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损伤的威胁，导致DNA损伤的产生。这种损伤触发DNA损伤反应（DDR），其中一个复杂信号网络的激活协调下游事件，包括细胞周期阻滞、DNA修复、衰老或凋亡。该系统的完整性至关重要，因为在许多癌细胞类型中发现的受损DDR网络允许它们绕过这种损伤感知或逃避诱导凋亡。DDR通路包括检测损伤的传感器分子、放大信号的信号换能器和招募/激活介导下游事件的效应分子。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。长期以来，人们一直认为可逆磷酸化是DDR中的一个关键调控事件，但是尽管人们对介导信号事件的主要丝氨酸/苏氨酸激酶了解甚多，但令人惊讶的是，人们对它们的主要对偶物（包括丝氨酸/苏氨酸磷酸酶PP1和PP2A）同样重要的作用知之甚少。

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