Grant: Discovery Grants Program - Individual

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