分析了两个主要的抑癌基因产物p27^&lt；Kip1&gt；和Rb蛋白的蛋白质降解的分子机制。首先，我们试图鉴定Rb蛋白的泛素连接酶，并分析其在恶性转化过程中的作用(2)。另一项工作是确定p27^&lt；Kip1&gt；的下调机制(1)。(1)p27；；kip1&gt；的下调机制。我们和其他研究小组报道，p27^&lt；Kip1&gt；在人类肿瘤中被有效降解，预后较差。靶向阻断p27^&lt；Kip1&gt；泛素连接酶Skp2导致p27^&lt；Kip1&gt；体内降解减少。然而，p27Kip1的另一种降解机制被提出。我们已经报道了两种机制下调，一种是泛素依赖途径，另一种是位点特异性切割。我们应该鉴定裂解酶。在本研究中，我们确定Ser10是p27^&lt；Kip1&gt；的主要磷酸化位点。(2)Rb蛋白泛素连接酶的鉴定我们建立了Rb蛋白的体外泛素化实验，并通过生化方法成功地获得了Rb蛋白泛素连接酶的候选蛋白。本研究通过体内泛素化分析和脉冲追逐实验证实了这一点。Rb蛋白的蛋白降解可能是其恶性转化的一种新机制。