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。适当的细胞周期转变是由两个主要的E3泛素连接酶复合体APC和SCF对关键细胞周期调节因子泛素依赖的降解的协调波驱动的。然而，调节SCF和APC有序活性的关键机制尚未确定。此前，我们证明了APC/CDH1复合体泛素化，从而针对SCF组分Skp2进行降解，从而对SCF和APC活性相互排斥的原因获得了重要的见解。最近，我们积累的证据表明，F-box蛋白？-TRCP可以靶向CDH1进行降解，从而创建一个负反馈环来抑制APC活性。这一发现扩大了我们对紧密协调SCF和APC复合体活动的潜在机制的理解。在具体目标#1中，我们将利用遗传和生化方法来探索？-TRCP控制CDH1丰度和活性的潜在分子机制。我们将进一步定义Cyclin A/CDK2和Plk1对CDH1的顺序磷酸化如何触发与？-TRCP的相互作用和随后的泛素化。这些研究有望揭示？-TrCP通过适时破坏CDH1在调控S进入时相中的重要作用。最近的研究表明，除了细胞周期调节功能外，β-TRCP还在S和G2DNA损伤反应检查点中发挥关键作用，主要是通过破坏其下游靶标CDc25A和Claspin。然而，要充分了解？-TRCP在DNA损伤反应中的功能，特别是在调节主要由P53途径调控的G1损伤反应检查点中的功能，还需要进一步的研究。MDM2是P53的主要负调控因子，在肿瘤中经常过表达，但其潜在机制尚不清楚。我们最近报道，SCF？-TRCP是一种新的E3泛素连接酶，以CK1依赖的方式靶向MDM2泛素化和破坏。但在很大程度上仍不清楚CK1是如何发挥作用的。在DNA损伤后被激活，以控制MDM2/P53途径。在特定的目标#2中，我们打算通过使用多学科方法来继续这项创新研究，以研究ATM如何通过调节CK1来调节MDM2的稳定性？活性和细胞定位。我们还将研究MDM2是否是主要的生理信号通路，通过？-TRCP调节p53通路来调控DNA损伤反应，以及不可降解的MDM2(？p1-23A)是否显示出高的致癌活性。此外，我们还将探索在临床上，MDM2破坏途径(？-TRCP/ATM/CK1？)的组成部分是否会中断。促进肿瘤的形成，这可能是肿瘤中常见的MDM2积聚的原因。我们提出的研究将为控制MDM2破坏的信号通路提供新的见解。这也为开发CKI和ATM激动剂作为抗癌药物提供了理论基础。

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.