Development of a proteome-wide assay to measure phosphatase substrate dephosphorylation rates in human cells (360G-Wellcome-211586_Z_18_Z)

Temporal ordering of cellular events during mitosis is crucial for accurate cell division. Temporal control is dynamically regulated by reversible phosphorylation by the opposing activities of kinases and phosphatases. Recent work in yeast has suggested that intrinsic differences in substrate phosphorylation rates by kinases are important for imposing temporal order. Compared to kinases, much less is understood about the full repertoire of phosphatase substrates in human cells and how dephosphorylation rates vary among substrates. To address this gap in knowledge, I will develop a screen to measure substrate dephosphorylation rates in cells using purified phosphatases and fixed human cells. These methods on fixed, permeabilised cells provide an opportunity to measure biochemical properties of proteins in the context of a static, but intact macromolecular environment. The project will be a collaborative effort between the Welburn and Ly laboratories. I will first express and purify the mitotic phosphatases in the Welburn lab and then perform phosphatase reactions on fixed human cells in the Ly lab. I will focus on PP1gamma and PP6, phosphatases that have been previously shown to be important in regulating mitotic progression. The assay will then be used for large-scale characterization of substrate dephosphorylation kinetics by mass spectrometry-based phosphoproteomics.