Pseudosubstrate Inhibition of the Anaphase Promoting Complex

细胞周期的正常运作及其对内部反应的调节 而外界刺激对于正常的生长、分裂和分化是必不可少的 细胞和组织。对细胞周期进程的主要控制是由泛素- 介导的蛋白质分解。例如，启动染色体分离(后期) 而细胞周期的完成需要降解多个细胞周期 调节蛋白质，如细胞周期蛋白、Securin(调节蛋白质持有量 染色体)和微管马达蛋白。泛素连接酶(或E3) 这种蛋白分解被称为后期促进复合体(APC)。蛋白质是 活性的两种底物识别蛋白cdc20和cdh1靶向APC 分别在有丝分裂晚期和G1期。监管中的一个新兴主题 APC涉及到假底物的作用，即本身不是APC的蛋白质 底物，但与底物竞争通过 在APC底物中发现销毁盒和肯盒序列。Acm1是一种最近的- 在芽殖酵母中鉴定出APCCDh1的假底物抑制物。除了 销毁盒子和KEN盒子，Acm1包含一个新的主题，使其 与CDH1的相互作用。Acm1也受到泛素介导的蛋白降解，这两种方式都是通过 APCCDc20在有丝分裂期间以及在整个细胞周期中以一种未知的机制表达，以及 通过磷酸化保护这两条途径不受影响。 为了加深我们对APC通过假底物抑制调控的理解， 我们提出了以下具体目标： 1)确定Acm1基序和细胞通路 降解和Acm1磷酸化在保护其免受降解中的作用(S)。 2)利用遗传筛选鉴定新的人和酵母假底物APC 抑制剂。 我们还将进一步探索脊椎动物Emi1蛋白作为 一种假底物抑制剂。

Proper functioning of the cell cycle and its regulation in response to both internal and external stimuli is essential for the normal growth, division, and differentiation of cells and tissues. Major control over cell cycle progression is exerted by ubiquitin- mediated proteolysis. For instance, the initiation of chromosome separation (anaphase) and completion of the cell cycle require the degradation of a number of cell cycle regulatory proteins such as cyclins, securin (which regulates the protein holding chromosomes together), and microtubule motor proteins. The ubiquitin ligase (or E3) for this proteolysis is termed the Anaphase Promoting Complex (APC). Proteins are targeted to the APC by two substrate recognition proteins, Cdc20 and Cdh1, active during late mitosis and in G1, respectively. An emerging theme in the regulation of the APC involves the actions of pseudosubstrates, proteins that are not themselves APC substrates but that compete with substrates for binding to Cdc20 and Cdh1 via the Destruction Box and KEN Box sequences found in APC substrates. Acm1 is a recently- identified pseudosubstrate inhibitor of APCCdh1 in budding yeast. In addition to a Destruction Box and a KEN box, Acm1 contains a novel motif that facilitates its interaction with Cdh1. Acm1 is also subject to ubiquitin-mediated proteolysis, both by APCCdc20 during mitosis and by an unidentified mechanism throughout the cell cycle, and is protected from both pathways via phosphorylation. To further our understanding of APC regulation via pseudosubstrate inhibition, we propose the following Specific Aims: 1) To determine the Acm1 motifs and cellular pathways responsible for Acm1 degradation and the role(s) of Acm1 phosphorylation in protecting it from degradation. 2) To use genetic screens to identify novel human and yeast pseudosubstrate APC inhibitors. We will also further explore the mechanism by which the vertebrate Emi1 protein acts as a pseudosubstrate inhibitor.