Regulation of DNA-damage response by phosphorylation clusters in the p53 signaling network

p53 信号网络中磷酸化簇调控 DNA 损伤反应

• 批准号: 446059690
• 负责人: Professor Dr. Alexander Loewer
• 金额: --
• 依托单位: Institut de Biologie Intégrative de la Cellule (I2BC) (UMR 9198)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/446059690?language=en
• 关键词: Regulation; DNA; damage; response; phosphorylation

The transcription factor p53 coordinates the cellular response to DNA damage. P53 protein level and activity are controlled by a signaling network comprising amongst others the DNA-damage response (DDR) kinases ATM/ATR/DNA-PK (PIKKs), the E3-ubiquitin ligase Mdm2, the phosphatase Wip1 and the kinase Chk2. The activity of the kinases/phosphatases and reciprocal feedbacks generate repeated p53 accumulation pulses, whose duration and number determine p53-mediated transcriptional responses and cell fate. The current view of the system is that i) constitutive expression of p53 and feedback regulation by its E3-ubiquitin ligase Mdm2 maintain low basal protein levels; ii) PIKKs activate p53 and inhibit Mdm2 as long as DNA-damage is present, iii) p53 accumulation triggers Mdm2 and Wip1 expression; iv) Wip1 reverses PIKK-mediated modifications. While these activities largely explain the observed p53 dynamics, recent studies using pharmacological perturbations and biochemical measurements provided evidence for additional regulatory mechanisms.Our groups have focused on unexplored phosphorylation mechanisms in the p53-DDR network: i) proteins show abundant, clustered (de)phosphorylation sites of PIKKs, Chk2, and Wip1, which affect the modification kinetics of key functional phosphosites; ii) sustained p53 pulses depend on Chk2 rather than PIKK activity. Based on these findings, we hypothesize that i) clustered modification sites act as buffers to set thresholds and molecular timers upon DNA-damage, and ii) ATM triggers p53 pulses in response to acute damage, while Chk2 is responsible to maintain p53 activity and enable cellular responses to sustained damage. We propose to validate this model by combining experiments at the biochemical and cellular levels. We will evaluate how the presence of clustered phosphosites shapes the kinetics of the acute and sustained DDR. To this end, we will focus on the modification of Mdm2, Chk2 and Wip1 and how it affects their activity. Specifically, we will i) delineate the competitive activities of PIKKs, Chk2, and Wip1 on p53, Mdm2, Chk2 and Wip1 in a site-specific and quantitative manner using NMR spectroscopy; ii) carry out structural studies to elucidate Mdm2, Chk2 and Wip1 inhibition/destabilization by their phosphorylation; iii) examine the functional role of individual phosphosites using Cas9-mediated genomic engineering and time-resolved live-cell microscopy; iv) evaluate how the balance between PIKKs, Chk2 and Wip1 activities shapes the DDR in cells. This will allow us to build mathematical models accounting for DNA-damage thresholds and long-term dynamics of the p53-driven DDR. Hence, integrating biochemical and structural information, real-time cellular signaling data and a systems biology approach, we will gain a better understanding of the p53-driven DDR and how to manipulate it in the context of cancer therapy.

转录因子p53协调细胞对DNA损伤的反应。P53蛋白水平和活性由信号传导网络控制，所述信号传导网络包括DNA损伤反应（DDR）激酶ATM/ATR/DNA-PK（PIKK）、E3-泛素连接酶Mdm 2、磷酸酶Wip 1和激酶Chk 2等。激酶/磷酸酶的活性和相互反馈产生重复的p53积累脉冲，其持续时间和数量决定p53介导的转录反应和细胞命运。该系统目前的观点是：i）p53的组成型表达和通过其E3-泛素连接酶Mdm 2的反馈调节维持低基础蛋白水平; ii）只要存在DNA损伤，PIKK就激活p53并抑制Mdm 2; iii）p53积累触发Mdm 2和Wip 1表达; iv）Wip 1逆转PIKK介导的修饰。虽然这些活性在很大程度上解释了所观察到的p53动力学，但最近的研究使用药理学扰动和生化测量提供了额外的调节机制的证据。我们的小组集中在p53-DDR网络中未探索的磷酸化机制：i）蛋白质显示出丰富的PIKKs、Chk 2和Wip 1的成簇（去）磷酸化位点，这些位点影响关键功能磷酸化位点的修饰动力学; ii）持续的p53脉冲依赖于Chk 2而不是PIKK活性。基于这些发现，我们假设i）成簇的修饰位点充当缓冲器以在DNA损伤时设置阈值和分子计时器，和ii）ATM触发p53脉冲以响应急性损伤，而Chk 2负责维持p53活性并使细胞能够响应持续损伤。我们建议通过结合生化和细胞水平的实验来验证这个模型。我们将评估聚集磷酸盐的存在如何塑造急性和持续DDR的动力学。为此，我们将重点关注Mdm 2，Chk 2和Wip 1的修饰以及它如何影响它们的活性。具体而言，我们将i）使用NMR光谱以位点特异性和定量的方式描绘PIKK、Chk 2和Wip 1对p53、Mdm 2、Chk 2和Wip 1的竞争活性; ii）进行结构研究以阐明Mdm 2、Chk 2和Wip 1通过其磷酸化的抑制/去稳定化; iii）使用Cas9介导的基因组工程和时间分辨活细胞显微镜检查单个磷酸化位点的功能作用; iv）评估PIKK、Chk 2和Wip 1活性之间的平衡如何塑造细胞中的DDR。这将使我们能够建立数学模型，解释DNA损伤阈值和p53驱动的DDR的长期动力学。因此，整合生物化学和结构信息，实时细胞信号数据和系统生物学方法，我们将更好地了解p53驱动的DDR以及如何在癌症治疗中操纵它。