Regulation of the Anaphase-Promoting Complex

描述(逐字摘自申请人的摘要)：通过 细胞周期依赖于调控蛋白的及时降解 泛素途径。后期促进复合体(APC)，与 它的激活剂Cdc2O或CDH1在有丝分裂中扮演泛素连接酶(E3)的角色。 降级系统。APCCdC2O泛素化后期抑制剂允许 姐妹染色单体的分离，而APCcdh1介导蛋白降解 有丝分裂周期蛋白，允许退出有丝分裂。在此提案中进行研究 主要研究APCcdh1泛素连接酶活性的细胞周期调节 在脊椎动物中。目的1研究哺乳动物纺锤体检查点的作用 MAD2及其新近发现的同系物MAD2b在细胞周期调控中的作用 APCcdh1.在前期，APCCdC2O的活性被MAD2阻断。 然而，目前还不清楚APCCDh1是否也被相同的途径抑制。 我们的初步数据表明，MAD2和Mad2b都抑制APCcdh1在 体外培养。过表达Mad2b会导致哺乳动物细胞的G2/M期延迟。这个 MAD2和Mad2b对Cdc2O或Cdh1的特异性将在 体外和体内。控制Mad2b的上行信号也将是 学习。目标2的重点是通过以下方式调节APCcdh1在后期的表达 HCdCl4磷酸酶。CDH1被cdc2磷酸化抑制APCCDh1，而 HCdCl4使CDH1去磷酸化并激活APCcdh1。HCdCl4蛋白 定位于中心体，在哺乳动物细胞中以500kD复合体的形式存在。 磷酸化抑制底物APCcdh1的机制 HCdCl4的特异性，以及hCdCl4对细胞周期的调节将是 已澄清。目标3是描述一种新的生物化学功能 HCdCl4结合蛋白(14BP1)。序列分析表明，I4BP1可能 定位于核仁，并在功能上与Bub2样蛋白相连。这个 14BP1对血管内皮细胞磷酸酶活性及细胞定位的影响 将对hCdCl4进行测试。与hCDCl4和14BP1相互作用的其他蛋白质 将会被确认。纺锤体组装检查点基因的突变 最近在人类癌症中发现，这个检查点的故障可能 导致肿瘤细胞遗传不稳定。关于规制的若干问题研究 APCcdh1将提供关于有丝分裂检查点如何停止细胞的见解 循环并呈现用于设计以下试剂的新型分子靶标 干扰这条路径。

DESCRIPTION (Verbatim from the applicant's abstract): Progression through the cell cycle depends on the timely degradation of regulatory proteins through the ubiquitin pathway. The anaphase-promoting complex (APC), in association with its activators Cdc2O or Cdh1, acts as the ubiquitin ligase (E3) in the mitotic degradation system. APCCdC2O ubiquitinates anaphase inhibitors to allow the separation of sister-chromatids, while APCcdh1 mediates the proteolysis of mitotic cyclins, permitting the exit from mitosis. Research in this proposal focuses on the cell cycle-regulation of the APCcdh1 ubiquitin ligase activity in vertebrates. Aim 1 studies the roles of the mammalian spindle checkpoint proteins Mad2 and its newly identified homolog, Mad2b, in the regulation of APCcdh1. At pro-metaphase, the activity of APCCdC2O is blocked by Mad2. However, it is unclear whether APCCdh1 is also inhibited by the same pathway. Our preliminary data indicate that both Mad2 and Mad2b inhibit APCcdh1 in vitro. Overexpression of Mad2b causes a G2/M delay in mammalian cells. The specificity of Mad2 and Mad2b toward Cdc2O or Cdh1 will be further analyzed in vitro and in vivo. The upstream signals that control Mad2b will also be studied. The focus of Aim 2 is the regulation of APCcdh1 in late anaphase by the hCdcl4 phosphatase. Phosphorylation of Cdh1 by cdc2 inhibits APCCdh1 while hCdcl4 dephosphorylates Cdh1 and activates APCcdh1. The hCdcl4 protein localizes to the centrosomes and exists as a 500 kD complex in mammalian cells. The mechanism by which phosphorylation inhibits APCcdh1, the substrate specificity of hCdcl4, and the cell cycle-regulation of hCdcl4 will be elucidated. Aim 3 is to characterize the biochemical function of a novel hCdcl4-binding protein (14BP1). Sequence analysis reveals that I4BP1 might localize to the nucleolus and be functionally linked to Bub2-like proteins. The effects of 14BP1 on the phosphatase activity and the cellular localization of hCdcl4 will be tested. Additional proteins that interact with hCdcl4 and 14BP1 will be identified. Mutations of spindle assembly checkpoint genes have recently been found in human cancers, and malfunction of this checkpoint may contribute to genetic instability of tumor cells. Research on the regulation of APCcdh1 will provide insights into how the mitotic checkpoint halts the cell cycle and present novel molecular targets for the design of agents that interfere with this pathway.