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

转录因子p53协调细胞对DNA损伤的反应。P53蛋白水平和活性由一个信号网络控制，该信号网络包括dna损伤反应（DDR）激酶ATM/ATR/DNA-PK （PIKKs）、e3 -泛素连接酶Mdm2、磷酸酶Wip1和激酶Chk2。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了p53介导的转录反应和细胞命运。目前关于该系统的观点是：1)p53的组成性表达及其e3 -泛素连接酶Mdm2的反馈调节维持了较低的基础蛋白水平；ii)只要存在dna损伤，PIKKs激活p53并抑制Mdm2， iii) p53积累触发Mdm2和Wip1的表达；iv) Wip1逆转pikk介导的修饰。虽然这些活动在很大程度上解释了观察到的p53动力学，但最近使用药理学扰动和生化测量的研究为其他调节机制提供了证据。我们的团队专注于p53-DDR网络中尚未探索的磷酸化机制：i)蛋白质显示大量的PIKKs， Chk2和Wip1的簇状（去）磷酸化位点，这影响了关键功能磷酸位点的修饰动力学；ii)持续的p53脉冲依赖于Chk2而不是PIKK活性。基于这些发现，我们假设i)聚集修饰位点作为缓冲物，在dna损伤时设置阈值和分子计时器，ii) ATM在急性损伤时触发p53脉冲，而Chk2负责维持p53活性并使细胞对持续损伤作出反应。我们建议通过结合生化和细胞水平的实验来验证这一模型。我们将评估簇状磷的存在如何影响急性和持续DDR的动力学。为此，我们将重点关注Mdm2， Chk2和Wip1的修饰及其对其活性的影响。具体来说，我们将i)利用核磁共振光谱以位点特异性和定量的方式描述PIKKs、Chk2和Wip1对p53、Mdm2、Chk2和Wip1的竞争活动；ii)进行结构研究，阐明Mdm2、Chk2和Wip1的磷酸化抑制/不稳定；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位点的功能作用；iv)评估PIKKs、Chk2和Wip1活性之间的平衡如何影响细胞中的DDR。这将使我们能够建立数学模型来解释p53驱动的DDR的dna损伤阈值和长期动态。因此，整合生化和结构信息、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的背景下操纵它。

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.