Biology subjects

Duewell, B. R.

Publications and source records attributed to Duewell, B. R..

3 recordsLinked to original sources

bioRxiv2025

PIP4K attenuates PIP5K lipid kinase activity by disrupting membrane-mediated dimerization

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of enzymes generate most of the phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) lipids in eukaryotes. In solution, PIP5K exists in a weak monomer-dimer equilibrium but undergoes membrane-mediated dimerization, which potentiates lipid kinase activity. In vivo, however, PI(4,5)P2 levels are held remarkably constant due to the homeostatic regulation of PIP5K by PIP4K. We hypothesized that mechanisms that regulate PIP5K dimerization could function to buffer lipid kinase activity, thus providing a mechanism for maintaining relatively constant PI(4,5)P2 levels at the plasma membrane. Due the transient nature and density dependence of PIP5K dimerization, deciphering how other proteins modulate PIP5K dimerization has not been feasible. To address this limitation, we established a single molecule FRET assay to visualize membrane-mediated homodimerization and heterodimerization of PIP5K paralogs on supported lipid bilayers using Total Internal Reflection Fluorescence Microscopy (TIRF-M). Using this approach, we find that PIP4K attenuates PIP5K lipid kinase activity by disrupting membrane-mediated dimerization. Guided by structure prediction, we generated PIP4K mutants that are unable to disrupt PIP5K membrane-mediated dimerization thus preventing the attenuation lipid kinase activity. In vivo, mutations that disrupt the PIP4K-PIP5K interaction similarly prevent PIP4K-mediated inhibition of the PIP5K activity. Overall, this work reveals the molecular basis of the PIP4K-mediated inhibition of PIP5K, which underlies PI(4,5)P2 lipid homeostasis. Creation of this PIP5K dimerization FRET biosensor also establishes a novel tool for deciphering how proteins modulate membrane-mediated dimerization of PIP5K in the future.

biochemistry↗

bioRxiv2023

Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P2 with positive feedback

The ability for cells to localize and activate peripheral membrane binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes in eukaryotic cells 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) catalyze the phosphorylation of PI(4)P to generate most PI(4,5)P2 lipids found in cells. 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. Here, we examine how two PIP5K motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis. 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 PIP lipids. Attaching the PIP5K specificity loop to other peripheral membrane binding proteins can enhance their membrane binding dynamics. After correctly orienting on the membrane, PIP5K transitions to binding PIP lipids in a structural 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 PIP lipids depends on the membrane density and the extent phosphorylation on the inositol head group. We propose 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.

biochemistry↗

bioRxiv2023

Molecular dissection of PI3Kβ synergistic activation by receptor tyrosine kinases, GβGγ, and Rho-family GTPases

The class 1A phosphoinositide 3-kinase (PI3K) beta (PI3K{beta}) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), heterotrimeric guanine nucleotide-binding protein (G-protein)-coupled receptors (GPCRs), and Rho-family GTPases. The mechanism by which PI3K{beta} prioritizes interactions with various membrane tethered signaling inputs, however, remains unclear. Previous experiments have not been able to elucidate whether interactions with membrane-tethered proteins primarily control PI3K{beta} localization versus directly modulate lipid kinase activity. To address this gap in our understanding of PI3K{beta} regulation, we established an assay to directly visualize and decipher how three distinct protein interactions regulate PI3K{beta} when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling membrane localization of PI3K{beta}, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3K{beta} prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either G{beta}G{gamma} or Rac1(GTP). Although pY peptides strongly localize PI3K{beta} to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/G{beta}G{gamma} or pY/Rac1(GTP), PI3K{beta} activity is dramatically enhanced beyond what can be explained by simply increasing the strength of membrane localization. Instead, PI3K{beta} is synergistically activated by pY/G{beta}G{gamma} and pY/Rac1(GTP) through a mechanism consistent with allosteric regulation.

biochemistry↗