Proteolytic Control of Early Events in Mitosis

描述（由申请人提供）：有丝分裂事件的时间对确保准确的染色体分离和基因组稳定性至关重要。对这些定时事件至关重要的是后期促进复合体，这是一种E3泛素连接酶，它指导细胞周期蛋白A、染色体分离调节因子Securin和细胞周期蛋白b的有序破坏。APC活性的已知控制器是纺锤体组装检查点和锌结合蛋白Emi1的组成部分。Emi1的作用是抑制S和G中的APC，从而允许细胞周期蛋白的积累。Emi1在G1期通过细胞周期蛋白D/Rb/E2F途径转录激活，并在SCFI3TrCP泛素连接酶磷酸化特异性结合后的早期有丝分裂中被破坏。这项拨款的目的之一是确定触发Emi1破坏的关键事件，包括触发Emi1破坏的激酶。我们也开始表征大量的Emi1相互作用蛋白（kIPs），以更好地了解控制APC的调控网络。在这里，我们发现由Evi5致癌基因编码的蛋白与Emi1相互作用，并在Emi1上游起作用，指导Emi1和细胞周期蛋白a的积累。Evi5是小鼠t细胞淋巴瘤中常见的前病毒插入位点，在人类神经母细胞瘤中发生突变。此外，它在多种肿瘤中高度表达。Evi5蛋白被预测为一种GTPase激活蛋白，但GTPase仍然未知。我们发现Evi5是G1早期Emi1和细胞周期蛋白A积累所必需的，新的证据表明它可能参与了泛素化后泛素化蛋白向蛋白酶体传递的调节步骤。因此，Evi5可能通过一种以前未知的机制促进肿瘤的发生。我们的目标是：(1)确定在G1-S和有丝分裂中控制Evi5功能的因子；(2)确定Evi5控制蛋白水解的机制；(3)鉴定Evi5调控的关键小GTPase；(4)确定有丝分裂中控制Emi1破坏的因素。

DESCRIPTION (provided by applicant): The timing of events in mitosis is critical to ensure accurate chromosome segregation and genomic stability. Critical to these timing events is the Anaphase Promoting Complex, an E3 ubiquitin ligase that directs the ordered destruction of cyclin A, the chromosome segregation regulator Securin, and cyclin B. The known controllers of APC activity are the components of the spindle assembly checkpoint and the zinc binding protein Emi1. Emi1 functions to restrain the APC in S and G, thereby allowing the accumulation of cyclins. Emi1 is transcriptionally activated in G1 by the cyclin D/Rb/E2F pathway and destroyed in early mitosis following phosphorylation specific binding of the SCFI3TrCP ubiquitin ligase. One aim of this grant is to identify the critical events triggering Emi1 destruction including the kinases that trigger Emi 1 destruction. We have also begun to characterize a large number of Emi1 interacting proteins (kIPs) to better understand the network of regulation controlling the APC. Here, we find the protein encoded by the Evi5 oncogene interacts with Emi1 and functions upstream of Emi1 to direct the accumulation of both Emi1 and cyclin A. Evi5 is a frequent site of proviral insertion in mouse T-cell lymphomas and is mutated in human neuroblastoma. Additionally, it is highly expressed in a variety of tumors. The Evi5 protein is predicted to be a GTPase activating protein, but the GTPase remains unknown. We find that Evi5 is required for accumulation of Emi1 and cyclin A in early G1 and new evidence suggests that it may participate in steps regulating the post-ubiquitination delivery of ubiquitinated proteins to the proteasome. Thus, Evi5 may promote oncogenesis by a previously unknown mechanism. Our aims here are to (1) define factors controlling Evi5 function at G1-S and in mitosis; (2) to identify the mechanism for Evi5 control of proteolysis; (3) to identify the critical small GTPase regulated by Evi5; and (4) to define the factors controlling Emi1 destruction in mitosis.