Regulation of Mdm2 compartmentalization

Mdm2 区室化的调控

• 批准号: 7064306
• 负责人: LINDSEY D MAYO
• 金额: $ 23.61万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-11 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7064306
• 关键词: DNA damage; apoptosis; biological signal transduction; epidermal growth factor; insulinlike growth factor; kinase inhibitor; mitogen activated protein kinase; mitogens; neoplasm /cancer genetics; neoplastic growth; nuclear proteins; nuclear transport; oncoproteins; p53 gene /protein; phosphorylation; posttranslational modifications; protein localization; site directed mutagenesis; small interfering RNA; tissue /cell culture

DESCRIPTION (provided by applicant): Mitogens play an integral role in resistance, proliferation, and survival of human tumors. Unraveling the intricate signal transduction pathways that regulate tumorigenesis is a critical step in identifying new therapeutic targets for disruption to impede the progression of cancer. It is not clear as to what extent tumor derived mitogens such as IGF-1 and EGF-1 signal to activate the oncogenic activity of the murine double minute protein (Mdm2). Mdm2 plays a role in tumorigenesis in part through the disruption of the tumor suppressor protein p53. It has been assumed that Mdm2 nuclear localization was a passive process requiring only the nuclear localization sequence (NLS). We show that IGF-1 activated PI3-K/Akt signaling induces the phosphorylation and nuclear translocation of Mdm2, which disrupts p53 activity and facilitates p53 degradation. The p53-Mdm2 complex is exported from the nucleus for both proteins to be degraded. Thus, it seems likely that there is a second signal transduction pathway responsible for mediating Mdm2 nuclear export. We hypothesize that Mdm2 nuclear export is regulated by post-translational modifications mediated by the mitogen activated protein kinase pathway, and culminates in the inactivation of p53. The specific aims in this proposal are designed to ascertain how signal transduction pathways directly regulate Mdm2 and its activity to block p53 function. [Aim 1] To elucidate the signal transduction pathway that mediates nuclear export of Mdm2 by post-translational modifications. We will use selective pharmacological inhibitors to signaling kinases, and dominant negative kinases to block nuclear export of Mdm2. We will determine post-translational modifications to Mdm2 by mass spectrometry in response to growth factor simulation and pharmacological blockade of growth factor stimulated signaling pathways. The identification of phosphorylation sites required for export will be alter to a non-phosphorylatable amino acids to confirm the requirement of these sites to promote Mdm2 nuclear export. [Aim 2] This specific aim is to characterize the effect of Mdm2 nuclear export on p53 activity and stability in response to growth factors and DNA damage. Preventing Mdm2 nuclear export should have a dramatic effect on p53 activity. We will examine p53 transcriptional activity when Mdm2 is unable to exit the nucleus. We will also determine if p53 ubiquitation mediated by Mdm2 occurs in the nucleus or cytoplasm and in what compartment the p53-Mdm2 complex is degraded. Biologically, we will test if Mdm2 rendered in the nucleus can attenuate p53 dependent apoptosis in response to DNA damage. Considering that nuclear Mdm2 is prevalent in human cancer, upon completion of this proposal we will understand the contribution of signal transduction pathways in promoting Mdm2 nuclear export and potentially how to regulate export of nuclear Mdm2 to increase p53 dependent apoptosis.

描述(申请人提供)：有丝分裂原在人类肿瘤的耐药、增殖和存活中起着不可或缺的作用。解开调控肿瘤发生的复杂信号转导通路是确定新的治疗靶点的关键一步，通过干扰来阻止癌症的进展。目前尚不清楚肿瘤来源的丝裂原如IGF-1和EGF-1信号在多大程度上激活了小鼠双微小蛋白(MDM2)的致癌活性。MDM2在肿瘤发生中的作用部分是通过破坏肿瘤抑制蛋白P53来实现的。人们认为MDM2的核定位是一个被动的过程，只需要核定位序列(NLS)。我们发现IGF-1激活的PI3-K/Akt信号诱导MDM2的磷酸化和核转位，从而破坏P53的活性，促进P53的降解。P53-MDM2复合体从细胞核中排出，使两种蛋白质都被降解。因此，似乎有可能存在第二条信号转导途径，负责调节MDM2核输出。我们假设MDM2核输出是由丝裂原活化蛋白激酶途径介导的翻译后修饰调控的，最终导致P53失活。这项建议的具体目的是确定信号转导通路如何直接调节MDM2及其阻断P53功能的活性。[目的1]阐明翻译后修饰介导MDM2核输出的信号转导途径。我们将使用选择性的药物抑制剂来信号转导激酶，并使用显性负性激酶来阻断MDM2的核输出。我们将通过质谱学确定MDM2的翻译后修饰，以响应生长因子模拟和药物阻断生长因子刺激的信号通路。确定出口所需的磷酸化位点将改为非磷酸化氨基酸，以确认这些位点促进MDM2核出口的要求。[目的2]研究MDM2核出口对P53活性和稳定性的影响，以应对生长因子和DNA损伤。阻止MDM2核出口应该会对P53活性产生巨大影响。当MDM2无法退出细胞核时，我们将检测P53的转录活性。我们还将确定MDM2介导的P53泛化是否发生在细胞核或细胞质中，以及P53-MDM2复合体在哪个隔室降解。从生物学上讲，我们将测试在细胞核中呈现的MDM2是否可以减弱DNA损伤引起的p53依赖的细胞凋亡。考虑到核MDM2在人类癌症中的普遍存在，在完成这一提议后，我们将了解信号转导通路在促进MDM2核输出中的贡献，以及潜在地如何调节核MDM2的输出以增加P53依赖的细胞凋亡。