Dynamics of Signaling Pathways: Mechanism and Function

描述（由申请人提供）：尽管p53信号通路可能是人类细胞中研究得最好的信号通路之一，但其调控和功能仍有许多悬而未决的问题。我们对通路中特定的蛋白质-蛋白质相互作用了解很多，但我们对输入（损伤和应激信号）和输出（从细胞生长到细胞死亡的细胞结果）之间的总体关系知之甚少，以及决定这些关系的反应动力学。我在这里申请的补充资金将允许我的实验室采用两种新颖的方法来解决这些问题。在Aim 1中，我们将跟进最近的发现，使用长期单细胞成像，p53在没有外部DNA损伤的情况下显示自发脉冲。这些脉冲似乎与细胞周期有关，因此我们怀疑它们反映了DNA复制或有丝分裂过程中的自发损伤。我们会问是什么触发了p53自发脉冲，是什么控制了它们的形状和时间。我们将使用我们开发的系统来量化活细胞中双链DNA断裂的数量，以确定p53自发脉冲是否与细胞生长和分裂过程中的自发DNA损伤相关。我们将使用化学和遗传扰动来测试哪些调节因子启动和控制p53自发脉冲。了解p53自发脉冲的调控将为我们提供操纵它们的工具，并开发了解其功能的方法。这些自发的p53脉冲可能在防止非应激细胞中DNA损伤的积累方面起着重要作用——这是p53活性的一个以前未被认识到的特征。我们有初步的数据显示，在非应激条件下p53脉冲不会激活p21，只有一部分细胞在辐照下激活p21。我们怀疑这是由于这些细胞中p53翻译后修饰的差异。在目标2中，我们将使用化学和遗传扰动来强制或阻止特定的p53修饰，并使用活细胞成像测试对p21动力学和细胞命运的影响。我们还将在第一次和第二次脉冲中确定p53的翻译后修饰，并将使用流式细胞术对激活p21的细胞和不激活p21的细胞进行分类和分离。然后，我们将使用质谱法来确定在这些不同的人群中发生了哪些p53修饰。这将使我们第一次能够采用一种无偏倚的方法，将p53修饰的组合与其特定靶基因的激活联系起来。

DESCRIPTION (provided by applicant): Although the p53 signaling pathway is perhaps one of the best studied pathways in the human cell, there are many open questions related to its regulation and function. We understand a great deal about specific protein- protein interactions in the pathway, but we understand little about the overall relationship between input (damage and stress signals) and output (cellular outcomes ranging from cell growth to cell death), and the kinetics of the response that determines these relationships. The supplementary funding I request here would allow my lab to take two novel approaches to these questions. In Aim 1, we will follow up on a recent discovery, made using long-term single-cell imaging, that p53 shows spontaneous pulses in the absence of external DNA damage. These pulses appear to be connected to the cell cycle and we therefore suspect that they reflect spontaneous damage during DNA replication or mitosis. We will ask what triggers p53 spontaneous pulses and what controls their shape and timing. We will use a system we have developed for quantifying the amount of double-stranded DNA breaks in live cells to determine whether p53 spontaneous pulses correlate with spontaneous DNA damage during cellular growth and division. We will use chemical and genetic perturbation to test which regulators initiate and control p53 spontaneous pulses. Understanding the regulation of p53 spontaneous pulses will give us the tools to manipulate them, and to develop approaches to understand their function. It is possible that these spontaneous p53 pulses have an important role in preventing the build-up of DNA damage in unstressed cells - a previously unappreciated feature of p53's activity. We have preliminary data showing that p53 pulses in non-stressed conditions do not activate p21 and only a subset of the cells activates p21 in response to irradiation. We suspect that this is due to differences in the post-translational modifications on p53 in these cells. In Aim 2 we will use chemical and genetic perturbations to force, or prevent specific p53 modifications and test the effect on p21 dynamics and on cell fate using live cell imaging. We will also determine the post-translational modification of p53 during the first and second pulse and will use flow cytometry to sort and isolate cells that activate p21 versus cells that do not. We will then use mass spectrometry to determine which p53 modifications occur in these different populations. This will allow us, for the first time, to take an unbiased approach for connecting combinations of p53 modifications with the activation of its specific target genes. PUBLIC HEALTH RELEVANCE: Our study will provide new insights into the control and manipulation of the p53 pathway, perhaps the most important pathway protecting human cells against the development of cancer. These studies will give a deeper understanding of the biological mechanisms and function of p53, and will provide a prototype for the analysis, description, and understanding of the dynamics of other signaling pathways in single living human cells.