Temporal resolution of phosphorylation-mediated signalling events in DNA Damage Repair

基因组的完整性不断受到内源性和外源性侮辱的威胁，这些侮辱会导致DNA损伤的产生。这种损伤触发了DNA损伤反应(DDR)，其中复杂信号网络的激活协调了下游事件，包括细胞周期停滞、DNA修复、衰老或凋亡。这个系统的完整性是至关重要的，因为在许多类型的癌细胞中发现的受损的DDR网络允许它们绕过这种损伤感知或逃避诱导细胞凋亡。DDR途径包括检测损伤的传感器分子、放大信号的信号转导器和被招募/激活以调节下游事件的效应器分子。不同的网络被激活取决于损伤的类型和发生损伤的细胞周期阶段。可逆的磷酸化早已被认为是DDR中的一个关键调控事件，但尽管人们对介导信号事件的丝氨酸/苏氨酸蛋白激酶的原理知之甚少，但令人惊讶的是，人们对它们的初级同源蛋白的同样重要的作用知之甚少，其中包括丝氨酸/苏氨酸磷酸酶PP1和PP2A。 PP1和PP2A介导的去磷酸化事件的分析由于其独特的调控机制而变得复杂，其中一个共同的催化亚基动态地分布在一个共享的调节亚单位池之间，形成具有特定定位和底物特异性的全酶复合体。我们已经开发了一些策略，使我们能够监测全酶复合体之间催化亚基的动态亚细胞分布，并以高特异性改变单个复合体的水平或活性。我们将荧光成像与定量相互作用组图相结合，为我们提供了核PP1分布的全面空间图，并确定了几个针对DDR底物的复合体。标准工作流程包括互补定量亲和纯化/质谱仪(AP/MS)和邻近标记方法，以高分辨率绘制时空相互作用图，基于CRISPR/Cas9的突变和定量磷酸蛋白质组图，以确定底物中的特定残基。 为了更全面地了解这一复杂途径的信号动力学，以下三个短期目标将使我们能够以前所未有的详细程度从功能上剖析PP1和PP2A磷酸酶复合体对DDR的贡献： 1.核全酶复合体中PP1和PP2A催化亚基在不同类型DDR反应中稳态分布的MAP变化。 2.构建DDR靶向的PP1和PP2A全酶复合体在损伤后早、中、晚三个时间点的时空相互作用网络图。 3.评估特定复合体的破坏对损伤检测、途径选择、修复时机和DDR相关的去磷酸化事件的影响

The integrity of the genome is constantly threatened by endogenous and exogenous insults that lead to generation of DNA damage. This damage triggers a DNA damage response (DDR) in which activation of a complex signaling network coordinates downstream events that can include cell cycle arrest, DNA repair, senescence or apoptosis. Integrity of this system is critical, as compromised DDR networks found in many cancer cell types allow them to either bypass this damage sensing or evade induction of apoptosis. The DDR pathway comprises sensor molecules that detect the damage, signal transducers that amplify the signal and effector molecules that are recruited/activated to mediate downstream events. Distinct networks are activated depending on the type of damage and cell cycle stage at which it occurs. Reversible phosphorylation has long been known to be a key regulatory event in DDR, but although much is known about the principle Ser/Thr kinases that mediate signalling events, surprisingly little is known about the equally essential roles of their primary counterparts, which include the Ser/Thr phosphatases PP1 and PP2A. Analysis of PP1- and PP2A-mediated dephosphorylation events is complicated by their unique regulatory mechanism, whereby a common catalytic subunit is dynamically distributed between a shared pool of regulatory subunits to form holoenzyme complexes with specific localizations and substrate specificities. We have developed strategies that allow us to monitor the dynamic subcellular distribution of catalytic subunits between holoenzyme complexes and alter the level or activity of individual complexes with high specificity. Our combination of fluorescence imaging with quantitative interactome mapping has armed us with a comprehensive spatial map of nuclear PP1 distribution and identified several complexes directed toward DDR substrates. Standard workflows include complementary quantitative affinity purification/mass spectrometry (AP/MS) and proximity labeling approaches to map spatiotemporal interactomes with high resolution, CRISPR/Cas9-based mutagenesis and quantitative phosphoproteomic mapping to identify the specific residues targeted in substrates. With a long-term goal of building a more complete picture of the signalling dynamics of this complex pathway, the following three short-term objectives will allow us to functionally dissect the contribution of PP1 and PP2A phosphatase complexes to DDR in unprecedented detail: 1. Map changes in the steady-state distribution of PP1 and PP2A catalytic subunits between nuclear holoenzyme complexes in response to distinct type of DDR. 2. Assemble a network map of the spatiotemporal interactomes of DDR-targeted PP1 and PP2A holoenzyme complexes at early, mid and late time points following damage. 3. Assess the effects of disruption of specific complexes on damage detection, pathway choice, repair timing and DDR-associated dephosphorylation events