Novel approaches for the discovery of dephosphorylation control in oocyte meiosis

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PROJECT SUMMARY Precise regulation of M-Phase in oocyte meiosis is essential for successful embryo development and female fertility. Highlighting this, ~1% of women are subfertile/infertile due to meiotic failure. Despite this, the precise molecular mechanisms governing M-Phase is only partly understood in oocytes. This research will focus on the phosphatase PP1 (Protein Phosphatase 1). PP1 is an important regulator of mitotic M-Phase and is responsible for ~50% of all dephosphorylations. However, the specific roles of PP1 in mammalian oocytes is unclear. PP1 is a holoenzyme consisting of a catalytic subunit (PP1c) and one to two PP1-interacting proteins (PIPs). In mammalian cells, there are three PP1cs (α, β, and γ) and >180 PIPs. Historically, PP1 research has focused on PP1c in isolation, leading to the misconception that PP1c is promiscuous. However, PIPs control localization, enzyme activity, and substrates of PP1c. Additionally, disease states mediated by aberrant PP1 function (e.g., heart disease) result from changes in the PP1c interactome, and not PP1c. Furthermore, studies of PP1c in oocytes, using dual PP1/PP2A inhibitors, overexpression, and anti-PP1c antibody injections, have yielded conflicting outcomes. Importantly, my preliminary data using a specific and novel PIP-based approach to inhibit PP1c has found PP1 activity is essential for meiosis I completion. To gain further insights into oocyte meiosis, this proposed research will: (1) determine the essential roles of PP1 in oocyte meiosis and; (2) develop novel tools for the specific and temporal control of PP1c throughout M-Phase to resolve controversies and unknowns about the roles of PP1 in oocytes. Aim 1 will determine the function of PP1c in oocyte meiosis with a new PIP-based manipulation approach combined with inducible protein degradation. In Aim 2, a novel method for reversible small molecule-mediated caging will be developed -- auxin-controllable caging (ACC). ACC will be used to specifically and temporally regulate PP1c, further defining the roles of PP1c in oocyte meiosis. Of note, this new state-of-the-art tool can be used to study any protein-of-interest. Finally, Aim 3 will use both hypothesis-driven and discovery-driven approaches to determine PP1 holoenzyme function and formation during meiosis. Specifically, this aim will establish the function of two PP1 holoenzymes (PP1c:PNUTS and PP1c:NIPP1) during oocyte meiosis, and map the PP1c:PIP interactome throughout oocyte meiosis. Overall, this project will shed light on a poorly understood, but health-relevant phosphatase, PP1, elucidating its functions in oocytes and more broadly M-Phase regulation. Ultimately, this work will develop a highly valuable tool for the research community, and inform the development of novel treatments for M-Phase based diseases including infertility.