Molecular basis of phosphatidylinositol 4-kinase alpha (PI4KA) regulation by regulatory protein partners and post-translational modifications

Type III phosphatidylinositol 4-kinase alpha (PI4KA) is a lipid kinase that generates the primary pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane (PM). It is a master regulator of PM asymmetry and identity and provides PI4P substrate for downstream PI(4,5)P₂/PIP₃ lipid signaling pathways. The regeneration of PI4P by PI4KA is tightly spatiotemporally regulated; however, the mechanism by which this is achieved is poorly defined. How PI4KA activity and PM recruitment are modulated, particularly by membrane-anchored regulators like EFR3A/B and the serine/threonine phosphatase calcineurin, and by post-translational modifications (PTMs), remains unclear. Using cryogenic electron microscopy, hydrogen deuterium exchange mass spectrometry, AI-enabled modelling, binding and kinetic assays, and supported lipid bilayer TIRF microscopy, my dissertation reveals how these membrane-associated regulators interact with the PI4KA complex and how phosphorylation affects PI4KA function. Specifically, it reveals the molecular basis of EFR3-mediated PM recruitment through direct contacts with the PI4KA regulatory proteins TTC7 and FAM126 and describes the development and characterization of a nanobody that blocks this interaction. It also identifies a novel phosphorylation site in PI4KA that inhibits kinase activity. Finally, it defines the PI4KA complex-calcineurin interfaces, providing insight into how calcium signaling directly regulates regeneration of the PM PI4P pool. Given the roles of PI4KA in viral infection and neurological, immunological, and gastrointestinal diseases, these findings provide a molecular framework for understanding how PI4KA activity is tuned in cells and how it may be therapeutically manipulated. More broadly, our identification of a functional phosphorylation site within the PI4KA complex points to a larger, yet unresolved, regulatory landscape governing complex assembly and activity.