Regulation of Mdm2 compartmentalization

Mdm2 区室化的调控

• 批准号: 7229474
• 负责人: LINDSEY D MAYO
• 金额: $ 22.64万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7229474
• 关键词: Affect; Alanine; Amino Acids; Apoptosis; Aspartic Acid; Attenuated; Cell Death; Cell Nucleus; Complex; Cytoplasm; DNA Damage; Data; Disruption; Dominant-Negative Mutation; Double Minutes; Embryo; Event; Feedback; Fibroblasts; Genes; Goals; Growth Factor; Growth Factor Receptors; Half-Life; Human; In Vitro; Induction of Apoptosis; Insulin-Like Growth Factor I; Kinetics; Knock-out; MAP Kinase Gene; MAPK Signaling Pathway Pathway; MAPK14 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mitogen-Activated Protein Kinases; Mitogens; Mus; Mutation; Nuclear; Nuclear Export; Nuclear Import; Nuclear Translocation; Oncogenic; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Translational Protein Processing; Process; Protein Overexpression; Protein p53; Proteins; Receptor Signaling; Regulation; Resistance; Ribosomal Protein S6 Kinase; Role; Serine; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Site-Directed Mutagenesis; TP53 gene; Testing; Tumor Suppressor Proteins; Tumor-Derived; Ubiquitin; base; design; in vivo; inhibitor/antagonist; mdm2 protein; novel therapeutics; prevent; response; simulation; therapeutic target; tumor; tumor progression; tumorigenesis; ubiquitin-protein ligase

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（由申请人提供）：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。揭示调节肿瘤发生的复杂信号转导途径是确定新的治疗靶点以阻止癌症进展的关键一步。目前尚不清楚肿瘤来源的丝裂原如IGF-1和EGF-1在多大程度上激活小鼠双分钟蛋白（Mdm2）的致癌活性。Mdm2部分通过破坏肿瘤抑制蛋白p53在肿瘤发生中发挥作用。一直认为Mdm2核定位是一个被动过程，只需要核定位序列（NLS）。我们发现IGF-1激活的PI3-K/Akt信号可诱导Mdm2磷酸化和核易位，从而破坏p53活性并促进p53降解。p53-Mdm2复合体从细胞核输出，使两种蛋白降解。因此，似乎有第二个信号转导通路负责介导Mdm2核输出。我们假设Mdm2核输出受丝裂原活化蛋白激酶途径介导的翻译后修饰调控，并最终导致p53失活。本提案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]阐明通过翻译后修饰介导Mdm2核输出的信号转导途径。我们将对信号激酶和显性负激酶使用选择性药物抑制剂来阻断Mdm2的核输出。我们将通过质谱法确定Mdm2的翻译后修饰，以响应生长因子模拟和生长因子刺激信号通路的药物阻断。出口所需磷酸化位点的鉴定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]这个特定的目的是表征Mdm2核输出在应对生长因子和DNA损伤时对p53活性和稳定性的影响。阻止Mdm2核输出对p53活性有显著影响。当Mdm2不能离开细胞核时，我们将检测p53的转录活性。我们还将确定由Mdm2介导的p53泛素化是否发生在细胞核或细胞质中，以及p53-Mdm2复合物在哪个区室被降解。生物学上，我们将测试Mdm2在细胞核中是否可以减轻p53依赖的细胞凋亡对DNA损伤的反应。考虑到核Mdm2在人类癌症中普遍存在，在完成本提案后，我们将了解信号转导途径在促进Mdm2核输出中的贡献，以及潜在的如何调节核Mdm2的输出以增加p53依赖性细胞凋亡。