Cdc25A Regulation by Cell Cycle Signalling and DNA Damage

描述(申请人提供)：细胞分裂是由调控细胞周期转变的细胞周期依赖性蛋白激酶(CDK)控制的。与CDKs相反的是使CDKs磷酸化和失活的Wee1激酶。CDC25蛋白磷酸酶家族可以解除Wee1施加的负调控，使CDK2和CDc2去磷酸化并激活。CDK是DNA损伤反应的主要靶点，在DNA损伤导致抗辐射的DNA合成后，CDK没有被抑制，这是S阶段检查点缺陷的一个标志。CDC25A对DNA损伤的失活对S期内检查点至关重要。我们已经确定了导致CDc25A在间期和DNA损伤反应中降解的信号转导途径。CDC25A的降解需要涉及Chk1和一种尚未确定的激酶的多激酶级联反应。这些激酶共同在CDc25A上产生一个磷酸降解子，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。识别与CDc25A降解有关的新的激酶，并确定它们如何调节以应对DNA损伤，是这项提案的中心目标。CDC25A也被认为是一种癌基因，其水平在人类癌细胞中通过转录和转录后机制升高。然而，去调控的CDC25A作为癌基因发挥作用的能力从未得到严格的证明。组织培养和体内实验都将被用来严格评估非调控形式的CDC25A作为癌基因发挥作用的能力。尽管CDC25A转录在一组癌症中被激活，但关于调节其表达的机制相对知之甚少。CDC25A调控通路中的正负向成分可能是肿瘤中去调控点。组织培养细胞中的遗传方法将被用来识别正向和负向调节CDC25A表达的蛋白质。此外，全基因组的shRNA筛选将被用来识别DNA损伤途径的新成分，将CDC25A的破坏作为检查点功能的标志。总之，这些实验将确定CDC25A调控的潜在机制，并将增强我们对CDC25A去调控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.