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

在这项研究中，我们分析了两种主要的肿瘤抑制基因产物如p27^和RB蛋白的蛋白水解降解的分子机制<Kip1>。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功能（2）。另一个项目是确定p27的下调机制^<Kip1>[1]。(1)p27的下调机制我们<Kip1>以及其他研究小组报道，p27<Kip1>在预后不良的人类肿瘤中被有效降解。靶向破坏Skp2（一种p27 α的泛素连接酶<Kip1>）导致p27 α在体内的降解减少<Kip1>。然而，p27Kip1降解的另一种机制被强烈地提出。我们已经报道了通过两种机制下调，一种是泛素依赖的途径，另一种是位点特异性切割。我们应该能找到裂解酶。在这项研究中，我们确定Ser10是p27 α的主要磷酸化位点<Kip1>。Ser10的磷酸化稳定了<Kip1>培养细胞中的p27 α。(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，并利用生化方法成功获得了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踪实验证实了这一点。RB蛋白的降解可能是其恶性转化的新机制。

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.