Functional analysis of beta-TRCP in cell cycle control and DNA damage response

描述（由申请人提供）：细胞周期进程的异常调节导致基因组不稳定，最终导致癌症发展。适当的细胞周期转换是由两个主要的E3泛素连接酶复合物APC和SCF对关键细胞周期调节因子的泛素依赖性降解的协调波驱动的。然而，介导有序SCF和APC活动的关键机制尚未确定。以前，我们证明了APC/Cdh 1复合物泛素化，从而靶向SCF组分Skp 2降解，从而获得重要的见解，为什么SCF和APC活动是相互排斥的。最近，我们积累的证据表明，F盒蛋白？TRCP可以靶向Cdh 1降解，从而产生负反馈回路来抑制APC活性。这一发现扩展了我们对紧密协调SCF和APC复合物活性的潜在机制的理解。在具体目标#1中，我们将利用遗传和生物化学方法来探索潜在的分子机制，通过这些机制？TRCP控制Cdh 1的丰度和活性。我们将进一步确定细胞周期蛋白A/Cdk 2和Plk 1如何顺序磷酸化Cdh 1，触发相互作用，并随后泛素化，通过？TRCP。这些研究有望揭示？- TRCP通过及时破坏Cdh 1来管理S相进入。最近的研究表明，除了细胞周期调节功能外，？TRCP也已成为S和G2 DNA损伤反应检查点的关键参与者，主要通过破坏其下游靶标Cdc 25 A和Claspin。然而，进一步的调查将是必要的，以充分了解的功能？- TRCP在DNA损伤反应中，特别是在G1期损伤反应检查点的调控中，主要受p53通路的调控。mdm 2是p53的主要负调控因子，在肿瘤中经常过度表达，但其潜在机制尚不清楚。我们最近报道了SCF？TRCP是一种新型的E3泛素连接酶，靶向Mdm 2，在CK 1？依赖的方式。但它仍然在很大程度上未知如何CK 1？在DNA损伤后被激活，以控制Mdm 2/p53通路。在具体目标#2，我们打算继续这项创新的研究，使用多学科的方法来研究如何ATM调制Mdm 2的稳定性，通过调节CK 1？活性和细胞定位。我们还将研究Mdm 2是否是主要的生理信号通路？TRCP调节p53通路以控制DNA损伤反应，以及不可降解的Mdm 2（p1- 23 A）是否显示出升高的致癌活性。此外，我们将探讨是否在临床上，破坏Mdm 2破坏途径的组成部分（？- TRCP/ATM/CK 1？）促进肿瘤发生，这可能是肿瘤中常见的Mdm 2积累的原因。我们提出的研究将为控制Mdm 2破坏的信号通路提供新的见解。它还提供了开发CKI和ATM激动剂作为抗癌剂的基本原理。

DESCRIPTION (provided by applicant): Aberrant regulation of cell cycle progression results in genomic instability that ultimately leads to cancer development. Proper cell cycle transitions are driven by coordinated waves of ubiquitin-dependent degradation of key cell cycle regulators by APC and SCF, the two major E3 ubiquitin ligase complexes. However, the critical mechanisms mediating ordered SCF and APC activities have not yet been identified. Previously we demonstrated that the APC/Cdh1 complex ubiquitinates and thus targets the SCF component Skp2 for degradation, hence gaining important insight as to why SCF and APC activities are mutually exclusive. More recently, we accumulated evidence suggesting that the F-box protein ?-TRCP can target Cdh1 for degradation, thereby creating a negative feedback loop to repress APC activity. This finding extends our understanding of the underlying mechanisms that tightly orchestrate the activity of the SCF and APC complexes. In Specific Aim #1, we will utilize both genetic and biochemical approaches to explore the underlying molecular mechanisms through which ?-TRCP controls Cdh1 abundance and activity. We will further define how sequential phosphorylation of Cdh1 by Cyclin A/Cdk2 and Plk1 triggers the interaction with, and subsequent ubiquitination, by ?-TRCP. The proposed studies are expected to reveal the important function of ?-TRCP in governing S phase entry via timely destruction of Cdh1. Recent studies indicate that beside its cell cycle regulatory function, ?-TRCP has also emerged as a critical player in S and G2 DNA damage response checkpoints, mainly through destruction of its downstream targets Cdc25A and Claspin. However, further investigation will be necessary to fully understand the function of ?-TRCP in DNA damage response, especially in the regulation of the G1 damage response checkpoint, primarily regulated by the p53 pathway. Mdm2 is the major negative regulator of p53 and frequently overexpressed in tumors, yet the underlying mechanisms are unclear. We recently reported that SCF?-TRCP is a novel E3 ubiquitin ligase targeting Mdm2 for ubiquitination and destruction in a CK1?-dependent manner. But it remains largely unknown how CK1? is activated following DNA damage to govern the Mdm2/p53 pathway. In Specific Aim #2, we intend to continue this innovative research by using multi-disciplinary approaches to investigate how ATM modulates Mdm2 stability by regulating CK1? activity and cellular localization. We will also examine whether Mdm2 is the primary physiological signaling pathway by which ?-TRCP regulates the p53 pathway to govern the DNA damage response, and whether non-degradable Mdm2 (¿p1-23A) displays elevated oncogenic activities. Moreover, we will explore whether clinically, disruption of the components of Mdm2 destruction pathways (?-TRCP/ATM/CK1?) facilitates tumorigenesis, which may be responsible for Mdm2 accumulation often seen in tumors. Our proposed studies will provide new insight into the signaling pathways controlling Mdm2 destruction. It also provides the rationale for developing CKI and ATM agonists as anti-cancer agents.