Mechanism of mitotic exit in budding yeast

描述(申请人提供)：所有真核细胞必须通过有丝分裂才能分裂。有丝分裂的退出是通过使驱动细胞进入有丝分裂的同一有丝分裂周期蛋白/细胞周期蛋白依赖性激酶(CDK)失活来实现的。在发芽酵母中，有丝分裂周期蛋白/CDK在有丝分裂结束时通过两种不同的机制下调：Swi5诱导CDK抑制剂SIC1，Hct1(又名CDH1)和后期促进环体复合体(APC)泛素连接酶促进有丝分裂周期蛋白的周转。在有丝分裂的早期，有丝分裂的Cyclin/CDK通过在Swi5和Hctl上附着抑制性磷酸盐来阻止其拮抗剂。在后期-末期，蛋白磷酸酶CDC14通过逆转这些抑制的磷酸化作用而使有丝分裂退出。有丝分裂退出的一个分水岭事件是CDC14的激活，这是通过从核仁锚定蛋白Net1释放CDC14来实现的。因此，了解细胞如何退出有丝分裂的关键是了解在有丝分裂结束时，CDC14是如何从核仁中动员起来的。这种动员涉及两条信号通路：CDC 14早期后期释放(FARE)网络和有丝分裂退出网络(MEN)。恐惧网络在早期后期激发了cdc14从核仁中的瞬时释放，而男性则以某种方式将这种短暂的核游动转化为cdc14在细胞中更持续的扩散，从而能够驱动有丝分裂的退出。本申请中描述的三个具体目标的目标是了解恐惧网络和男性如何导致CDC14的激活。在第一个目标中，我们将试图确定恐惧网络和有丝分裂周期蛋白/CDK对Net1的磷酸化是如何合作带来恐惧的。在第二个目标中，我们将解决这样的假设，即男性的一个关键功能是通过恐惧网络的作用改变从核仁释放的CDC14的核质分布。在第三个目标中，我们将寻找作为MAN组件Dbf2底物的蛋白质，以了解这种蛋白激酶如何导致CDC14在细胞中的持续扩散。考虑到恐惧网络和人类蛋白质的保守性，这里描述的工作具有很好的潜力，可以深入了解正常和癌症人类细胞的生长和分裂，从而突出抗癌药物的新候选靶点。我们的工作也可能为理解其他使用可逆核仁闭锁的通路-例如ARF-MDM2-P53电路-是如何调控的提供了一个有用的范例。

DESCRIPTION (provided by applicant): All eukaryotic cells must exit mitosis to divide. Exit from mitosis is achieved by inactivation of the same mitotic cyclin/cyclin-dependent kinase (CDK) enzyme that drives cells into mitosis. Mitotic cyclin/CDK is down-regulated at the end of mitosis by two different mechanisms in budding yeast: induction of the CDK inhibitor Sic1 by Swi5 and the turnover of mitotic cyclin promoted by Hctl (aka Cdhl) and the anaphasepromoting complex-cyclosome (APC) ubiquitin ligase. Early in mitosis, mitotic cyclin/CDK keeps its antagonists at bay by attaching inhibitory phosphates on Swi5 and Hctl. During anaphase-telophase, the protein phosphatase Cdc14 enables the exit from mitosis by reversing these inhibitory phosphorylations. A watershed event in the exit from mitosis is the activation of Cdc14, which is achieved by its release from the nucleolar anchor protein Net1. Thus, a key to understanding how cells exit mitosis is to understand how Cdc14 is mobilized from the nucleolus at the end of mitosis. This mobilization involves two signaling pathways: the Cdc Fourteen Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN). The FEAR network instigates the transient release of Cdc14 from the nucleolus during early anaphase, whereas the MEN somehow transforms this brief nuclear furlough into a more sustained dispersal of Cdc14 throughout the cell that is able to drive exit from mitosis. The goal of the 3 specific aims described in this application is to understand how the FEAR network and the MEN bring about the activation of Cdc14. In the first aim we will seek to determine how the FEAR network and phosphorylation of Net1 by mitotic cyclin/CDK collaborate to bring about FEAR. In the second aim we will address the hypothesis that a key function of the MEN is to alter the nucleocytoplasmic distribution of Cdc14 released from the nucleolus through the action of the FEAR network. In the third aim, we will seek proteins that serve as substrates of the MEN component Dbf2 to understand how this protein kinase brings about sustained dispersal of Cdc14 throughout the cell. Given the conservation of the FEAR network and MEN proteins, the work described here has excellent potential to shed insight into the growth and division of normal and cancerous human cells, and thereby highlight new candidate targets for anti-cancer drugs. Our work may also provide a useful paradigm for understanding how other pathways that employ reversible nucleolar sequestra - such as the ARF-Mdm2- p53 circuit - are regulated.