Cdc25A Regulation by Cell Cycle Signalling and DNA Damage

描述（由申请人提供）：细胞分裂由调节细胞周期转换的细胞周期蛋白依赖性激酶（Cdks）控制。与Cdks相反的是磷酸化和磷酸化Cdks的Wee 1激酶。Wee 1施加的负调节被Cdc 25蛋白磷酸酶家族解除，Cdc 25蛋白磷酸酶家族使Cdk 2和Cdc 2去磷酸化并激活Cdk 2和Cdc 2。 Cdk是DNA损伤反应的主要靶标，并且DNA损伤后Cdk抑制的缺乏导致抗辐射DNA合成，这是S期内检查点缺陷的标志。Cdc 25 A在DNA损伤中的失活对S期内检查点至关重要。我们已经确定的信号转导途径，导致降解的Cdc 25 A在间期和响应DNA损伤。Cdc 25 A降解需要涉及Chkl和尚未鉴定的激酶的多激酶级联。这些激酶共同在Cdc 25 A上产生磷酸降解决定子，这是其被SCF-B-TRCP泛素连接酶快速泛素化和被蛋白酶体降解所必需的。鉴定参与Cdc 25 A降解的新型激酶，并确定它们如何响应DNA损伤而受到调控，是本提案的中心目标。Cdc 25 A也被认为是一种致癌基因，其水平在人类癌细胞中通过转录和转录后机制升高。然而，去调控Cdc 25 A作为癌基因发挥功能的能力从未得到严格证明。基于组织培养的实验和体内实验都将用于严格评估Cdc 25 A的失调形式作为癌基因发挥功能的能力。虽然Cdc 25 A转录在癌症的一个子集中被激活，但关于调节其表达的机制知之甚少。Cdc 25 A调节通路中的阳性和阴性成分是癌症中失调的可能点。组织培养细胞中的遗传方法将用于鉴定正向和负向调节Cdc 25 A表达的蛋白质。此外，全基因组shRNA筛选将用于鉴定DNA损伤途径的新组分，使用Cdc 25 A的破坏作为检查点功能的标记。总之，这些实验将定义Cdc 25 A调控的机制，并将增强我们对Cdc 25 A失调参与人类癌症的理解。

DESCRIPTION (provided by applicant): Cell division is controlled by cyclin-dependent kinases (Cdks) which regulate cell cycle transitions. Acting in opposition to Cdks are the Wee1 kinases that phosphorylate and inactivate Cdks. Negative regulation imposed by Wee1 is relieved by the Cdc25 family of protein phosphatases, which dephosphorylate and activate Cdk2 and Cdc2. Cdks are a primary target of the DNA damage response and the absence of Cdk inhibition after DNA damage leads to radio-resistant DNA synthesis, a hallmark of the defects in the intra-S-phase checkpoint. Inactivation of Cdc25A in response to DNA damage is critical to the intra-S-phase checkpoint. We have determined the signal transduction pathway that leads to degradation of Cdc25A during both interphase and in response to DNA damage. Cdc25A degradation requires a multi-kinase cascade involving Chkl and an as yet unidentified kinase. Together, these kinases generate a phosphodegron on Cdc25A that is required for its rapid ubiquitination by the SCF-B-TRCP ubiquitin ligase and degradation by the proteasome. The identification of novel kinases involved in Cdc25A degradation and a determination of how they are regulated in response to DNA damage are central aims of this proposal. Cdc25A has also been suggested to be an oncogene and its levels are elevated in human cancer cells through both transcriptional and post-transcriptional mechanisms. However, the ability of deregulated Cdc25A to function as an oncogene has never been rigorously demonstrated. Both tissue culture based and in vivo experiments will be used to rigorously assess the ability of deregulated forms of Cdc25A to function as oncogenes. Although Cdc25A transcription is activated in a subset of cancers, relatively little is known concerning the mechanisms that regulate its expression. Positive and negative components in the Cdc25A regulatory pathway are possible points of deregulation in cancer. Genetic approaches in tissue culture cells will be used to identify proteins that positively and negatively regulate Cdc25A expression. Moreover, genome-wide shRNA screens will be used to identify new components of the DNA damage pathway using destruction of Cdc25 A as a marker for checkpoint function. In total, these experiments will define the mechanisms underlying Cdc25A regulation and will enhance our understanding of the involvement of Cdc25A deregulation in human cancer.