Regulation of Mdm2 compartmentalization

描述（由申请人提供）：有丝分裂原在人类肿瘤的耐药性、增殖和存活中起着不可或缺的作用。解开调节肿瘤发生的复杂信号转导通路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有丝分裂原如IGF-1和EGF-1信号在多大程度上激活鼠双微蛋白（Mdm 2）的致癌活性。mdm 2通过破坏肿瘤抑制蛋白p53在肿瘤发生中发挥作用。Mdm 2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PI 3-K/Akt信号转导诱导Mdm 2的磷酸化和核转位，这破坏了p53活性并促进p53降解。p53-Mdm 2复合物从细胞核输出，用于降解两种蛋白质。因此，似乎有可能是第二个信号转导途径负责介导Mdm 2核输出。我们假设Mdm 2核输出受有丝分裂原活化蛋白激酶途径介导的翻译后修饰的调节，并最终导致p53的失活。该提案的具体目标旨在确定信号转导途径如何直接调节Mdm 2及其活性以阻断p53功能。[Aim 1]阐明Mdm 2通过翻译后修饰介导核输出的信号转导途径。我们将使用选择性药物抑制剂的信号激酶，和显性负性激酶阻断核输出的Mdm 2。我们将通过质谱法确定Mdm 2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理学阻断。输出所需的磷酸化位点的鉴定将改变为不可磷酸化的氨基酸，以确认这些位点促进Mdm 2核输出的要求。[Aim[2]这个特定的目的是表征Mdm 2核输出对响应于生长因子和DNA损伤的p53活性和稳定性的影响。阻止Mdm 2核输出应该对p53活性产生巨大影响。我们将检查当Mdm 2不能离开细胞核时p53的转录活性。我们还将确定由Mdm 2介导的p53泛素化是否发生在细胞核或细胞质中，以及p53-Mdm 2复合物在什么样的隔室中降解。在生物学上，我们将测试在细胞核中呈现的Mdm 2是否可以减弱响应于DNA损伤的p53依赖性细胞凋亡。考虑到核Mdm 2在人类癌症中普遍存在，完成本提案后，我们将了解信号传导途径在促进Mdm 2核输出中的贡献，以及可能如何调节核Mdm 2的输出以增加p53依赖性细胞凋亡。

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.