MECHANISMS OF MEMBRANE TARGETING AND ACTIVATION OF PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-KINASES (PIP5Ks)
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Date
2025-02-24
Authors
Duewell, Benjamin
Journal Title
Journal ISSN
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Publisher
University of Oregon
Abstract
The ability for cells to localize and activate peripheral membrane binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyzes the phosphorylation of PI(4)P to generate most of the PI(4,5)P2 lipids found in eukaryotic plasma membrane. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P2. In Chapter II of this dissertation, we examine how two motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis of PIP5K. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P2. We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PI(4,5)P2 lipids. After correctly orienting on the membrane, PIP5K transitions to binding PI(4,5)P2 lipids near the active site through a motif previously referred to as the substrate or PIP binding motif (PIPBM). Our data reveals that the PIPBM has broad specificity for anionic lipids and serves a critical role in regulating membrane association in vitro and in vivo. The strength of the interaction between the PIPBM and various PIP lipids depends on both the membrane density and the extent phosphorylation on the inositol head group. Overall, our data supports a two-step membrane binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P2 production. In Chapter III, we follow up on a recent study that showed PIP5K exist in a weak monomer-dimer equilibrium in solution but can shift to a dimeric state following membrane association. Dimerization potentiates PIP5K function, increasing lipid kinase activity 20-fold, providing a possible mechanism for the rapid PI(4,5)P2 generation seen during signaling events. In Chapter III we established a novel FÖrster Resonance Energy Transfer (FRET) biosensor to detect and quantify PIP5K dimerization on supported lipid bilayer technology using Total Internal Reflection Fluorescence Microscopy (TIRF-M). This FRET biosensor allows for the frequency and duration of PIP5K dimerization to be quantified with high resolution. We used this FRET biosensor to demonstrate that human PIP5K paralogs (α, β, and γ) are able to heterodimerize. Previous studies have shown that PIP4K enzymes inhibit PIP5K enzymes by an unknown mechanism. Here, we use the FRET biosensor to demonstrate the mechanism of inhibition is via blocking the dimer interface. The creation of this PIP5K dimerization FRET biosensor establishes a novel assay for examining how proteins and peptides modulate membrane-mediated dimerization of PIP5K, which will be critical for elucidating the mechanisms that control cellular PI(4,5)P2 lipid homeostasis in the future.
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Keywords
FRET, PIP lipids, PIP5K, Single particle tracking