Mechanism of Mitotic Checkpoint in Mammalian Cells

描述(申请人提供)：我的实验室的长期目标是获得对非整倍体在肿瘤发生中的作用的机械性见解。我们目前的重点是研究关键的机械和调控事件，这些事件指定了人类细胞中准确的染色体分离，对于理解致癌机制和开发新的杀死癌细胞的方法具有直接的重要意义。我们一直专注于鉴定和表征着丝粒的分子组成，以此作为了解准确染色体分离的分子要求的主要努力。这项提议的目标是研究这些蛋白质中的一些如何共同作用，形成一个将动粒：微管相互作用连接到有丝分裂检查点途径的功能动粒。我们将使用分子、生化和微观方法来实现我们的目标。这项建议将集中在进化保守的检查点激酶，hBUB 1和MPS 1，以及后期促进复合体(APC)的CDC27亚单位。我们将研究hBUB 1指定动粒亚域组装的机制，该亚域由检查点蛋白hBUBR1、MAD1、MAD2和CDC20组成。这些研究对于理解检查点蛋白和动点蛋白之间相互作用的动态性质至关重要。我们将通过检测HMPS 1在有丝分裂检查点通路中的重要性以及在将信号从独立的动点传递到APC中的重要性来表征它在有丝分裂检查点通路中的作用。最后，我们有了新的发现，APC的CDC27亚基促进了对APC的检查点抑制，在人类和萌芽的酵母中都是如此。我们对CDC27如何完成这一过程的分析提供了一种独特的“自下而上”的方法来研究信号通路，该通路允许具有甚至单个未附着的染色体的细胞过早退出有丝分裂。

DESCRIPTION (provided by applicant): The long-term objective of my lab is to obtain mechanistic insights into the role of aneuploidy in tumorogenesis. Our current focus on studying the key mechanical and regulatory events that specify accurate chromosome segregation in human cells is of direct importance towards understanding mechanisms that cause cancer and for the development of novel approaches to kill cancer cells. We have focused on identifying and characterizing the molecular components of the kinetochore as a major effort towards understanding the molecular requirements for accurate chromosome segregation. The goals of this proposal are to examine how some of these proteins work together to make a functional kinetochore that links kinetochore:microtubule interactions to the mitotic checkpoint pathway. We will use molecular, biochemical and microscopic approaches to accomplish our goals. This proposal will focus on the evolutionary conserved checkpoint kinases, hBUB 1 and MPS 1, and the Cdc27 subunit of the Anaphase Promoting Complex (APC). We will examine the mechanism by which hBUB 1 specifies the assembly of subdomain of the kinetochore that consists of checkpoint proteins hBUBR1, MAD1, MAD2 and Cdc20. These studies will be critical for understanding the dynamic nature of the interactions between checkpoint proteins and kinetochores. We will characterize the role of the hMPS 1 kinase in the mitotic checkpoint pathway by examining its importance at kinetochores and in transducing the signal from unattached kinetochores to the APC. Lastly, we have made the novel discovery that the Cdc27 subunit of the APC facilitates checkpoint inhibition of the APC in both humans and budding yeast. Our analysis of how Cdc27 accomplishes this provides a unique "bottoms-up" approach to study the signaling pathway that allows cells with even a single unattached chromosome from prematurely exiting mitosis.